madman
Super Moderator
Testosterone recovery therapy targeting dysfunctional Leydig cells(2022)
Samuel Garza and Vassilios Papadopoulos
Abstract
Reduced serum testosterone affects millions of men across the world and has been linked to several comorbidities, metabolic dysfunctions, and quality of life changes. The standard treatment for testosterone deficiency remains testosterone replacement therapy. However, limitations on its use and the risk of significant adverse effects make alternative therapeutics desirable. Studies on the mechanisms regulating and synthesizing testosterone formation in testicular Leydig cells demonstrate numerous endogenous targets that could increase testosterone biosynthesis, which could alleviate reduced testosterone effects. Testosterone biosynthesis is facilitated by a conglomerate of cytosolic and mitochondrial proteins that facilitate cholesterol translocation into the mitochondria, the rate-limiting step in steroidogenesis. An effective therapeutic approach would be to increase endogenous testosterone formation by enhancing steroidogenesis in Leydig cells. Numerous ligands for steroidogenic proteins have been developed that increase steroid hormone formation. However, off-target effects on neurosteroid and adrenal steroid formation may limit their clinical use. First-in-class biologics, such as voltage-dependent anion channel peptides and transplantation of induced human Leydig-like cells offer advances in the development of specific strategies that could be used to enhance endogenous steroid formation in hormone deficient patients.
Introduction
Although some testosterone decline is normal in men of middle and advanced age, some men have significantly decreased testosterone levels known as hypogonadism. Hypogonadism is a condition characterized by severe testosterone deficiency and affects nearly 5 million men in the United States1. While hypogonadism is most commonly associated with infertility, it has also been correlated with other numerous conditions, such as cardiovascular disease, depression, fatigue, reduced bone mineral density, increased body fat, metabolic syndrome, and declining muscle mass2,3. Hypogonadism can be separated into two categories: primary hypogonadism and secondary hypogonadism. Primary hypogonadal patients present depleted testosterone levels due to a suboptimal response to luteinizing hormone (LH) stimulation; whereas secondary hypogonadism is characterized by low LH levels or low gonadotropin-releasing hormone (GnRH) levels, leading to insufficient steroid hormone biosynthesis4. Moreover, primary hypogonadal patients display increased LH, suggesting that Leydig cell mechanisms are disrupted5. The primary causes of secondary hypogonadism are associated with the pituitary or hypothalamus4. These can be congenital, acquired, or caused by damage to gonadotrophs4.
Given testosterone’s essential role in spermatogenesis, hypogonadal patients suffer from infertility6. Furthermore, androgen metabolites levels, such as dihydrotestosterone (DHT) and 3αandrostenediol glucuronide (3α-ADG), become imbalanced and cause alterations in secondary sex characteristics, including muscle mass, body mass index, and facial hair5. Patients may present with fatigue and declining mood, given the ability of neurosteroids to act as positive or negative regulators of the GABA receptor7. There are also numerous congenital and acquired origins of hypogonadism that may manifest throughout the male lifespan8. Therapeutic strategies for endogenous targets to treat hypogonadism from all origins are highly sought.
