49 research outputs found
α1- and β-adrenergic receptors in brown adipose tissue and the adrenergic regulation of thyroxine 5'-deiodinase
No full text
Tarmo Areda – neuroteaduste filosoofiadoktor
No full text28. septembril 2006 kaitses Tartu Ülikooli neuroteaduse kraadinõukogu ees doktoriväitekirja Tarmo Areda. Doktoritöö “Kassilõhna tekitatud ärevuse mehhanismide käitumuslik ja neurogeneetiline uurimus närilistel” (“Behavioural and neurogenetic study of mechanisms related to cat odour induced anxiety in rodents”) valmis Tartu Ülikooli neuroteaduste doktorikoolis. Töö juhendajateks olid professorid Toomas Asser ja Alar Karis ning vanemteadur Sulev Kõks. Tööd oponeeris dotsent Atso Raasmaja Helsingi Ülikooli farmaatsiateaduskonna farmakoloogia ja toksikoloogia instituudist. Oponent märkis tunnustavalt, et tegemist on väga heal tasemel originaalse ning tervikliku eksperimentaalse uurimistööga
Transkriptoomilised ja metaboolsed muutused WFS1-puudulikkusega hiiremudelis
No full textVäitekirja elektrooniline versioon ei sisalda publikatsiooneWolframi sündroom (WFS) on haruldane pärilik haigus, mida iseloomustavad lapseeas algav insuliinist sõltuv suhkurtõbi, nägemisnärvi kahjustus, magediabeet, kurtus ja mitmed neuroloogilised häired. Haigust põhjustavad mutatsioonid WFS1 geenis. WFS1 omab rakus mitmeid ülesandeid, osaledes endoplasmaatilises retiikulumis (ER) membraanitranspordis, valkude töötlemises ja raku vastuses voltumata valkude kogunemisele. Üha rohkem on teavet selle kohta, et ER homöostaasi kõrvalekalded ja krooniline ER stress on olulised mitmete haiguste arengus, näiteks neurodegeneratiivsed haigused ja diabeet. WFS1 tase ER stressi korral tavaliselt tõuseb ning WFS1 puudumine soodustab kroonilist ER stressi ja raku surma. See omakorda põhjustab haigusele iseloomulike sümptomite avaldumist ja süvenemist.
Käesoleva töö eesmärgiks oli leida WFS hiiremudelis WFS1 puudulikkusest tingitud võimalikke transkriptoomilisi ja metaboolseid muutuseid, mis soodustavad WFS sümptomite arengut. WFS1 puudulikkus põhjustab transkriptoomilisi muutusi hüpotaalamuses, hipokampuses ja pankrease Langerhansi saartes, mis soodustavad haigusega seotud mitmesuguste endokriinsete, neurodegeneratiivsete ja käitumuslike sümptomite teket ja süvenemist. WFS1 puudulikkusse korral väheneb ka pankrease Langerhansi saarte arv ja häirub insuliini sekretsioon. WFS1 puudulikkus põhjustab ka metaboolseid kõrvalekaldeid, sest võrreldes oma normaalsete pesakonnakaaslastega kaaluvad Wfs1 mutansed hiired oluliselt vähem ja neil on lühem eluiga. Kõrge rasvasisaldusega toit põhjustab ühe toimiva Wfs1 geeni koopiaga hiirtel mitmesuguseid metaboolseid muutuseid, nagu kaalutõus, insuliini sekretsiooni muutused ning ER stressiga seotud geenide ekspressiooni muutused. Wfs1 heterosügootsus põhjustab aga variatsiooni nii metaboolsete tüsistuste kui ka ER stressi vahendavate geenide ekspressiooni mustrites, mis on seotud ebasoodsate metaboolsete häirete ja Wolframi sündroomiga seotud sümptomite kujunemisega.Wolfram syndrome (WFS) is a rare inherited disease characterized by juvenile-onset insulin-dependent diabetes, optic nerve atrophy, diabetes insipidus, deafness and several neurological abnormalities. The diseases caused by mutations in the WFS1 gene. WFS1 plays a number of roles in the cell, participating in membrane transport, protein processing, and the cellular response to accumulation of unfolded proteins in the endoplasmic reticulum (ER). There is growing evidence that abnormalities in ER homeostasis and chronic ER stress are important in the development of many diseases, such as neurodegenerative diseases and diabetes. WFS1 levels in case of ER stress usually increase, and the absence of WFS1 contributes to chronic ER stress and cell death. This in turn causes the symptoms of the disease to develop and worsen.
