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: 8762445
• 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/8762445
• 关键词: 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; Equilibrium; Excision; Fertility; Free Radical Formation; General Population; Gland; Goals; Gonadal Steroid Hormones; Gonadal structure; Health; 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; antioxidant enzyme; 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; 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)： 人类和实验动物的年龄增长与类固醇激素的合成和分泌发生了深刻的变化。这一问题的原因似乎是衰老大鼠模型中的肾上腺皮质细胞或间质细胞无法有效地将胆固醇输送到细胞内的线粒体膜，在那里发生了类固醇激素产生的最初步骤。虽然，控制这种衰老缺陷的各种细胞和分子机制还没有确定，但有相当多的证据表明 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)对细胞器调节关键蛋白、STAR、PBR/TSPO和其他可能参与胆固醇运输到线粒体膜的STARD蛋白的功能表达。这项建议中概述的研究将进一步探讨这些问题，并将具体测试 假设增加ROS的形成和随之而来的氧化损伤导致类固醇转移蛋白，STAR和PBR/TSPO蛋白在类固醇合成组织中的表达发生变化，在衰老动物模型和遗传性小鼠氧化应激增加的模型中，这导致下游，这导致较少的胆固醇转移到发生胆固醇侧链断裂的线粒体膜内区，并且形成了第一种类固醇，孕烯醇酮。我们还测试了第二个假设，即使用小分子质量的合成抗氧化剂进行药物干预，降低氧化损伤的程度，将防止、逆转或减弱与年龄相关的和氧化应激依赖的类固醇激素合成反应的丧失。为了解决这些假设，本文提出了三个具体目标。目的1建立衰老加速小鼠倾向8(SAMP8)小鼠作为类固醇合成受损的新的衰老模型。利用老年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)激素对类固醇生成的调节，细胞内储存的胆固醇的利用，激素诱导的胆固醇向线粒体的运输以及STAR和PBR/TSPO的表达；b)细胞抗氧化水平，氧化损伤和抗氧化状态；以及c)ASK1-p38 MAPK信号级联的变化。目的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作用。将进行的第一组研究将包括监测肾上腺(肾上腺皮质细胞)和从易氧化应激小鼠分离的间质细胞中类固醇合成、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向线粒体转运的动力学。第二组研究将集中于使用从抗氧化应激的铜、锌-SODTg、锰-SODTg和GPX1Tg小鼠分离的肾上腺细胞和间质细胞来测量这些不同参数的变化。最后一组研究将考察这两种抗氧化酶同时过表达对氧化应激诱导的类固醇合成抑制和胆固醇向线粒体膜内侧链裂解的损害的影响。目的3是利用药理学策略逆转或防止衰老诱导和氧化应激相关的类固醇合成丧失。这一部分的研究将评估SAMR-1、SAMP-8、A/T-Mn-SOD-/-(或Mn-SOD+/-)、GPX-1-/-和铜、锌-SOD-/-以及对照组小鼠与各种低分子抗氧化剂(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合成抑制)对a)类固醇合成的影响；b)STAR、PBR/TSPO和STARD蛋白的mRNA表达；C)激素诱导的胆固醇传递到线粒体并与线粒体一起传递；d)ASK1-p38 MAPK信号转导和e)关键氧化应激标志物的水平，使用肾上腺(肾上腺皮质细胞)和睾丸间质细胞。