Testosterone replacement therapy (TRT)9 and aromatase inhibitors10 have been used to elevate serum testosterone and alleviate symptoms of hypogonadism. TRT involves administering exogenous testosterone at appropriate intervals, both daily-acting, intermediate-acting (1-3 weeks), and long-acting (2-6 months)9. However, this exogenous testosterone leads to hypothalamic-pituitary-gonadal axis (HPG) imbalance and suppresses the release of gonadotropins11. This represses Leydig cell testosterone biosynthesis, a critical driver of spermatogenesis, and leads to reduced fertility9,11. Moreover, intermediate and long-acting injections may produce serious adverse events (SAEs) including pulmonary microembolism, anaphylaxis, and polycythaemia9,12,13, and an increased risk of cardiovascular disease and stroke may exist in older men receiving TRT as indicated in recent studies14,15, resulting in the FDA and medical societies cautioning its use3. Numerous alternatives to TRT have been considered16. The testosterone metabolite DHT is also used strategically to treat hypogonadism in some countries17. DHT binds to androgen receptors with a greater affinity than testosterone and provides some relief from symptoms of hypogonadism18. The disadvantages of DHT are its price, increased hemoglobin, increased red blood cell count, and inferior clinical results when compared to TRT17,18. Aromatase inhibitors are also used to prevent aromatase from converting testosterone to estrogen, thereby, maintaining testosterone levels19. In clinical studies with aromatase inhibitors used for hypogonadal patients, LH levels, free testosterone, and sexual desire increased20. Moreover, aromatase inhibitors may be suitable for hypogonadal patients with increased estrogen levels18. However, concerns regarding the effect of aromatase inhibitors on bone minerals still remain after treatment with the inhibitor letrozole led to vertebrae deformities in 45% of adolescent males with delayed puberty21. The selective estrogen receptor modulators clomiphene citrate and tamoxifen are also used off-label for the treatment of primary hypogonadism due to their ability to induce the release of GnRH by the hypothalamus and subsequently increase the production of the gonadotropins LH and FSH by the anterior pitutary16
*Testosterone regulation and formation
*Mechanisms of Leydig cell dysfunction
-Reductions in steroidogenic enzymes
-Imbalanced antioxidant and reactive oxygen species production
-Reduced mitochondrial function of Leydig cells
*Endogenous targets for testosterone recovery therapy
The role of numerous SITE proteins and steroidogenic regulators has been investigated to identify endogenous therapeutic targets that induce steroid hormone formation. Several proteins within the cytosol and mitochondria mediate cholesterol translocation from intracellular stores to the OMM where the SITE complex resides38. Rone et al. investigated the role of numerous steroidogenic and mitochondrial dynamic proteins to elucidate their role in steroidogenesis38. Such investigations revealed that knocking down OPA1, VDAC1, and ATAD3A had no effect on membrane permeable steroid formation. However, VDAC1 and ATAD3A knockdowns did reduce hormone-induced steroidogenesis, suggesting that OPA1 is not critical for hormone-induced steroidogenesis38. Recently it was shown that upregulating OPA1 via pharmacological and transfection methods increased TSPO expression and steroid hormone formation in both basal and hormone-stimulated dysfunctional Leydig cells, suggesting that OPA1 may play a role in the regulation and formation of the SITE complex76. Progress has been made in targeting SITE proteins to ameliorate testosterone decline. Studies on TSPO ligands and 14-3-3ε peptides (VDAC1 peptides) have offered potential therapeutic strategies for inducing endogenous testosterone formation. While TSPO ligands enhance cholesterol translocation, VDAC1 peptides are designed to block the negative regulation of steroidogenesis34,51,77- 80.
TSPO ligands
Engagement of the OMM protein TSPO via a drug ligand-induced activation stimulates steroid hormone production in vitro and in vivo in rats and mice48,61,77,78,81,82. TSPO possesses a high affinity for cholesterol binding, which leads to its subsequent translocation to the IMM for side-chain cleavage by CYP11A1-producing pregnenolone83,84. TSPO’s C terminus plays a key role in the uptake of cholesterol from the cytosol and translocation into the mitochondria85,86, and disruption of the protein within steroidogenic cells disrupts mitochondrial cholesterol transport and steroid formation84,87. Steroidogenesis and TSPO expression correlate with one another as shown by disruption of steroidogenesis with TSPO’s age-related decline in vivo and its ablation in vitro62,87. Moreover, transfection of TSPO into TSPO-disrupted cells restores steroid formation, demonstrating its indispensable role in steroidogenesis87.