The aim of this study was to identify possible transcriptomic and metabolic changes due to WFS1 deficiency in a WFS mouse model that contribute to the development of WFS symptoms. WFS1 deficiency causes transcriptomic changes in the hypothalamus, hippocampus, and pancreatic islets of Langerhans, which contribute to the development and exacerbation of a variety of endocrine, neurodegenerative, and behavioral symptoms associated with the disease. WFS1 deficiency reduces the number of pancreatic islets and impairs insulin secretion. WFS1 deficiency also causes metabolic abnormalities, because Wfs1 mutant mice weigh significantly less and have a shorter lifespan than their normal littermates. A high fat diet causes a variety of metabolic changes in mice with a single copy of the active Wfs1 gene, such as weight gain, changes in insulin secretion, and changes in ER stress-related gene expression. However, the heterozygosity of Wfs1 causes variation in the expression patterns of both metabolic complications and ER stress-mediating genes associated with the development of adverse metabolic disorders and symptoms related to Wolfram syndrome.https://www.ester.ee/record=b552398
Cellular Stress and p53-Associated Apoptosis by Juniperus communis L. Berry Extract Treatment in the Human SH-SY5Y Neuroblastoma Cells
No full textPlant phenolics have shown to activate apoptotic cell death in different tumourigenic cell lines. In this study, we evaluated the effects of juniper berry extract (Juniperus communis L.) on p53 protein, gene expression and DNA fragmentation in human neuroblastoma SH-SY5Y cells. In addition, we analyzed the phenolic composition of the extract. We found that juniper berry extract activated cellular relocalization of p53 and DNA fragmentation-dependent cell death. Differentially expressed genes between treated and non-treated cells were evaluated with the cDNA-RDA (representational difference analysis) method at the early time point of apoptotic process when p53 started to be activated and no caspase activity was detected. Twenty one overexpressed genes related to cellular stress, protein synthesis, cell survival and death were detected. Interestingly, they included endoplasmic reticulum (ER) stress inducer and sensor HSPA5 and other ER stress-related genes CALM2 and YKT6 indicating that ER stress response was involved in juniper berry extract mediated cell death. In composition analysis, we identified and quantified low concentrations of fifteen phenolic compounds. The main groups of them were flavones, flavonols, phenolic acids, flavanol and biflavonoid including glycosides of quercetin, apigenin, isoscutellarein and hypolaetin. It is suggested that juniper berry extract induced the p53-associated apoptosis through the potentiation and synergism by several phenolic compounds.Peer reviewe
The Water Extract of Juniperus communis L. Induces Cell Death and Sensitizes Cancer Cells to Cytostatic Drugs through p53 and PI3K/Akt Pathways
No full textJuniper (Juniperus communis L.) is a northern coniferous plant generally used as a spice and for nutritional purposes in foods and drinks. It was previously reported that juniper extract (JE) affects p53 activity, cellular stress, and gene expression induced cell death in human neuroblastoma cells. Therefore, the effects of juniper on p53 and Akt signaling was examined further in A549 lung, 22RV1 and DU145 prostate, and HepG2 liver cancer cells using Western blot, confocal microscopy, and MTT analysis. We found that juniper simultaneously decreased cell viability, activated the p53 pathway, and inactivated the PI3K/Akt pathway. The p53 activation was associated with increased nuclear p53 level. Akt was dephosphorylated, and its inactivation was associated with increased levels of PHLPP1 and PHLPP2 phosphatases. Parallel increases of PARP suggest that JE decreased cell viability by activating cell death. In addition, JE potentiated the effects of gemcitabine and 5-fluorouracil anticancer drugs. Thus, JE can activate cell death in different cancer cell lines through p53 and Akt pathways.Peer reviewe
Analysis of metabolic effects of menthol on WFS1-deficient mice
No full textIn this study, we investigated the physiological regulation of energy metabolism in wild-type (WT) and WFS1-deficient (Wfs1KO) mice by measuring the effects of menthol treatment on the O2 consumption, CO2 production, rectal body temperature, and heat production. The basal metabolism and behavior was different between these genotypes as well as TRP family gene expressions. Wfs1KO mice had a shorter life span and weighed less than WT mice. The food and water intake of Wfs1KO mice was lower as well as the body temperature when compared to their WT littermates. Furthermore, Wfs1KO mice had higher basal O2 consumption, and CO2 and heat production than WT mice. In addition, Wfs1KO mice showed a higher response to menthol administration in comparison to WT mice. The strongest menthol effect was seen on different physiological measures 12 h after oral administration. The highest metabolic response of Wfs1KO mice was seen at the menthol dose of 10 mg/ kg. Menthol increased O2 consumption, and CO2 and heat production in Wfs1KO mice when compared to their WT littermates. In addition, the expression of Trpm8 gene was increased. In conclusion, our results show that the Wfs1KO mice develop a metabolic phenotype characterized with several physiological dysfunctions.Peer reviewe
Dopaminergic modulation of reward-guided decision making in alcohol-preferring AA rats
