Wen Guo

Testosterone Induces Erythrocytosis via Increased Erythropoietin and Suppressed Hepcidin: Evidence for a New Erythropoietin/Hemoglobin Set Point

BACKGROUNDnThe mechanisms by which testosterone increases hemoglobin and hematocrit remain unclear.nnnMETHODSnWe assessed the hormonal and hematologic responses to testosterone administration in a clinical trial in which older men with mobility limitation were randomized to either placebo or testosterone gel daily for 6 months.nnnRESULTSnThe 7%-10% increase in hemoglobin and hematocrit, respectively, with testosterone administration was associated with significantly increased erythropoietin (EPO) levels and decreased ferritin and hepcidin levels at 1 and 3 months. At 6 months, EPO and hepcidin levels returned toward baseline in spite of continued testosterone administration, but EPO levels remained nonsuppressed even though elevated hemoglobin and hematocrit higher than at baseline, suggesting a new set point. Consistent with increased iron utilization, soluble transferrin receptor (sTR) levels and ratio of sTR/log ferritin increased significantly in testosterone-treated men. Hormonal and hematologic responses were similar in anemic participants. The majority of testosterone-treated anemic participants increased their hemoglobin into normal range.nnnCONCLUSIONSnTestosterone-induced increase in hemoglobin and hematocrit is associated with stimulation of EPO and reduced ferritin and hepcidin concentrations. We propose that testosterone stimulates erythropoiesis by stimulating EPO and recalibrating the set point of EPO in relation to hemoglobin and by increasing iron utilization for erythropoiesis.

The Effects of a Single Developmentally Entrained Pulse of Testosterone in Female Neonatal Mice on Reproductive and Metabolic Functions in Adult Life

Early postnatal exposures to sex steroids have been well recognized to modulate predisposition to diseases of adulthood. There is a complex interplay between timing, duration and dose of endocrine exposures through environmental or dietary sources that may alter the sensitivity of target tissues to the exogenous stimuli. In this study, we determined the metabolic and reproductive programming effects of a single developmentally entrained pulse of testosterone (T) given to female mice in early postnatal period. CD-1 female mice pups were injected with either 5 μg of T enanthate (TE) or vehicle (control [CON] group) within 24 hours after birth and followed to adult age. A total of 66% of T-treated mice exhibited irregular cycling, anovulatory phenotype, and significantly higher ovarian weights than vehicle-treated mice. Longitudinal nuclear magnetic resonance measurements revealed that TE group had greater body weight, whole-body lean, and fat mass than the CON group. Adipose tissue cellularity analysis in TE group revealed a trend toward higher size and number than their littermate CONs. The brown adipose tissue of TE mice exhibited white fat infiltration with down-regulation of several markers, including uncoupling protein 1 (UCP-1), cell death-inducing DNA fragmentation factor, α-subunit-like effector A, bone morphogenetic protein 7 as well as brown adipose tissue differentiation-related transcription regulators. T-injected mice were also more insulin resistant than CON mice. These reproductive and metabolic reprogramming effects were not observed in animals exposed to TE at 3 and 6 weeks of age. Collectively, these data suggest that sustained reproductive and metabolic alterations may result in female mice from a transient exposure to T during a narrow postnatal developmental window.

Testosterone Supplementation Improves Anemia in Aging Male Mice

Whether aging alone causes anemia is still controversial. In this study, we show that 28-month-old male C57BL/6 mice, maintained in a pathogen-free environment, had significantly lower hemoglobin, hematocrit, and erythrocyte counts than young mice. The anemic condition aggravated further from 28 to 30 months. Old mice displayed increased erythropoietic activity, evidenced by an increase in reticulocyte counts, serum erythropoietin, and splenic expression of erythropoietic genes. An increase in late-stage erythroid progenitors was detected in spleen but not in bone marrow of the old mice. However, old mice also had lower serum iron and transferrin saturation, as well as lower erythrocyte iron incorporation rate. Testosterone supplementation restored serum iron status in old mice to levels similar to that of young adults, further upregulated splenic expression of erythropoietic genes, increased splenic erythroid progenitors, and significantly improved the red cell index. In conclusion, we found that mice can become anemic at very old age without apparent illness. The endogenous compensatory erythropoietic activity was insufficient to normalize the red cell index in old mice, either due to impaired iron homeostasis, ineffective erythropoiesis, or other unknown factors. Testosterone supplementation normalized the iron status and further stimulated splenic erythropoietic activity; both may contribute to improve the anemic condition in the old mice.