Numerous studies have shown that drug ligands targeting TSPO produce enhanced steroid levels in both MA-10 tumorigenic Leydig cells and isolated primary Leydig cells, as well as increased serum testosterone levels77,78,81. However, serum LH levels may also become increased following TSPO drug ligand treatment likely due to an effect of the ligand on brain TSPO88,89, suggesting that using this target may enhance testosterone biosynthesis by either stimulating the Leydig cell steroidogenic machinery and/or by elevating LH release77. TSPO-specific ligands are also known to increase glucocorticoid and corticosteroid levels61 and have been shown to affect neurosteroid production90-92. Accordingly, the use of TSPO ligands as a therapeutic approach to treat neurological and psychiatric disorders has also been investigated93. Similarly, the use of TSPO ligands may also induce anxiolytic-like responses, as ligand treatment has been shown to counteract panic attacks in rodents94. While molecular entities targeting TSPO elevate serum testosterone levels, adrenal steroids and neurosteroids are also affected. Therefore, TSPO ligands have been proposed as therapeutic agents for the regulation of steroid hormones in the testis and brain. However, this lack of specificity remains an issue, as TSPO is expressed in numerous tissues.
-VDAC1 peptides
New insights into the role of 14-3-3ε in the regulation of steroidogenesis have made it a promising therapeutic target. 14-3-3 proteins regulate target proteins by altering activity, posttranslational modifications, and subcellular localization95. LHR stimulation initiates the translocation of 14-3-3ε to the OMM33 and its recruitment to the TSPO-VDAC1 complex at Ser167 on VDAC1. There it competes with TSPO for VDAC1 binding and thus reduces cholesterol import51. Blocking the interaction between 14-3-3ε and VDAC1 using cell-penetrating peptides induces steroid formation in vivo and ex vivo80. Aghazadeh et al. fused a component of the HIV transcription factor 1 (TAT) with the predicted Ser167 binding motif on 14-3-3ε, creating a cell-permeable VDAC1 peptide, TATVDAC1 containing Ser167 (TVS167), which competed with 14-3-3ε for VDAC1 binding79. This reduced the negative regulation of steroidogenesis by blocking the 14-3-3ε binding to VDAC1, which led to increased steroidogenesis in vitro and in vivo. Given the homologous mechanisms of 14-3-3ε between species, the TAT-based peptide offers a promising approach in humans. Although TAT peptides penetrate indiscriminately and 14-3-3ε is found in numerous tissues, the function is tissue-specific79. TVS167 treatment did not significantly increase corticosterone levels in rats treated with the compound, demonstrating specificity to testicular Leydig cells80. Additionally, the action of the TVS167 peptide induced steroidogenesis independent of LH and would offer a major improvement in safety when compared to TRT96. The minimal bioactive sequence of the peptide was recently identified, and we ultimately generated bioactive stable peptide derivatives that can be administered orally and induce T formation in normal and hypogonadal animal models (manuscript in preparation). Moreover, they demonstrate safety, efficacy, and target specificity34,51,79,80. In summary, these first-in-class biologics make an excellent candidate for the treatment of diseases caused by Leydig cell dysfunction over other pharmacologic or biologic strategies.
-Implantation of human Leydig-like cells
The generation of transplantable testosterone-producing cells offers another alternative for treating pathologies related to Leydig cell dysfunction. Previously, it was shown that mesenchymal stem cells (MSCs) were able to differentiate into testosterone-producing Leydig cells, suggesting that healthy Leydig cell populations could be transplanted into hypogonadal patients97. However, MSC isolation produces limited cell numbers and reduces the clinical application of this method. Recent developments have revealed human Leydig-like cells (hLLCs) can be generated from human induced pluripotent stem cells (hiPSCs), which are highly expandable in cell culture98,99. Li et al. demonstrated that hLLCs producing steroidogenic gene expression, steroidogenic enzymes, and testosterone could be generated by differentiating early mesenchymal progenitors from hiPSCs while overexpressing steroidogenic factor 1 (SF-1) in culture with dibutyryl-cAMP (dbcAMP), recombinant desert hedgehog, and human chorionic gonadotropin (hCG)98. Given their clinical viability, the implantation of hLLCs would represent a monumental step forward in treating diseases related to Leydig cell dysfunction. This strategy could restore testosterone levels by replenishing testosterone-producing-cell populations in the testicular environment, leading to the production of endogenous testosterone formation.
