Testosterone supplementation increases muscle mass and decreases fat mass in healthy young men, hypogonadal men, and in HTV-infected men with weight loss; these observations have prompted clinical trials of testosterone supplementation in MTV-infected men with weight loss or fat redistribution syndromes. However, the molecular mechanisms by which testosterone regulates muscle and fat mass are unknown and are the objective of this investigation. We hypothesize that testosterone and dihydrotestosterone (DHT), two prototypical androgens, promote the differentiation of adult, pluripotent mesenchymal stem cells into myogenic lineage and inhibit the differentiation of these cells into adipogenic lineage through an androgen receptor pathway. These studies will determine whether testosterone and DHT promote myogenic commitment and inhibit adipogenic differentiation of mesenchymal, pluripotent cells by activation of Wnt signaling pathway. We will employ two validated in vitro models of adult, mesenchymal, stem cell commitment and differentiation, namely, adult, pluripotent stem cells isolated from lipoaspirates, and muscle stem cells isolated from regenerating skeletal muscle of adult mice after an injury. We will determine the effects of graded doses of testosterone and DHT on myogenic and adipogenic differentiation of adult, mesenchymal, stem cells in vitro, and evaluate whether the effects of testosterone are blocked by bicalutamide, an androgen receptor antagonist. Promyogenic effects of testosterone on differentiation and commitment of mesenchymal stem cells will be studied in a mouse model of muscle injury and regeneration. In this model, we will determine the effects of surgical orchiectomy without and with testosterone replacement on the number of mesenchymal precursor cells (CD34+, Pax7), satellite cells (CD34+, Pax7+, located inside the basal lamina), and number of donor cells fused with muscle fibers during regeneration following muscle injury. To demonstrate Wnt activation , we will first determine whether testosterone or DHT cause translocation of p-catenin into nucleus, a hallmark of Wnt activation, and decrease the amount of phosphorylated p-catenin. The interaction and co-localization of p-catenin and androgen receptor in the nucleus will be analyzed. We will evaluate whether inhibition of beta-catenin by a silencer RNA blocks the activation of TCF4 and MyoD transcription by testosterone and DHT. We will also assess whether testosterone and DHT induce TCF4 transcription, and upregulate Pax 7 and MyoD expression, while downregulating adipogenic differentiation markers, PPARgamma2 and C/EBPalpha. We will ascertain whether inhibition of TCF4 by a dominant negative TCF4 cDNA blocks upregulation of Pax 7 and MyoD and downregulation of PPARgamma2 and C/EBPa, by testosterone and DHT. These studies will provide novel insights into the mechanisms of androgen action on the skeletal muscle and have direct relevance to the proposed applications of testosterone for the treatment of HTV-associated weight loss and fat redistribution syndromes and for the development of selective androgen receptor modulators.
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