No full textR**esults from animal gambling models have highlighted the importance of dopaminergic neurotransmission in modulating decision making when large sucrose rewards are combined with uncertainty. The majority of these models use food restriction as a tool to motivate animals to accomplish operant behavioral tasks, in which sucrose is used as a reward. As enhanced motivation to obtain sucrose due to hunger may impact its reward-seeking effect, we wanted to examine the decision-making behavior of rats in a situation where rats were fed ad libitum. For this purpose, we chose alcohol-preferring AA (alko alcohol) rats, as these rats have been shown to have high preference for sweet agents. In the present study, AA rats were trained to self-administer sucrose pellet rewards in a two-lever choice task (one pellet vs. three pellets). Once rational choice behavior had been established, the probability of gaining three pellets was decreased over time (50%, 33%, 25% then 20%). The effect of D-amphetamine on decision making was studied at every probability level, as well as the effect of the dopamine D-1 receptor agonist SKF-81297 and D-2 agonist quinpirole at probability levels of 100% and 25%. D-Amphetamine increased unprofitable choices in a dose-dependent manner at the two lowest probability levels. Quinpirole increased the frequency of unprofitable decisions at the 25% probability level, and SKF-82197 did not affect choice behavior. These results mirror the findings of probabilistic discounting studies using food-restricted rats. Based on this, the use of AA rats provides a new approach for studies on reward-guided decision making. (C) 2017 Elsevier B.V. All rights reserved.Peer reviewe
Distribution, regulation and physiological roles of catechol-O-methyltransferase (COMT) in rodents
No full textCatechol-O-methyltransferase (COMT) metabolises catecholamines such as dopamine, noradrenaline and adrenaline, which are well-characterised neurotransmitters that play important roles in the regulation of physiological processes. The COMT enzyme exists in membrane-bound (MB-COMT) and soluble (S-COMT) forms that are both encoded by the same gene. Presently, the necessity and physiological importance of the existence of two COMT isoenzymes remains partially unknown. The aim of this thesis was to provide new insights into the specific distribution patterns of MB-COMT and S-COMT, to verify the proposed hormonal regulation of COMT and to investigate its possible physiological role in feeding behaviour.
We used two different study designs to address the distribution of COMT in the brain. First, we utilised dopaminergic and noradrenergic toxins to assess the presence of COMT in presynaptic neurons originating from selected brain areas. Successful lesioning of the neurons was confirmed by measuring dopamine and noradrenaline levels in their respective projection areas. However, despite successful lesioning, no changes in COMT protein expression or activity could be noted, indicating that COMT is not present in presynaptic dopaminergic and noradrenergic neurons. The specific distribution patterns of MB-COMT and S-COMT were studied in an S-COMT-deficient mouse line (lacking the S-COMT form of COMT). By comparing these mice to wild type mice, we found that the general distribution patterns of both COMT isoforms were similar between the genotypes. Notably, S-COMT-deficient mice did not exhibit nuclear COMT staining, indicating that MB-COMT is not present in cell nuclei. Furthermore, S-COMT-deficient mice allowed us to examine the association of MB-COMT with the plasma membrane. Contrary to previous assumptions, MB-COMT is not associated with the plasma membrane, but instead may be attached to intracellular cytosolic membranes.
The proposed regulatory function of oestrogens on COMT activity was studied by subjecting male and female Wistar rats to different hormonal treatments over a two-week period. Antagonising/modulating the effects of oestrogen via the administration of tamoxifen increased COMT activity and expression. Unexpectedly, increasing oestradiol levels in male rats also led to the upregulation of COMT expression in several tissues, underscoring the importance of COMT in the clearance of biologically active oestrogen metabolites.
Finally, we used female rodents (rats and mice) to address the possible role of COMT in feeding behaviour. Our studies revealed that acute COMT inhibition affects the feeding pattern of rodents; COMT-inhibited animals have significantly more ''long'' meals (lasting 300 sec or more) than vehicle-treated animals. Notably, a relatively short starvation period of 16 h induced a slight, albeit not significant, decrease in prefrontal and striatal COMT activity in mice. This finding is interesting, especially as COMT activity is thought to be rather robust, and may suggest the increased importance of COMT during altered physiological status.
In conclusion, although the specific roles of MB-COMT and S-COMT remain partly obscure, their absence from presynaptic dopaminergic and noradrenergic neuron terminals suggests a secondary (and perhaps more modulatory) role of COMT in neurotransmitter metabolism in the brain. Furthermore, the observed decrease in COMT activity after a relatively short starvation period suggests that COMT has an increased importance during certain physiological states. Finally, the observed regulatory function of oestrogens on COMT activity and protein expression in vivo may be of clinical importance as COMT inhibitors are used as adjuncts in the treatment of PD.Katekoli-O-metyylitransferaasi (COMT) hajottaa katekoliamiineja, kuten dopamiinia, noradrenaliinia ja adrenaliinia, jotka toimivat välittäjäaineina useiden fysiologisten toimintojen säätelyssä. Entsyymiä esiintyy kahta muotoa, kalvoihin sitoutuvaa (MB-COMT) ja liukoista (S-COMT), jotka ovat saman geenin tuotteita. Näiden kahden erillisen isoentsyymin fysiologista merkitystä ja tarpeellisuutta ei vielä tunneta tarkasti.