Conclusions and future directions
Testosterone deficiency impacts the quality of life and well-being of millions of men worldwide, with only limited treatments having undesirable off-target effects16. A new understanding of the molecular interactions producing testosterone has laid the foundation for the development of novel therapeutic strategies. Identification of the hormonally regulated multiprotein SITE complex38 and the deeper understanding of hormonal stimulation and cholesterol translocation from cytosolic stores across the OMM and into the IMM for side-chain cleavage demonstrates numerous therapeutic targets for various indications related to hormone insufficiency (Table 1)16,18. However, their effects on neurosteroids, adrenal steroids, and the HPG axis have remained a barrier to the safe and efficacious treatment of testosterone deficiency. Apart from VDAC1 peptides, existing strategies have lacked specificity for testicular Leydig cells and, therefore, have raised concerns regarding off-target effects (Fig. 2). VDAC1 peptides are first-in-class biologics that offer a novel approach for rescuing intratesticular and serum testosterone formation in hormonally mediated diseases79,80. These therapeutics could be used to restore endogenous testosterone formation and restore well-being for millions of aging men worldwide.
There are additional mechanisms to uncover. The movement of cholesterol between the mitochondrial membranes, the relationship between aging and the Leydig cell oxidative environment, and age-dependent protein-protein interactions remain elusive and are active areas of research24. With more information, we may determine the cause of reduced testosterone and develop interventions that may maintain Leydig cell function. Moreover, targeting the molecular deteriorations that differ between aging Leydig cells and other aging steroidogenic tissues could lead to additional testis-specific strategies.
Samuel Garza and Vassilios Papadopoulos
Abstract
Reduced serum testosterone affects millions of men across the world and has been linked to several comorbidities, metabolic dysfunctions, and quality of life changes. The standard treatment for testosterone deficiency remains testosterone replacement therapy. However, limitations on its use and the risk of significant adverse effects make alternative therapeutics desirable. Studies on the mechanisms regulating and synthesizing testosterone formation in testicular Leydig cells demonstrate numerous endogenous targets that could increase testosterone biosynthesis, which could alleviate reduced testosterone effects. Testosterone biosynthesis is facilitated by a conglomerate of cytosolic and mitochondrial proteins that facilitate cholesterol translocation into the mitochondria, the rate-limiting step in steroidogenesis. An effective therapeutic approach would be to increase endogenous testosterone formation by enhancing steroidogenesis in Leydig cells. Numerous ligands for steroidogenic proteins have been developed that increase steroid hormone formation. However, off-target effects on neurosteroid and adrenal steroid formation may limit their clinical use. First-in-class biologics, such as voltage-dependent anion channel peptides and transplantation of induced human Leydig-like cells offer advances in the development of specific strategies that could be used to enhance endogenous steroid formation in hormone deficient patients.
Introduction
Although some testosterone decline is normal in men of middle and advanced age, some men have significantly decreased testosterone levels known as hypogonadism. Hypogonadism is a condition characterized by severe testosterone deficiency and affects nearly 5 million men in the United States1. While hypogonadism is most commonly associated with infertility, it has also been correlated with other numerous conditions, such as cardiovascular disease, depression, fatigue, reduced bone mineral density, increased body fat, metabolic syndrome, and declining muscle mass2,3. Hypogonadism can be separated into two categories: primary hypogonadism and secondary hypogonadism. Primary hypogonadal patients present depleted testosterone levels due to a suboptimal response to luteinizing hormone (LH) stimulation; whereas secondary hypogonadism is characterized by low LH levels or low gonadotropin-releasing hormone (GnRH) levels, leading to insufficient steroid hormone biosynthesis4. Moreover, primary hypogonadal patients display increased LH, suggesting that Leydig cell mechanisms are disrupted5. The primary causes of secondary hypogonadism are associated with the pituitary or hypothalamus4. These can be congenital, acquired, or caused by damage to gonadotrophs4.
