Gene Profiling of Testosterone-Regulated Genes in the Skeletal Muscle of Hiv-Infected Men Experiencing Weight Loss. Homo sapiens

Context: Although androgen treatment increases muscle mass in HIV-infected men, the underlying mechanisms are poorly understood. Analysis of genome-wide microarray data obtained from skeletal muscle biopsies can be used to profile androgen-regulated pathways. Objective: To identify genes and pathways associated with testosterone treatment and myogenesis in the context of HIV-infected men using genome-wide microarray analysis of skeletal muscle biopsies. Results: A significant weight gain was observed in subjects treated with testosterone compared to placebo (+2.05 kgs and –1.07 kgs, respectively; P = 0.003) as well as gains in DEXA lean mass (2.93 kgs vs. 0.35 kgs, respectively; P = 0.003). Microarray expression profiles and RTPCR validation of RNA after 14 days of treatment indicated that several gene sets including transcriptional control, myogenesis, adipogenesis, insulin signaling, apoptosis, cell cycle, chromatin remodeling, and stress response genes were differentially expressed with testosterone treatment. Protein expression analysis of myogenic differentiation and protein synthesis markers (MyoD, Myogenin, phosphorylated p38 MAPK and phosphorylated AKT) in muscle biopsies and skeletal muscle cells treated with DHT confirmed that testosterone engages a network of pro-myogenic genes. Conclusions: Testosterone-associated gain in muscle mass in HIV-infected men engaged a network of regulatory pathways involved in broad transcriptional control, myogenesis, insulin signaling, chromatin remodeling, and stress response. Further evaluation of precise signaling intermediates within androgen regulated pathways may help to better define improvements in muscle mass, both in healthy and HIV infected patients. Keywords: Dose Response, Myogenesis Overall design: Design, Setting, and Participants: 44 HIV+ men with weight loss were randomized to receive either 300mg testosterone enanthate or placebo injections IM weekly for 16 weeks. Muscle biopsies were obtained at baseline and on treatment day 14. A random subset of specimens was chosen for microarray analysis; changes in selected genes were confirmed by reverse transcriptase - polymerase chain reaction (RT-PCR), and western blot analysis. Human skeletal myogenic precursor cells (SkMCs) were cultured in vitro in the presence of dihydrotestosterone (DHT) to evaluate activation of myogenic protein expression. Main outcome measures: Relative RNA levels in skeletal muscle biopsies were measured by expression profiling with microarrays and expression levels were validated in samples using quantitative RT-PCR. Further confirmation of changes in key myogenic biomarkers was obtained in DHT treated SkMCs.

研究背景：尽管雄激素治疗可增加HIV感染男性的肌肉质量，但其潜在机制仍不甚明确。对骨骼肌活检获取的全基因组微阵列（genome-wide microarray）数据进行分析，可用于解析雄激素调控的通路。
研究目的：通过对HIV感染男性的骨骼肌活检样本进行全基因组微阵列分析，鉴定与睾酮治疗及肌生成相关的基因与通路。
研究结果：与安慰剂组相比，睾酮治疗组受试者体重显著增加（分别增加2.05kg与减少1.07kg；P=0.003），双能X线吸收法（DEXA）检测的瘦体重同样显著增加（分别为2.93kg与0.35kg；P=0.003）。治疗14天后的微阵列表达谱与逆转录-聚合酶链反应（reverse transcriptase-polymerase chain reaction, RT-PCR）验证结果显示，包括转录调控、肌生成、脂肪生成、胰岛素信号通路、细胞凋亡、细胞周期、染色质重塑以及应激反应相关基因在内的多个基因集在睾酮治疗后呈现差异表达。对睾酮处理后的骨骼肌活检样本及骨骼肌细胞中的肌分化与蛋白质合成标志物MyoD（MyoD）、肌细胞生成素（Myogenin）、磷酸化p38丝裂原活化蛋白激酶（phosphorylated p38 MAPK）与磷酸化蛋白激酶B（phosphorylated AKT）进行蛋白质表达分析，证实睾酮可激活促肌生成基因网络。
研究结论：HIV感染男性中与睾酮相关的肌肉质量增加，涉及广泛的转录调控、肌生成、胰岛素信号通路、染色质重塑以及应激反应相关的调控通路网络。进一步解析雄激素调控通路中的精准信号中间体，或有助于更清晰地阐明健康人群与HIV感染患者的肌肉质量改善机制。
关键词：剂量反应（Dose Response）、肌生成（Myogenesis）
整体研究设计：
研究设计、研究场景与研究对象：将44例体重下降的HIV阳性男性随机分为两组，每周分别接受300mg庚酸睾酮肌肉注射或安慰剂注射，持续16周。分别于基线期与治疗第14天采集骨骼肌活检样本。选取随机子集样本进行微阵列分析；通过逆转录-聚合酶链反应（RT-PCR）验证选定基因的表达变化，并采用蛋白质印迹（western blot）分析进行验证。在体外培养的人骨骼肌前体细胞（skeletal myogenic precursor cells, SkMCs）中加入二氢睾酮（dihydrotestosterone, DHT）处理，以评估肌源性蛋白表达的激活情况。
主要结局指标：通过微阵列表达谱分析检测骨骼肌活检样本中的相对RNA水平，并采用定量RT-PCR验证样本中的基因表达水平。在DHT处理的SkMCs中进一步验证关键肌源性生物标志物的表达变化。