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与类固醇激素的合成和分泌发生了深远的变化有关。看来该问题的原因是衰老大鼠模型的肾上腺皮质细胞或leydig细胞无力有效地将胆固醇传输到细胞的内部线粒体膜内，在这种线粒体膜中，在类固醇激素生产的初始步骤发生。虽然，尚未确定控制这种衰老缺陷的各种细胞和分子机制 实验室指出，过度自由基的形成和氧化损伤（尤其是终生持续加工胆固醇以用于类固醇生产），以调节关键蛋白，Star，PBR/TSPO的功能表达，PBR/TSPO以及可能涉及其他与恒星相关的（StARD）蛋白质中涉及胆固醇内部线粒体的胆固醇。该提案中概述的研究将进一步探讨这些问题，并专门测试 假设增加ROS形成并随之增加的氧化损伤导致固醇转移蛋白，星和PBR/TSPO蛋白的表达发生变化，在衰老动物的类固醇组织模型中，以及增加氧化应激的遗传小鼠模型，氧化应激和下游的遗传小鼠模型，这导致了较少的胆固醇的转移，从而将胆固醇转移到内部线粒体骨膜内部链纤维骨骼固定群，并将孕烯醇酮形成。我们还检验了第二个假设，即使用小分子量的合成抗氧化剂来降低氧化损伤程度将预防，反向或减弱与年龄相关的氧化应激损失，从而降低氧化损伤程度。为了解决这些假设，提出了三个具体目标。目的1是建立衰老加速鼠4（SAMP8）小鼠，成为一种新的类固醇生成的衰老模型。使用肾上腺（或肾上腺皮质细胞）和来自衰老的SAMP8小鼠（和对照SAMR1小鼠）的睾丸leydig细胞，我们将测量：a）a）荷尔蒙调节类固醇的荷尔蒙调节，从细胞内存储中胆固醇的利用，激素诱导的胆固醇诱导的胆固醇和胆固醇的胆固醇和糖果和pbr/pbr/pbr/pbr/pbr/pbr; b）细胞抗氧化剂水平，氧化损伤和抗氧化剂状态； c）Ask1-P38 MAPK信号级联反应中的改变。目的2是确定氧化应激在肾上腺和睾丸类固醇激素产生与年龄相关的下降中的因果作用。 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 - / - 和cu，zn-sod - / - 小鼠。第二个研究的第二项将着重于使用肾上腺细胞和从抗氧化应激的Cu，Zn-SODTG，MN-SODTG和GPX1TG小鼠中分离出来的肾上腺细胞和Leydig细胞来测量这些各种参数的变化。最后一组研究将检查两种抗氧化剂对氧化应激诱导的类固醇发生的抑制以及胆固醇转运到内部线粒体裂解的抑制作用的影响。目的3是采用药理学策略来逆转或防止衰老诱导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-SOD-/ - 和对照小鼠的治疗作用，具有各种低分子量抗氧化剂（NAC，CTMIO，TEMPOL，TEMPOL，METB，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK，EUK9999999999999999。抑制剂（SC-409，SD-169; p38 MAPK与氧化应激介导的类固醇生成的抑制作用有关）：a）a）类固醇生成； b）恒星，pBR/tspo和明星蛋白的mRNA表达； c）激素诱导的胆固醇在线粒体上和与线粒体的递送； d）使用肾上腺（肾上腺皮质细胞）和睾丸leydig细胞，ask1-p38 MAPK信号传导和e）关键氧化应激标志物的水平。