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Dugotrajna restrikcija hrane produžava životni vek i odlaže pojavu mnogih bolesti koje se javljaju sa starenjem. Brojni literaturni podaci ukazuju da kod životinja kojima je unos hrane smanjen izostaje karakterističan starosno-zavistan pad u kognitivnim funkcijama, međutim, mehanizam ovakvog neuroprotektivnog dejstva restrikcije hrane nije u potpunosti razjašnjen. U isto vreme, kod životinja na dugotrajnoj restrikciji hrane se povećava nivo kortikosterona u plazmi što ukazuje da ovaj tretman deluje kao blagi stresor. Do sada nije ispitivano do kakvih promena u signalnom putu glukokortikoida dovodi smanjeni unos hrane tokom starenja u mozgu. Centralno polje istraživanja procesa starenja neizbežno podrazumeva ispitivanje promena do kojih dolazi u korteksu i hipokampusu, strukturama mozga koje su ključne za kognitivne funkcije. Sa druge strane, iste strukture učestvuju u regulaciji HHA ose kao važni regioni delovanja negativne povratne sprege. Cilj doktorske disertacije je da se ispita uloga signalnog puta glukokortikoida u korteksu i hipokampusu pacova tokom starenja i pod uticajem dugotrajne restrikcije hrane. Eksperimentalne životinje (mužjaci pacova soja Wistar) starosti 6 meseci su podeljene u dve grupe. Prva grupa, označena kao ad libitum (AL), je imala neograničen pristup hrani, dok je druga grupa (označena kao DR) podvrgnuta režimu redukovane ishrane koji je podrazumevao dobijanje 100% dnevnog unosa hrane AL životinja svakog drugog dana. Životinje su analizirane kada su dostigle starost od 18 i 24 meseca. Životinje stare 6 meseci su predstavljale kontrolnu grupu. Glukokortikoidna signalizacija u ispitivanim strukturama je praćena na prereceptorskom nivou, kao i na nivou ekspresije i aktivacije glukokortikoidnog receptora primenom imunoesej, imunoblot i PCR metoda, kao i imunohistohemijske analize. Rezultati doktorske disertacije pokazuju da tokom starenja dolazi do povećanja koncentracije kortikosterona u korteksu pacova, dok dugotrajna restrikcija hrane poveća, Moderate restriction in food intake (dietary restriction, DR) extends the life-span and delays the onset of many age-related diseases. In addition, food-restricted animals exhibit attenuated cognitive deficits during aging as they perform better in learning and memory tasks than their ad libitum fed counterparts. At the same time, the animals on chronic food restriction have elevated levels of plasma corticosterone, which implies that this treatment could be considered as a mild stressor. So far, there are no literature data regarding the effects of food restriction on glucocorticoid signaling in the brain during aging and the mechanisms by which DR exhibits its neuroprotective effects are poorly understood. Two brain regions of interest are cortex and hippocampus, regions particularly prone to age-related changes. The same regions are also involved in cognition and regulation of the hypothalamic–pituitary–adrenal (HPA) axis activity. The aim of the present study was to evaluate the effect of long-term food restriction on glucocorticoid signaling pathway in the cortex and hippocampus of rats during aging. Experimental animals (male Wistar rats, 6 months old) were divided into two groups. The AL group was fed ad libitum, whereas the food restricted group (DR) received 100% of the mean daily intake of the AL animals every other day. The animals were examined at the age of 18 and 24 months. Six-month-old animals were used as a control. Glucocorticoid signaling in specific brain regions was examined at the pre-receptor level, as well as regarding the level of expression and glucocorticoid receptor activity by using immunoassays, Western blot, PCR and immunohistochemistry. The results of this study demonstrate an age-related increase in cortical corticosterone concentration. The increase in the hormone level was further detected in DR animals, in both brain regions examined. However, 11β-HSD1, a key enzyme in brain tissue that regulates the availability of corticosterone