Effects of Testosterone on Erythropoiesis in a Female Mouse Model of Anemia of Inflammation

The anemia of inflammation is a common problem in inflammatory and autoimmune diseases. We characterized a mouse model of anemia of chronic inflammation induced by repeated injections of low doses of heat-killed Brucella abortus (HKBA), and determined the effects of T administration on erythropoiesis in this model. Female C57BL/6NCrl mice were injected weekly with HKBA for 10 wk. Weekly injections of T or vehicle oil were started 4 wk later. Control mice were injected with saline and vehicle oil in parallel. HKBA-injected mice had significantly lower hemoglobin, hematocrit, mean corpuscular volume, reticulocyte hemoglobin, transferrin saturation (TSAT), and tissue nonheme iron in liver and spleen, enlarged spleen, and up-regulated hepatic expression of inflammatory markers, serum amyloid A1, and TNFα, but down-regulated IL-6, bone morphogenic protein 6, and hepcidin compared with saline controls. HKBA also reduced serum hepcidin and increased serum erythropoietin. Bone marrow erythroid precursors were substantially reduced in HKBA-injected mice. Cotreatment with T increased the percentage of late-stage erythroid precursors in the bone marrow relative to HKBA-injected and saline controls and reversed HKBA-induced suppression of hemoglobin and hematocrit. T also normalized serum erythropoietin, TSAT, and reticulocyte hemoglobin without correcting the expression of the hepatic inflammation markers. Conclusions are that low-dose HKBA induces moderate anemia characterized by chronic inflammation, decreased iron stores, and suppression of erythroid precursors in the bone marrow. T administration reverses HKBA-induced anemia by stimulating erythropoiesis, which is associated with a shift toward accelerated maturation of erythroid precursors in the bone marrow.

The effects of an ActRIIb receptor Fc fusion protein ligand trap in juvenile simian immunodeficiency virus-infected rhesus macaques

There are no approved therapies for muscle wasting in children infected with human immunodeficiency virus (HIV), which portends poor disease outcomes. To determine whether a soluble ActRIIb receptor Fc fusion protein (ActRIIB.Fc), a ligand trap for TGF‐β/activin family members including myostatin, can prevent or restore loss of lean body mass and body weight in simian immunodeficiency virus (SIV)‐infected juvenile rhesus macaques (Macaca mulatta). Fourteen pair‐housed, juvenile male rhesus macaques were inoculated with SIVmac239 and, 4 wk postinoculation (WPI) treated with intramuscular injections of 10 mg · kg‐1 · wk‐1 ActRIIB.Fc or saline placebo. Body weight, lean body mass, SIV titers, and somatometric measurements were assessed monthly for 16 wk. Age‐matched SIV‐infected rhesus macaques were injected with saline. Intervention groups did not differ at baseline. Gains in lean mass were significantly greater in the ActRIIB.Fc group than in the placebo group (P < 0.001). Administration of ActRIIB.Fc was associated with greater gains in body weight (P = 0.01) and upper arm circumference than placebo. Serum CD4+ T‐lymphocyte counts and SIV copy numbers did not differ between groups. Administration of ActRIIB.Fc was associated with higher muscle expression of myostatin than placebo. ActRIIB.Fc effectively blocked and reversed loss of body weight, lean mass, and fat mass in juvenile SIV‐infected rhesus macaques.—OConnell, K. E., Guo, W., Serra, C., Beck, M., Wachtman, L., Hoggatt, A., Xia, D., Pearson, C., Knight, H., OConnell, M., Miller, A. D., Westmoreland, S. V., Bhasin, S. The effects of an ActRIIb receptor Fc fusion protein ligand trap in juvenile simian immunodeficiency virus‐infected rhesus macaques. FASEB J. 29, 1165‐1175 (2015). www.fasebj.org