Tässä tutkimuksessa kartoitettiin COMT:n esiintymistä jyrsijöiden aivoissa kahdessa koeasetelmassa. Rottien aivojen dopamiini- ja noradrenaliinihermosolut tuhottiin selektiivisin hermomyrkyin, jotta COMT:n sijaintia näiden hermosolujen päätteissä tietyillä aivoalueilla voitiin arvioida. Vaikka hermosoluvauriot tuotettiin menestyksellisesti, COMT-proteiinin määrässä tai sen toiminnassa ei nähty muutoksia. Havainnosta pääteltiin, että kyseistä entsyymiä ei ilmeisesti esiinny dopamiini- ja noradrenaliinihermosolujen päätteissä. COMT-isoentsyymien esiintymistä aivoissa tutkittiin lisäksi hyödyntämällä S-COMT -puutteellista hiirikantaa. Verrattaessa näitä hiiriä saman kannan villityypin hiiriin havaittiin COMT-isoentsyymien jakaumien olevan samankaltaiset molemmissa genotyypeissä. S-COMT -puutteellisilta hiiriltä ei kuitenkaan löydetty COMT:a tumista lainkaan, mikä kertoo pelkän liukoisen muodon ilmenevän solujen tumissa. S-COMT -puutteellisia hiiriä hyödynnettiin myös tutkimuksessa, jossa selvitettiin MB-COMT:n liittymistä solukalvoon. Vastoin aiempia oletuksia liittymistä ei kuitenkaan havaittu, vaan ilmeisesti kyseinen muoto sijaitsee solunsisäisten solulimakalvostojen yhteydessä.
Väitöstyössä selvitettiin myös estrogeenien kykyä säädellä COMT:n toimintaa altistamalla Wistar rottanaaraita ja -koiraita kahden viikon ajan erilaisille hormonaalisille käsittelyille. Estrogeenin vaikutusten estäminen tai muunteleminen tamoksifeenilla lisäsi COMT:n aktiivisuutta ja ilmentymistä naarasrotilla. Vastoin odotuksia myös estrogeenin annostelu koirasrotille lisäsi COMT:n ilmentymistä useissa kudoksissa, mikä painottaa COMT:n tärkeää tehtävää estrogeenin biologisesti aktiivisten hajoamistuotteiden poistamisessa.
Lisäksi väitöstyössä tutkittiin COMT:n mahdollista osuutta syömiskäyttäytymisessä naarashiirillä ja rotilla. Tutkimuksessa selvisi, että COMT:n toiminnan lyhytkestoinen esto vaikutti jyrsijöiden tapaan syödä: COMT-estäjää saaneiden eläinten havaittiin ruokailevan pitkään (aterian kesto yli 300 s) useammin kuin verrokkiryhmän eläinten. Lisäksi suhteellisen lyhyt 16 tunnin paastottaminen aiheutti lievän COMT-aktiivisuuden laskun hiirten aivojuoviossa ja etuaivokuorella, joskaan tulos ei ollut tilastollisesti merkitsevä. Tämä mielenkiintoinen havainto viittaa kuitenkin siihen, että COMT:n merkitys saattaa korostua fysiologisen tilan muutoksissa, vaikka COMT:n aktiivisuuden on aiemmin ajateltu olevan hyvin vakaa.
Yhteenvetona tutkimuksista voidaan todeta, että MB-COMT:n ja S-COMT:n samankaltainen jakauma sekä puuttuminen dopaminergisista ja noradrenergisista hermopäätteistä antavat ymmärtää, että COMT:lla on toissijainen (tai muunteleva) rooli hermovälittäjäaineiden aineenvaihdunnassa aivoissa. Havainnoilla estrogeenien vaikutuksesta COMT:n toiminnan säätelyyn sekä sen ilmentymisen kompensatoriseen lisääntymiseen saattaa olla kliinistä merkitystä, sillä COMT-estäjiä käytetään Parkinsonin taudin lääkkeinä. Lisäksi jo suhteellisen lyhyen paaston aiheuttama COMT-aktiivisuuden lasku antaa vihjeitä entsyymin mahdollisesta piilevästä tehtävästä fysiologisissa muutostiloissa. Vaikka COMT:n toiminnan esto ei muuta katekoliamiinien pitoisuuksia perustilanteessa, COMT:n merkitys saattaa kasvaa tietyissä fysiologisissa ja patologisissa tiloissa.ei saavutettav
