Role of Cholesterol in Age-related Decline in Steroidogenesis

胆固醇在与年龄相关的类固醇生成下降中的作用

• 批准号: 8624522
• 负责人: Salman Azhar
• 金额: --
• 依托单位: VETERANS ADMIN PALO ALTO HEALTH CARE SYS
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-10-01 至 2016-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8624522
• 关键词: Acetylcysteine; Address; Aging; Animal Genetics; Animal Model; Animals; Antioxidants; Attenuated; Biochemical; Blood Pressure; Bone Growth; Carrier Proteins; Cell Aging; Cells; Cellular biology; Cholesterol; Defect; Development; Diabetes Mellitus; Disease; Down-Regulation; EUK-189; Elderly; Enzymes; Equilibrium; Excision; Fertility; Free Radical Formation; General Population; Gland; Goals; Gonadal Steroid Hormones; Gonadal structure; Heart Diseases; Hormones; Human; Immunity; In Vitro; Inner mitochondrial membrane; Intervention; Kinetics; Laboratories; Life; Link; MAP Kinase Gene; MAPK14 gene; Manganese Superoxide Dismutase; Measures; Mediating; Mitochondria; Mitogen-Activated Protein Kinase Inhibitor; Modeling; Molecular; Molecular Biology Techniques; Molecular Target; Molecular Weight; Monitor; Mus; Muscle; Neurodegenerative Disorders; Oxidants; Oxidative Stress; Pathogenesis; Play; Pregnenolone; Process; Production; Proteins; Rattus; Resistance; Role; Sex Characteristics; Side; Signal Transduction; Site; Sodium Chloride; Steroid biosynthesis; Steroids; Sterols; Testing; Testosterone; Tissue Model; Veterans; Water; age related; attenuation; carbohydrate metabolism; cell age; ebselen; hormone regulation; leydig interstitial cell; lipid metabolism; mRNA Expression; men; mimetics; mouse model; oxidative damage; prevent; protein expression; protein metabolism; public health relevance; response; senescence; sex; steroid hormone; steroidogenic acute regulatory protein; tempol; treatment effect

DESCRIPTION (provided by applicant): Advancing age in human and experimental animals is associated with a profound change in the synthesis and secretion of steroid hormones. It appears that the cause of this problem is the inability of adrenocortical cells or Leydig cells of the aging rat model to effectively transport cholesterol to the cell's inner mitochondrial membrrane where the initial steps in steroid hormone production take place. Although, the various cellular and molecular mechanisms controlling this aging defect have not been definitely identified, considerable evidence from this laboratory points to excessive free radical formation and oxidative damage (especially from life-long continued processing of cholesterol for steroid production) to the cell machinery regulating the functional expression of crucial proteins, StAR, PBR/TSPO and potentially other StAR-related (StarD) proteins involved in cholesterol transport to inner mitochondrial membrane. The studies outlined in this proposal will explore these issues further and will specifically test the hypothesis that increased ROS formation and ensuing oxidative damage leads to changes in expression of sterol transfer proteins, StAR, and PBR/TSPO proteins in steroidogenic tissues model of aging animals and genetic mouse models of increased oxidative stress, and downstream, this leads to the transfer of less cholesterol to the inner mitochondrial membrane sites where cholesterol side chain cleavage takes place, and the first steroid, pregnenolone is formed. We also test a second hypothesis that pharmacological intervention with the use of small molecular weight synthetic antioxidants that reduce the degree of oxidative damage will prevent, reverse, or attenuate the age-related and oxidative stress dependent loss of steroidogenic response. To address these hypotheses, three Specific Aims are proposed. Aim 1 is to establish Senescence-Accelerated-Mouse-Prone 8 (SAMP8) mice as a new aging model of impaired steroidogenesis. Using adrenal (or adrenocortical cells) and testicular Leydig cells from aging SAMP8 mice (and control SAMR1 mice) we will measure: a) hormonal regulation of steroidogensis, utilization of cholesterol from intracellular stores, hormone-induced cholesterol transport to mitochondria and expression of StAR and PBR/TSPO; b) cellular antioxidant levels, oxidative damage and antioxidant status; and c) alteration in the ASK1- p38 MAPK signaling cascade. Aim 2 is to establish the causal role of oxidative stress in age-related decline in adrenal and testicular steroid hormone production. The first set of studies to be conducted will include monitoring changes in in vitro steroidogenesis, mRNA expression of StAR, PBR/TSPO and StarD proteins, levels of oxidants and markers of oxidative damage and the kinetics of hormone-induced cholesterol transport to mitochondria in adrenal (adrenocortical cells) and Leydig cells isolated from oxidative stress-prone A/T-Mn-SOD-/- (or Mn-SOD+/-), GPX1-/- and Cu,Zn-SOD-/- mice. The second of set studies will focus on measuring changes in these various parameters using adrenal cells and Leydig cells isolated from oxidative stress resistant Cu,Zn-SODTg, Mn-SODTg, and GPX1Tg mice. The final set of studies will examine the impact of simultaneous over-expression of the two antioxidant enzymes on oxidative stress-induced inhibition of steroidogenesis and impaired cholesterol transport to the inner mitochondrial membrane for side-chain cleavage. Aim 3 is to employ pharmacological strategies to reverse or prevent aging-induced and oxidative stress-associated loss of steroidogenesis. Studies proposed in this section will evaluate the effect of treatment of SAMR-1, SAMP-8, A/T-Mn-SOD-/- (or Mn-SOD+/-), GPX-1-/-, and Cu,Zn-SOD-/-, and control mice, with various low molecular weight antioxidants (NAC, CTMIO, tempol, MnTBAP, EUK-189, ebselen) or p38 MAPK inhibitors (SC-409, SD-169; p38 MAPK is implicated in oxidative stress-mediated inhibition of steroidogenesis) on: a) steroidogenesis; b) mRNA expression of StAR, PBR/TSPO and StarD proteins; c) hormone-induced cholesterol delivery to and with the mitochondria; d) ASK1-p38 MAPK signaling and e) levels of key oxidative stress markers, using adrenal (adrenocortical cells) and testicular Leydig cells.

