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Acute cold exposure is a significant stressor activating heat production by shivering after the prolonged exposure cellular oxidative stress increases. Chronic exposure to cold lasting at least 2 weeks leads to the development of cold acclimatization. The main thermogenic role is taken over by non-shivering thermogenesis taking place in brown adipose tissue, which significantly increases its weight due to cold. Cardiac hypertrophy, hypertension and impaired renal function are frequently observed pathologies of acclimatization at 4-5 řC. Our laboratory recently introduced a model of mild chronic cold acclimatization at 8 řC, during which no damage to the heart or kidneys occurs and has proven cardioprotective effect on reducing infarct size. Hence, the influence of this cold acclimatization model on the other cellular and molecular processes needs to be investigated. The cardioprotective effect of cold acclimatization includes changes in β-AR signaling, activation of anti-apoptotic pathways or augmentation of the antioxidant system. The aim of this thesis was to investigate the effect of cold acclimation and subsequent reacclimation on proteins regulating Ca2+ levels in the rat heart (SERCA2 and phospholamban) and on the stimulation of regulatory proteins β-arrestin 1/2 and protein kinase PDPK1. The...

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京都大学 / 新制・課程博士 / 博士(理学) / 甲第24463号 / 理博第4962号 / 新制||理||1708(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 森 和俊, 教授 川口 真也, 准教授 船山 典子 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

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Indiana University-Purdue University Indianapolis (IUPUI) / Diabetes mellitus is a group of metabolic diseases characterized by disordered insulin secretion from the pancreatic β cell and chronic hyperglycemia. In order to maintain adequate levels of insulin secretion, the β cell relies on a highly developed and active endoplasmic reticulum (ER). Calcium localized in this compartment serves as a cofactor for key proteins and enzymes involved in insulin production and maturation and is critical for ER health and function. The ER Ca2+ pool is maintained largely through activity of the sarco-endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) pump, which pumps two Ca2+ ions into the ER during each catalytic cycle. The goal of our research is to understand the molecular mechanisms through which SERCA2 maintains β cell function and whole body glucose metabolism. Our previous work has revealed marked dysregulation of β cell SERCA2 expression and activity under diabetic conditions. Using a mixture of pro-inflammatory cytokines to model the diabetic milieu, we found that SERCA2 activity and protein stability were decreased through nitric oxide and AMP-activated protein kinase (AMPK)mediated signaling pathways. Moreover, SERCA2 expression, intracellular Ca2+ storage, and β cell death under diabetic conditions were rescued by pharmacologic or genetic inhibition of AMPK. These findings provided novel insight into pathways leading to altered β cell Ca2+ homeostasis and reduced β cell survival in diabetes. To next define the role of SERCA2 in the regulation of whole body glucose homeostasis, SERCA2 heterozygous mice (S2HET) were challenged with high fat diet (HFD). Compare to wild-type controls, S2HET mice had lower serum insulin and significantly reduced glucose tolerance with similar adiposity and systemic and tissue specific insulin sensitivity, suggesting an impairment in insulin secretion rather than insulin action. Consistent with this, S2HET mice exhibited reduced β cell mass, decreased β cell proliferation, increased ER stress, and impaired insulin production and processing. Furthermore, S2HET islets displayed impaired cytosolic Ca2+ oscillations and reduced glucose-stimulated insulin secretion, while a small molecule SERCA2 activator was able to rescue these defects. In aggregate, these data suggest a critical role for SERCA2 and the maintenance of ER Ca2+ stores in the β cell compensatory response to diet induced obesity.