Dugotrajna restrikcija hrane produžava životni vek i odlaže pojavu mnogih bolesti koje sejavljaju sa starenjem. Brojni literaturni podaci ukazuju da kod životinja kojima je unos hranesmanjen izostaje karakterističan starosno-zavistan pad u kognitivnim funkcijama, međutim,mehanizam ovakvog neuroprotektivnog dejstva restrikcije hrane nije u potpunosti razjašnjen. Uisto vreme, kod životinja na dugotrajnoj restrikciji hrane se povećava nivo kortikosterona uplazmi što ukazuje da ovaj tretman deluje kao blagi stresor. Do sada nije ispitivano do kakvihpromena u signalnom putu glukokortikoida dovodi smanjeni unos hrane tokom starenja u mozgu.Centralno polje istraživanja procesa starenja neizbežno podrazumeva ispitivanje promena dokojih dolazi u korteksu i hipokampusu, strukturama mozga koje su ključne za kognitivnefunkcije. Sa druge strane, iste strukture učestvuju u regulaciji HHA ose kao važni regionidelovanja negativne povratne sprege.Cilj doktorske disertacije je da se ispita uloga signalnog puta glukokortikoida u korteksu ihipokampusu pacova tokom starenja i pod uticajem dugotrajne restrikcije hrane.Eksperimentalne životinje (mužjaci pacova soja Wistar) starosti 6 meseci su podeljene u dvegrupe. Prva grupa, označena kao ad libitum (AL), je imala neograničen pristup hrani, dok jedruga grupa (označena kao DR) podvrgnuta režimu redukovane ishrane koji je podrazumevaodobijanje 100% dnevnog unosa hrane AL životinja svakog drugog dana. Životinje su analiziranekada su dostigle starost od 18 i 24 meseca. Životinje stare 6 meseci su predstavljale kontrolnugrupu. Glukokortikoidna signalizacija u ispitivanim strukturama je praćena na prereceptorskomnivou, kao i na nivou ekspresije i aktivacije glukokortikoidnog receptora primenom imunoesej,imunoblot i PCR metoda, kao i imunohistohemijske analize.Rezultati doktorske disertacije pokazuju da tokom starenja dolazi do povećanjakoncentracije kortikosterona u korteksu pacova, dok dugotrajna restrikcija hrane povećava nivokortikosterona, Moderate restriction in food intake (dietary restriction, DR) extends the life-span anddelays the onset of many age-related diseases. In addition, food-restricted animals exhibitattenuated cognitive deficits during aging as they perform better in learning and memory tasksthan their ad libitum fed counterparts. At the same time, the animals on chronic food restrictionhave elevated levels of plasma corticosterone, which implies that this treatment could beconsidered as a mild stressor. So far, there are no literature data regarding the effects of foodrestriction on glucocorticoid signaling in the brain during aging and the mechanisms by whichDR exhibits its neuroprotective effects are poorly understood. Two brain regions of interest arecortex and hippocampus, regions particularly prone to age-related changes. The same regions arealso involved in cognition and regulation of the hypothalamic–pituitary–adrenal (HPA) axisactivity.The aim of the present study was to evaluate the effect of long-term food restriction onglucocorticoid signaling pathway in the cortex and hippocampus of rats during aging.Experimental animals (male Wistar rats, 6 months old) were divided into two groups. The ALgroup was fed ad libitum, whereas the food restricted group (DR) received 100% of the meandaily intake of the AL animals every other day. The animals were examined at the age of 18 and24 months. Six-month-old animals were used as a control. Glucocorticoid signaling in specificbrain regions was examined at the pre-receptor level, as well as regarding the level of expressionand glucocorticoid receptor activity by using immunoassays, Western blot, PCR andimmunohistochemistry.The results of this study demonstrate an age-related increase in cortical corticosteroneconcentration. The increase in the hormone level was further detected in DR animals, in bothbrain regions examined. However, 11β-HSD1, a key enzyme in brain tissue that regulates theavailability of corticosterone to its receptors, was

2007年度～2009年度科学研究費補助金（基盤研究(Ｂ)）研究成果報告書, 研究概要（和文）：肥満の脂肪組織で細胞内グルココルチコイド活性化酵素、11β-HSD1 の活性が亢進するメカニズムに関して 肥満脂肪組織で普遍的に観察される 炎症シグナルの亢進、セラミドシグナルの亢進、酸化ストレスの亢進との関連において ヒト、マウスの培養脂肪細胞と脂肪組織で検証した。前駆脂肪細胞における11β-HSD1 活性は種々の炎症性サイトカインやセラミド系によって誘導され、11β-HSD1 の阻害は細胞における炎症シグナルを有意に抑制した。また、ヒト、マウスの肥満脂肪組織では11β-HSD1 の最大活性化に必要な補因子、NADPH を供給し、それ自体が酸化ストレス誘導因子、中性脂肪合成を担うG6PD などペントース経路を構成する一連のNADPH 供給酵素の発現や活性が上昇しており、培養脂肪細胞系におけるヒト型Ｇ６ＰＤの過剰発現が11β-HSD1 酵素活性を顕著に誘導し、培地中へのコルチゾル分泌を有意に増加させることが明らかとなった。本研究により、肥満脂肪組織の機能異常に これらの酵素ネットワーク系の機能異常が関与することが初めて明らかとなった。