Given testosterone’s essential role in spermatogenesis, hypogonadal patients suffer from infertility6. Furthermore, androgen metabolites levels, such as dihydrotestosterone (DHT) and 3αandrostenediol glucuronide (3α-ADG), become imbalanced and cause alterations in secondary sex characteristics, including muscle mass, body mass index, and facial hair5. Patients may present with fatigue and declining mood, given the ability of neurosteroids to act as positive or negative regulators of the GABA receptor7. There are also numerous congenital and acquired origins of hypogonadism that may manifest throughout the male lifespan8. Therapeutic strategies for endogenous targets to treat hypogonadism from all origins are highly sought.
Testosterone replacement therapy (TRT)9 and aromatase inhibitors10 have been used to elevate serum testosterone and alleviate symptoms of hypogonadism. TRT involves administering exogenous testosterone at appropriate intervals, both daily-acting, intermediate-acting (1-3 weeks), and long-acting (2-6 months)9. However, this exogenous testosterone leads to hypothalamic-pituitary-gonadal axis (HPG) imbalance and suppresses the release of gonadotropins11. This represses Leydig cell testosterone biosynthesis, a critical driver of spermatogenesis, and leads to reduced fertility9,11. Moreover, intermediate and long-acting injections may produce serious adverse events (SAEs) including pulmonary microembolism, anaphylaxis, and polycythaemia9,12,13, and an increased risk of cardiovascular disease and stroke may exist in older men receiving TRT as indicated in recent studies14,15, resulting in the FDA and medical societies cautioning its use3. Numerous alternatives to TRT have been considered16. The testosterone metabolite DHT is also used strategically to treat hypogonadism in some countries17. DHT binds to androgen receptors with a greater affinity than testosterone and provides some relief from symptoms of hypogonadism18. The disadvantages of DHT are its price, increased hemoglobin, increased red blood cell count, and inferior clinical results when compared to TRT17,18. Aromatase inhibitors are also used to prevent aromatase from converting testosterone to estrogen, thereby, maintaining testosterone levels19. In clinical studies with aromatase inhibitors used for hypogonadal patients, LH levels, free testosterone, and sexual desire increased20. Moreover, aromatase inhibitors may be suitable for hypogonadal patients with increased estrogen levels18. However, concerns regarding the effect of aromatase inhibitors on bone minerals still remain after treatment with the inhibitor letrozole led to vertebrae deformities in 45% of adolescent males with delayed puberty21. The selective estrogen receptor modulators clomiphene citrate and tamoxifen are also used off-label for the treatment of primary hypogonadism due to their ability to induce the release of GnRH by the hypothalamus and subsequently increase the production of the gonadotropins LH and FSH by the anterior pitutary16
*Testosterone regulation and formation
*Mechanisms of Leydig cell dysfunction
-Reductions in steroidogenic enzymes
-Imbalanced antioxidant and reactive oxygen species production
-Reduced mitochondrial function of Leydig cells
*Endogenous targets for testosterone recovery therapy
The role of numerous SITE proteins and steroidogenic regulators has been investigated to identify endogenous therapeutic targets that induce steroid hormone formation. Several proteins within the cytosol and mitochondria mediate cholesterol translocation from intracellular stores to the OMM where the SITE complex resides38. Rone et al. investigated the role of numerous steroidogenic and mitochondrial dynamic proteins to elucidate their role in steroidogenesis38. Such investigations revealed that knocking down OPA1, VDAC1, and ATAD3A had no effect on membrane permeable steroid formation. However, VDAC1 and ATAD3A knockdowns did reduce hormone-induced steroidogenesis, suggesting that OPA1 is not critical for hormone-induced steroidogenesis38. Recently it was shown that upregulating OPA1 via pharmacological and transfection methods increased TSPO expression and steroid hormone formation in both basal and hormone-stimulated dysfunctional Leydig cells, suggesting that OPA1 may play a role in the regulation and formation of the SITE complex76. Progress has been made in targeting SITE proteins to ameliorate testosterone decline. Studies on TSPO ligands and 14-3-3ε peptides (VDAC1 peptides) have offered potential therapeutic strategies for inducing endogenous testosterone formation. While TSPO ligands enhance cholesterol translocation, VDAC1 peptides are designed to block the negative regulation of steroidogenesis34,51,77- 80.