Abstract Melatonin is an important modulator of several physiological and behavioural processes, and it influences the function of many different tissues. Melatonin has effective antioxidative properties, but some of its actions in mammals are also mediated through the MT1 and MT2 melatonin receptors. Antioxidative properties are seen especially when the melatonin concentration is high (≥ nM), and melatonin's affinity for its receptors appears at lower concentrations (pM). Recently, the involvement of melatonin in protecting the heart against cardiac diseases, including myocardial infarction (MI), has been brought out. MI alters the structure and function of myocardium, attenuating for example cardiac contractility by affecting the amount and function of the essential Ca2+ handling proteins, dihydropyridine receptor (DHPR), ryanodine receptor (RyR2) and sarco-endoplasmic reticulum (SR) Ca2+-ATPase2 (SERCA2). MI also evokes many adaptive responses in organisms, such as elevated production of atrial and brain natriuretic peptides (ANP and BNP). In this thesis, the expression of MT1 and MT2 receptor mRNAs was investigated in several rat tissues. Furthermore, the effect of MI and exogenous melatonin on the rat endogenous melatonin and on the expression of cardiac MT1, MT2, DHPR, RyR2 and SERCA2 proteins was evaluated. The concentrations of ANP and BNP were also measured after post-MI melatonin administration. The results show the expression of MT1 and/or MT2 receptor mRNAs in the hypothalamus, retina, small intestine, liver and heart, which indicates that at least some effects of melatonin could be mediated through the receptors in these tissues. Melatonin synthesis in the pineal gland increased rapidly in response to MI, supporting an important role of endogenous melatonin in protecting the heart after MI. Furthermore, exogenous melatonin altered the mRNA expression of DHPR, RyR2 and SERCA2 after MI, suggesting that melatonin might contribute to the post-infarction cardiac contractile function. The results also revealed a novel, positive relationship between melatonin and ANP, and thereby bring out one more possible way of melatonin to protect the heart against MI-induced injuries. Taken together, the present thesis (i) supports the notion that melatonin is an important endogenous protective agent of the organism, and (ii) extends our knowledge of melatonin's post-infarction cardioprotective actions. / Tiivistelmä Melatoniini osallistuu monien fysiologisten toimintojen ja käyttäytymisen säätelyyn sekä vaikuttaa useiden eri kudosten toimintaan. Melatoniini on tehokas antioksidantti, mutta jotkut sen vaikutuksista välittyvät myös MT1 ja MT2 melatoniini reseptorien kautta. Antioksidatiiviset vaikutukset tulevat esiin erityisesti silloin, kun melatoniinin pitoisuus on korkea (≥ nM). Alhaisemmilla pitoisuuksilla (pM) on puolestaan havaittavissa melatoniinin sitoutuminen reseptoreihinsa. Viime aikoina on tullut esille melatoniinin mahdollinen suojavaikutus sydänsairauksia, kuten sydäninfarkteja, vastaan. Sydäninfarkti muuttaa sydänlihaksen rakennetta ja toimintaa esimerkiksi vaikuttamalla supistuksen kannalta välttämättömien proteiinien, dihydropyridiini reseptorin (DHPR), ryanodiini reseptorin (RyR2) ja sarko-endoplasmakalvoston Ca2+-ATPaasi2:n (SERCA2) lukumääriin ja toimintaan, ja sitä kautta muun muassa heikentää sydämen supistuvuutta. Sydäninfarkti laukaisee elimistössä myös monia adaptiivisia vasteita, kuten eteispeptidin (ANP) ja aivojen natriureettisen peptidin (BNP) lisääntyneen erityksen. Tässä väitöstyössä tutkittiin MT1 ja MT2 reseptorien mRNAn ilmentymistä useissa rotan eri kudoksissa. Lisäksi tutkittiin sydäninfarktin ja eksogeenisen melatoniinin vaikutuksia rotan endogeeniseen melatoniiniin sekä sydämen MT1, MT2, DHPR, RyR2 ja SERCA2 proteiinien ekspressioon. Myös ANP ja BNP pitoisuudet mitattiin. Tulokset osoittivat MT1 ja/tai MT2 reseptori mRNAn ilmentymisen hypotalamuksessa, silmän verkkokalvolla, ohutsuolessa, maksassa ja sydämessä, minkä perusteella ainakin osa melatoniinin vaikutuksista saattaisi olla reseptorivälitteisiä näissä kudoksissa. Tulosten mukaan käpyrauhasen melatoniinisynteesi lisääntyi nopeasti sydäninfarktin jälkeen, mikä tukee käsitystä endogeenisen melatoniinin tärkeästä roolista infarktin jälkeisessä sydämen suojauksessa. Lisäksi eksogeeninen melatoniini muutti DHPR:n, RyR2:n ja SERCA2:n mRNA ekspressiota infarktin jälkeen, mikä voisi merkitä, että melatoniini saattaa vaikuttaa infarktin jälkeiseen sydämen supistuvuuteen. Tulosten osoittama positiivinen riippuvuus melatoniinin ja ANP:n välillä tuo puolestaan esille yhden uuden mahdollisen keinon, jonka kautta melatoniini voisi suojata sydäntä infarktin aiheuttamia vaurioita vastaan. Yhteenvetona voidaan todeta, että tämä väitöstyö (i) tukee käsitystä, että endogeenisella melatoniinilla on tärkeä merkitys elimistön suojaamisessa, ja (ii) laajentaa tietämystämme infarktin jälkeisistä melatoniinin sydäntä suojaavista vaikutuksista.

Indiana University-Purdue University Indianapolis (IUPUI) / Diabetes mellitus affects an estimated 285 million people worldwide, and a central component of diabetes pathophysiology is diminished pancreatic islet beta cell function resulting in the inability to manage blood glucose effectively. The beta cell is a highly specialized metabolic factory that possesses a number of specialized characteristics, chief among these a highly developed endoplasmic reticulum (ER). The sarco endoplasmic reticulum Ca2+ ATPase 2b (SERCA2b) pump maintains a steep Ca2+ gradient between the cytosol and ER lumen, and while the Pancreatic and duodenal homeobox 1 (Pdx-1) transcription factor is known to play an indispensable role in beta cell development and function, recent data also implicate Pdx-1 in the maintenance of ER health. Our data demonstrates that a decrease of beta cell Pdx-1 occurs in parallel with decreased SERCA2b expression in models of diabetes, while in silico analysis of the SERCA2b promoter reveals multiple putative Pdx-1 binding sites. We hypothesized that Pdx-1 loss under inflammatory and diabetic conditions leads to decreased SERCA2b with concomitant alterations in ER health. To test this, siRNA-mediated knockdown of Pdx-1 was performed in INS-1 cells. Results revealed reduced SERCA2b expression and decreased ER Ca2+, which was measured using an ER-targeted D4ER adenovirus and fluorescence lifetime imaging microscopy. Co-transfection of human Pdx-1 with a reporter fused to the human SERCA2 promoter increased luciferase activity three-fold relative to the empty vector control, and direct binding of Pdx-1 to the proximal SERCA2 promoter was confirmed by chromatin immunoprecipitation. To determine whether restoration of SERCA2b could rescue ER stress induced by Pdx-1 loss, Pdx1+/- mice were fed high fat diet for 8 weeks. Isolated islets from these mice demonstrated increased expression of spliced Xbp1, signifying ER stress, while subsequent SERCA2b overexpression in isolated islets reduced spliced Xbp1 levels to that of wild-type controls. These results identify SERCA2b as a direct transcriptional target of Pdx-1 and define a novel role for altered ER Ca2+ regulation in Pdx-1 deficient states.

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