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Impact of stable ghrelin receptor agonists and antagonists on food intake regulation
Holubová, Martina ; Maletínská, Lenka (advisor) ; Kopecký, Jan (referee) ; Sobotka, Luboš (referee)
The thesis is focused on the effect of ghrelin receptor (GHS-R1a) agonists and antagonist on food intake regulation. Ghrelin is the only known periferally produced orexigenic hormone and the only known acylated hormone. GHS-R1a agonists and antagonists could be useful in the treatment of cachexia and obesity, respectively. In the first part of the thesis, newly designed peptidic GHS-R1a agonists were characterized. The agonists were stabilized by replacing octanoylated Ser3 with a fatty acid coupled to diaminopropionic acid by a stable amide bond. Other noncoded amino acids were also incorporated. Ghrelin analogs were modified by replacing the octanoyl group with another fatty acid, incorporation of the second fatty acid or shortening the peptide chain. Most of the tested GHS-R1a agonists were found to possess high affinities for GHS-R1a (Ki = 10-9 - 10-10 nM) and to activate signaling pathways of ghrelin. After subcutaneous (SC) administration to mice, agonists showed significant and prolonged orexigenic effect. In the second part of the thesis, acute and long-term effects of pseudopeptide GHS-R1a agonist JMV1843 were tested in lean C57BL/6 mice. Acute SC administration of JMV1843 to fed mice increased food intake in a dose-dependent manner (ED50 = 1.94 mg/kg). JMV1843 was stable in blood serum in...
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Characterization of CART peptide analogs in vitro and in vivo
Nagelová, Veronika ; Maletínská, Lenka (advisor) ; Vybíral, Stanislav (referee)
Peptide CART (cocaine- and amphetamine- regulated transcript) is a neuropeptide acting in the hypothalamus to reduce food intake (anorexigenic peptide). Despite all efforts the receptor and the mechanism of action is still unknown. This peptide has two biologically active forms, CART(55-102) and CART(61-102). Peptide CART is able to bind to pheochromocytoma cells PC12. PC12 cells differentiated in neuronal phenotype with NGF (nerve growth factor) showed a higher number of binding sites (11250 ± 2520 binding sites/cell) compared to undifferentiated cells (3600 ± 570 binding sites/cell). PC12 cells differentiated by dexamethasone to chromaffin cells showed high non-specific binding. Peptide CART contains three disulfide bridges. To clarify the importance of each disulfide bridge to maintain biological activity, analogues with one (analogue 3, 4 and 5) or two (2, 6, 7 and 8) disulfide bridges and a peptide analogue of CART (61-102), which has methionin at position 67 replaced with norleucine were synthesized. We showed that biological activity was unchanged at analogue 1 and analogue 7 containing disulfide bridges in positions 74-94 and 88-101. When investigating cell signaling in PC12 cells, we tested if peptide CART activate of c-Fos, c-Jun, phosphorylated ERK1/2, CREB, JNK and p38. CART peptide...
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New pharmacological interventions influencing food intake focused on effects of CART (cocaine- and amphetamine-regulated transcript) peptide and prolactin-releasing peptide
Maixnerová, Jana ; Maletínská, Lenka (advisor) ; Zorad, Štefan (referee) ; Skálová, Lenka (referee)
The thesis was focused on characterization of biological activities of two recently discovered anorexigenic neuropeptides: CART (cocaine- and amphetamine-regulated transcript) peptide and prolactin-releasing peptide (PrRP). In order to find a pharmacophore of CART peptide, shorter fragments of CART(61- 102) peptide were tested for binding to PC12 cells and inhibition of food intake in fasted mice. The results showed that a compact structure of CART peptide containing three disulphide bridges is necessary for preservation of its full biological activity. In the second part of the thesis, synergistic and long-lasting effect of centrally administered peptide CART and peripherally administered cholecystokinin (CCK) is described. In fasted C57BL/6 mice, the anorexigenic effect of CART was enhanced by a subthreshold dose of CCK, while CCK1 receptor antagonist devazepide blocked the effect of CART peptide on food intake. In the third part of the thesis, food intake in fed lean and MSG (monosodiumglutamate treated) obese male mice with lesions in nucleus arcuatus (ARC) was followed. Anorexigenic action of CART peptide was preserved but satiety effect of CCK was completely lost in MSG obese mice and therefore, effective leptin signaling in ARC is necessary for satiety effect of CCK. Finally, the PrRP...