TSPO ligands
Engagement of the OMM protein TSPO via a drug ligand-induced activation stimulates steroid hormone production in vitro and in vivo in rats and mice48,61,77,78,81,82. TSPO possesses a high affinity for cholesterol binding, which leads to its subsequent translocation to the IMM for side-chain cleavage by CYP11A1-producing pregnenolone83,84. TSPO’s C terminus plays a key role in the uptake of cholesterol from the cytosol and translocation into the mitochondria85,86, and disruption of the protein within steroidogenic cells disrupts mitochondrial cholesterol transport and steroid formation84,87. Steroidogenesis and TSPO expression correlate with one another as shown by disruption of steroidogenesis with TSPO’s age-related decline in vivo and its ablation in vitro62,87. Moreover, transfection of TSPO into TSPO-disrupted cells restores steroid formation, demonstrating its indispensable role in steroidogenesis87.
Numerous studies have shown that drug ligands targeting TSPO produce enhanced steroid levels in both MA-10 tumorigenic Leydig cells and isolated primary Leydig cells, as well as increased serum testosterone levels77,78,81. However, serum LH levels may also become increased following TSPO drug ligand treatment likely due to an effect of the ligand on brain TSPO88,89, suggesting that using this target may enhance testosterone biosynthesis by either stimulating the Leydig cell steroidogenic machinery and/or by elevating LH release77. TSPO-specific ligands are also known to increase glucocorticoid and corticosteroid levels61 and have been shown to affect neurosteroid production90-92. Accordingly, the use of TSPO ligands as a therapeutic approach to treat neurological and psychiatric disorders has also been investigated93. Similarly, the use of TSPO ligands may also induce anxiolytic-like responses, as ligand treatment has been shown to counteract panic attacks in rodents94. While molecular entities targeting TSPO elevate serum testosterone levels, adrenal steroids and neurosteroids are also affected. Therefore, TSPO ligands have been proposed as therapeutic agents for the regulation of steroid hormones in the testis and brain. However, this lack of specificity remains an issue, as TSPO is expressed in numerous tissues.