, 研究概要（英文） : Elevated expression and activity of the intracellular glucocorticoid reactivating enzyme, 11beta-hydroxysteroid dehydrogenase type 1 (11 β -HSD1), contribute to dysfunction of obese adipose tissue. Although the pathophysiologic role of 11β-HSD1 in obese adipose tissue has long been investigated, the mechanism whereby 11β-HSD1 is augmented in obese adipose tissue still remains obscure. In this context, in the present study, we investigated the possible involvement of chronic inflammation, oxidative stress, ceramide signals and NADPH provision from enzymes comprising of pentose pathways. In cultured adipocytes and SVF cells from obese adipose tissue from mice, enzyme activity and expression of 11 β -HSD1 was augmented by TNF α , IL-1 β , LPS and ceramide. Pharmacological inhibition of 11β-HSD1 and RNA interference against 11β-HSD1 reduced a line of pro-inflammatory molecules as well as substantially attenuated NF-kβ and MAPK inflammatory signaling in cultured adipocytes. Expression of enzymes comprising of pentose pathways was elevated in obese adipose tissue in proportion to the degree of obesity in both humans and mice. Forced expression of glucose-6-phosphate ehydrogenase (G6PD), a member of pentose pathways, in adipocytes augmented intracellular glucocorticoid reamplification and resultant secretion of cortisol into the media, indicating a functional link between 11β-HSD1 and NADPH provision in obese adipose tissue. Our data highlight an unexpected role of reamplified glucocorticoids and molecular basis for augmented 11β-HSD1 in obese adipose tissue.

Pancreatic β-cells sense the concentration of glucose in the systemic circulation through metabolism of the sugar molecule. Failure to correlate the blood sugar concentration to an appropriate metabolic signal disrupts the function of the β-cell as a controller of glucose homeostasis and may contribute to the development of type 2 diabetes mellitus. Release of insulin is pulsatile and this thesis presents data that support that metabolism drives such pulsatile release. It is also found that increase in insulin release in response to elevation of the glucose concentration is only seen when the rise in glucose induces a prompt and sustained increase in mitochondrial metabolism. Such activation of mitochondrial metabolism depended on the metabolic state of the β-cell prior to the glucose challenge. In this context, prolonged periods of elevated levels of fatty acids are harmful to the pancreatic β-cell. To study the protein expression changes induced by fatty acids a protocol for islet protein profiling and identification of differently expressed proteins were developed. By using this protocol it was discovered that oleate decreased the cellular level of the chaperone peptidyl-prolyl isomerase B. The protocol was also used to study protein expression in islets obtained from mice fed a high-fat and/or a high-sucrose diet. Excess of glucocorticoids in the systemic circulation also cause a diabetic phenotype. Tissue response to glucocorticoids is regulated by the intracellular concentration of the active form of glucocorticoids, which is formed from the inactive form by the enzyme 11β-hydroxysteroid dehydrogenase type 1. It was found that pancreatic islets produce 11β-HSD1 protein in relation to substrate availability and that the amount of islet 11β-HSD1 protein was negatively correlated with insulin secretion.

Pancreatic β-cells sense the concentration of glucose in the systemic circulation through metabolism of the sugar molecule. Failure to correlate the blood sugar concentration to an appropriate metabolic signal disrupts the function of the β-cell as a controller of glucose homeostasis and may contribute to the development of type 2 diabetes mellitus. Release of insulin is pulsatile and this thesis presents data that support that metabolism drives such pulsatile release. It is also found that increase in insulin release in response to elevation of the glucose concentration is only seen when the rise in glucose induces a prompt and sustained increase in mitochondrial metabolism. Such activation of mitochondrial metabolism depended on the metabolic state of the β-cell prior to the glucose challenge. In this context, prolonged periods of elevated levels of fatty acids are harmful to the pancreatic β-cell. To study the protein expression changes induced by fatty acids a protocol for islet protein profiling and identification of differently expressed proteins were developed. By using this protocol it was discovered that oleate decreased the cellular level of the chaperone peptidyl-prolyl isomerase B. The protocol was also used to study protein expression in islets obtained from mice fed a high-fat and/or a high-sucrose diet. Excess of glucocorticoids in the systemic circulation also cause a diabetic phenotype. Tissue response to glucocorticoids is regulated by the intracellular concentration of the active form of glucocorticoids, which is formed from the inactive form by the enzyme 11β-hydroxysteroid dehydrogenase type 1. It was found that pancreatic islets produce 11β-HSD1 protein in relation to substrate availability and that the amount of islet 11β-HSD1 protein was negatively correlated with insulin secretion.