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Impact of leptin and ghrelin on food intake and metabolic parameters in obese ovariectomized female mice
Matyšková, Resha ; Maletínská, Lenka (advisor) ; Jurčovičová, Jana (referee) ; Kuneš, Jaroslav (referee)
The thesis is focused on the effect of leptin and ghrelin receptor antagonists on food intake and metabolic parameters in ovariectomized (OVX) female mice on a high fat (HF) diet. In the first part of the thesis, diet-induced obesity was introduced in two strains of mice (NMRI and C57BL/6). Diet-induced obesity resulted from overconsumption of a HF diet containing 60 % of fat; a standard (St) diet contained only 9 % of fat. The strain C57BL/6 was more susceptible to a HF diet than the NMRI strain and was chosen for further experiments on food intake. In the second part of the thesis, OVX C57BL/6 female mice on a HF diet were introduced as a model of common obesity in women after menopause and overconsumption of high caloric food. OVX mice on a HF diet accumulated more than 4 times higher amount of the white adipose tissue compared to those on a St diet, had significantly elevated glucose, insulin and leptin levels and attenuated sensitivity to effect of centrally administered leptin, an adipokine that decreases food intake. Central leptin sensitivity and metabolic syndrome parameters were improved after 4 weeks of estradiol treatment. Because both ovariectomy and HF diet result in enhanced sensitivity to ghrelin, the hormone produced predominantly by the stomach that stimulates appetite, in the...
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Peptide hormones affecting the food intake and their analogs as potential drugs for treatment of obesity
Nagelová, Veronika ; Maletínská, Lenka (advisor) ; Vybíral, Stanislav (referee)
Obesity is nowadays a major global health problem. Every year amount of obese (BMI > 30 kg . m-2 ) and overweight (BMI > 25 kg . m-2 ) people increases. Obesity is not just a cosmetic problem, but it leads to many serious health complications, particularly cardiovascular diseases, metabolic diseases etc. We can define obesity as an excessive amount of body fat. The development of obesity is often influenced by energy intake, which overrides the energy expenditure. Many studies are currently describe the influence of various substances that could potentially act as antiobesity drugs. Peptide hormones, which are engaged in this work, can be divided to the long-term (leptin, insulin, ghrelin) and short-term (e.g. cholecystokinin, glucagon like peptide 1, peptide YY, CART peptides, melanocortin system, neuropeptide Y and melanin concentrating hormone) acting. Peptides can be also divided according to their effect on food intake to the anorexigenic and orexigenic. Anorexigenic peptides reduce food intake, orexigenic do the reverse.
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Mass Spectrometry Imaging of Metformin in Tissue Sections
Strnad, Štěpán ; Sýkora, D. ; Vrkoslav, Vladimír ; Cvačka, Josef ; Maletínská, Lenka ; Pirník, Zdenko
Mass spectrometry imaging is a powerful technique suitable for visualization of the distribution of a wide variety of compounds within tissue sections. The main aim of the study was the development and optimization of a sample preparation procedure allowing determination of the distribution of orally dosed metformin in mice kidney sections. Metformin is the first-line medication for the treatment of type 2 diabetes. The optimization of the sample preparation step before imaging experiments included the selection of a suitable matrix and the optimization of various parameters of MALDI analysis. 2,5-dihydroxybenzoic acid was identified as the best matrix providing highest sensitivity. A sublimation method was successfully used for the matrix deposition. The highest relative concentration of metformin was found in the inner zone of kidney 30 minutes after the drug administration.
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Specific binding of prolactin-releasing peptide analogues in pituitary cells
Maixnerová, Jana ; Špolcová, Andrea ; Matyšková, Resha ; Blechová, Miroslava ; Veselá, Iva ; Železná, Blanka ; Maletínská, Lenka
We report specific binding of 125I-PrRP31 to the following cell lines: GH3 cells (rat – somatotrophs), AtT20 cells (mouse – adenocorticotrophs) and RC-4B/C cells (rat- somatotrophs, lactrophs, adenocortitrophs and gonadotrophs). These results will serve as a basic knowledge for future structure-activity studie sof PrRP.
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