-VDAC1 peptides
New insights into the role of 14-3-3ε in the regulation of steroidogenesis have made it a promising therapeutic target. 14-3-3 proteins regulate target proteins by altering activity, posttranslational modifications, and subcellular localization95. LHR stimulation initiates the translocation of 14-3-3ε to the OMM33 and its recruitment to the TSPO-VDAC1 complex at Ser167 on VDAC1. There it competes with TSPO for VDAC1 binding and thus reduces cholesterol import51. Blocking the interaction between 14-3-3ε and VDAC1 using cell-penetrating peptides induces steroid formation in vivo and ex vivo80. Aghazadeh et al. fused a component of the HIV transcription factor 1 (TAT) with the predicted Ser167 binding motif on 14-3-3ε, creating a cell-permeable VDAC1 peptide, TATVDAC1 containing Ser167 (TVS167), which competed with 14-3-3ε for VDAC1 binding79. This reduced the negative regulation of steroidogenesis by blocking the 14-3-3ε binding to VDAC1, which led to increased steroidogenesis in vitro and in vivo. Given the homologous mechanisms of 14-3-3ε between species, the TAT-based peptide offers a promising approach in humans. Although TAT peptides penetrate indiscriminately and 14-3-3ε is found in numerous tissues, the function is tissue-specific79. TVS167 treatment did not significantly increase corticosterone levels in rats treated with the compound, demonstrating specificity to testicular Leydig cells80. Additionally, the action of the TVS167 peptide induced steroidogenesis independent of LH and would offer a major improvement in safety when compared to TRT96. The minimal bioactive sequence of the peptide was recently identified, and we ultimately generated bioactive stable peptide derivatives that can be administered orally and induce T formation in normal and hypogonadal animal models (manuscript in preparation). Moreover, they demonstrate safety, efficacy, and target specificity34,51,79,80. In summary, these first-in-class biologics make an excellent candidate for the treatment of diseases caused by Leydig cell dysfunction over other pharmacologic or biologic strategies.
-Implantation of human Leydig-like cells
The generation of transplantable testosterone-producing cells offers another alternative for treating pathologies related to Leydig cell dysfunction. Previously, it was shown that mesenchymal stem cells (MSCs) were able to differentiate into testosterone-producing Leydig cells, suggesting that healthy Leydig cell populations could be transplanted into hypogonadal patients97. However, MSC isolation produces limited cell numbers and reduces the clinical application of this method. Recent developments have revealed human Leydig-like cells (hLLCs) can be generated from human induced pluripotent stem cells (hiPSCs), which are highly expandable in cell culture98,99. Li et al. demonstrated that hLLCs producing steroidogenic gene expression, steroidogenic enzymes, and testosterone could be generated by differentiating early mesenchymal progenitors from hiPSCs while overexpressing steroidogenic factor 1 (SF-1) in culture with dibutyryl-cAMP (dbcAMP), recombinant desert hedgehog, and human chorionic gonadotropin (hCG)98. Given their clinical viability, the implantation of hLLCs would represent a monumental step forward in treating diseases related to Leydig cell dysfunction. This strategy could restore testosterone levels by replenishing testosterone-producing-cell populations in the testicular environment, leading to the production of endogenous testosterone formation.
Conclusions and future directions
Testosterone deficiency impacts the quality of life and well-being of millions of men worldwide, with only limited treatments having undesirable off-target effects16. A new understanding of the molecular interactions producing testosterone has laid the foundation for the development of novel therapeutic strategies. Identification of the hormonally regulated multiprotein SITE complex38 and the deeper understanding of hormonal stimulation and cholesterol translocation from cytosolic stores across the OMM and into the IMM for side-chain cleavage demonstrates numerous therapeutic targets for various indications related to hormone insufficiency (Table 1)16,18. However, their effects on neurosteroids, adrenal steroids, and the HPG axis have remained a barrier to the safe and efficacious treatment of testosterone deficiency. Apart from VDAC1 peptides, existing strategies have lacked specificity for testicular Leydig cells and, therefore, have raised concerns regarding off-target effects (Fig. 2). VDAC1 peptides are first-in-class biologics that offer a novel approach for rescuing intratesticular and serum testosterone formation in hormonally mediated diseases79,80. These therapeutics could be used to restore endogenous testosterone formation and restore well-being for millions of aging men worldwide.
There are additional mechanisms to uncover. The movement of cholesterol between the mitochondrial membranes, the relationship between aging and the Leydig cell oxidative environment, and age-dependent protein-protein interactions remain elusive and are active areas of research24. With more information, we may determine the cause of reduced testosterone and develop interventions that may maintain Leydig cell function. Moreover, targeting the molecular deteriorations that differ between aging Leydig cells and other aging steroidogenic tissues could lead to additional testis-specific strategies.