描述（由申请人提供）： 人类和实验动物的衰老与类固醇激素合成和分泌的深刻变化有关。造成这个问题的原因似乎是衰老大鼠模型的肾上腺皮质细胞或间质细胞无法有效地将胆固醇转运到细胞的线粒体内膜，类固醇激素产生的初始步骤在此发生。虽然控制这种衰老缺陷的各种细胞和分子机制尚未明确确定，但有大量证据表明 实验室指出，过度的自由基形成和氧化损伤（特别是终生持续加工胆固醇以产生类固醇）对调节关键蛋白质、StAR、PBR/TSPO 和其他潜在 StAR 相关蛋白（StarD）功能表达的细胞机制造成了损害。参与胆固醇转运至线粒体内膜的蛋白质。本提案中概述的研究将进一步探讨这些问题，并将专门测试 假设增加的 ROS 形成和随之而来的氧化损伤导致衰老动物的类固醇生成组织模型和氧化应激增加的遗传小鼠模型中甾醇转移蛋白、StAR 和 PBR/TSPO 蛋白表达的变化，以及下游，这导致转移较少的胆固醇到达线粒体内膜位点，胆固醇侧链在此处发生裂解，并形成第一种类固醇孕烯醇酮。我们还测试了第二个假设，即使用小分子量合成抗氧化剂进行药物干预以降低氧化损伤的程度，将预防、逆转或减轻与年龄相关和氧化应激依赖性的类固醇生成反应的丧失。为了解决这些假设，提出了三个具体目标。目标 1 是建立衰老加速小鼠倾向 8 (SAMP8) 小鼠作为类固醇生成受损的新衰老模型。使用来自衰老 SAMP8 小鼠（和对照 SAMR1 小鼠）的肾上腺（或肾上腺皮质细胞）和睾丸间质细胞，我们将测量：a）类固醇生成的激素调节、细胞内储存的胆固醇的利用、激素诱导的胆固醇转运至线粒体以及 StAR 的表达和 PBR/TSPO； b) 细胞抗氧化水平、氧化损伤和抗氧化状态； c) ASK1-p38 MAPK 信号级联的改变。目标 2 是确定氧化应激在与年龄相关的肾上腺和睾丸类固醇激素产生下降中的因果作用。即将进行的第一组研究将包括监测体外类固醇生成的变化、StAR、PBR/TSPO 和 StarD 蛋白的 mRNA 表达、氧化剂和氧化损伤标志物的水平以及激素诱导的胆固醇转运至肾上腺线粒体的动力学。 （肾上腺皮质细胞）和 Leydig 细胞分离自氧化应激倾向 A/T-Mn-SOD-/-（或 Mn-SOD+/-）、GPX1-/- 和Cu,Zn-SOD-/- 小鼠。第二组研究将重点使用从抗氧化应激的 Cu、Zn-SODTg、Mn-SODTg 和 GPX1Tg 小鼠中分离出的肾上腺细胞和 Leydig 细胞来测量这些不同参数的变化。最后一组研究将检查两种抗氧化酶同时过度表达对氧化应激诱导的类固醇生成抑制和胆固醇转运至线粒体内膜侧链裂解受损的影响。目标 3 是采用药理学策略来逆转或预防衰老引起的和氧化应激相关的类固醇生成丧失。本节提出的研究将评估 SAMR-1、SAMP-8、A/T-Mn-SOD-/-（或 Mn-SOD+/-）、GPX-1-/- 和 Cu,Zn 的治疗效果-SOD-/- 和对照小鼠，使用各种低分子量抗氧化剂（NAC、CTMIO、tempol、MnTBAP、EUK-189、ebselen）或 p38 MAPK 抑制剂（SC-409、 SD-169；p38 MAPK 参与氧化应激介导的类固醇生成抑制：a) 类固醇生成； b) StAR、PBR/TSPO和StarD蛋白的mRNA表达； c) 激素诱导的胆固醇递送至线粒体并与线粒体一起递送； d) ASK1-p38 MAPK 信号传导和 e) 关键氧化应激标记物的水平，使用肾上腺（肾上腺皮质细胞）和睾丸 Leydig 细胞。