The Effects of Short-Term and Long-Term Testosterone Supplementation on Blood Viscosity and Erythrocyte Deformability in Healthy Adult Mice

Testosterone treatment induces erythrocytosis that could potentially affect blood viscosity and cardiovascular risk. We thus investigated the effects of testosterone administration on blood viscosity and erythrocyte deformability using mouse models. Blood viscosity, erythrocyte deformability, and hematocrits were measured in normal male and female mice, as well as in females and castrated males after short-term (2 wk) and long-term (5-7 mo) testosterone intervention (50 mg/kg, weekly). Castrated males for long-term intervention were studied in parallel with the normal males to assess the effect of long-term testosterone deprivation. An additional short-term intervention study was conducted in females with a lower testosterone dose (5 mg/kg). Our results indicate no rheological difference among normal males, females, and castrated males at steady-state. Short-term high-dose testosterone increased hematocrit and whole-blood viscosity in both females and castrated males. This effect diminished after long-term treatment, in association with increased erythrocyte deformability in the testosterone-treated mice, suggesting the presence of adaptive mechanism. Considering that cardiovascular events in human trials are seen early after intervention, rheological changes as potential mediator of vascular events warrant further investigation.

Combined administration of testosterone plus an ornithine decarboxylase inhibitor as a selective prostate-sparing anabolic therapy

Because of its anabolic effects on muscle, testosterone is being explored as a function‐promoting anabolic therapy for functional limitations associated with aging; however, concerns about testosterones adverse effects on prostate have inspired efforts to develop strategies that selectively increase muscle mass while sparing the prostate. Testosterones promyogenic effects are mediated through upregulation of follistatin. We show here that the administration of recombinant follistatin (rFst) increased muscle mass in mice, but had no effect on prostate mass. Consistent with the results of rFst administration, follistatin transgenic mice with constitutively elevated follistatin levels displayed greater muscle mass than controls, but had similar prostate weights. To elucidate signaling pathways regulated differentially by testosterone and rFst in prostate and muscle, we performed microarray analysis of mRNAs from prostate and levator ani of castrated male mice treated with vehicle, testosterone, or rFst. Testosterone and rFst shared the regulation of many transcripts in levator ani; however, in prostate, 593 transcripts in several growth‐promoting pathways were differentially expressed after testosterone treatment, while rFst showed a negligible effect with only 9 transcripts differentially expressed. Among pathways that were differentially responsive to testosterone in prostate, we identified ornithine decarboxylase (Odc1), an enzyme in polyamine biosynthesis, as a testosterone‐responsive gene that is unresponsive to rFst. Accordingly, we administered testosterone with and without α‐difluoromethylornithine (DFMO), an Odc1 inhibitor, to castrated mice. DFMO selectively blocked testosterones effects on prostate, but did not affect testosterones anabolic effects on muscle. Co‐administration of testosterone and Odc1 inhibitor presents a novel therapeutic strategy for prostate‐sparing anabolic therapy.

Administration of an activin receptor IIB ligand trap protects male juvenile rhesus macaques from simian immunodeficiency virus-associated bone loss

HIV-infected individuals are at an increased risk of osteoporosis despite effective viral suppression. Observations that myostatin null mice have increased bone mass led us to hypothesize that simian immunodeficiency virus (SIV)-associated bone loss may be attenuated by blocking myostatin/TGFβ signaling. In this proof-of-concept study, pair-housed juvenile male rhesus macaques were inoculated with SIVmac239. Four weeks later, animals were treated with vehicle or Fc-conjugated soluble activin receptor IIB (ActR2B·Fc, iv. 10mg∗kg-1∗week-1) - an antagonist of myostatin and related members of TGFβ superfamily. Limb and trunk bone mineral content (BMC) and density (BMD) using dual-energy X-Ray absorptiometry, circulating markers of bone growth and turnover, and serum testosterone levels were measured at baseline and during the 12-week intervention period. The increase in BMC was significantly greater in the ActRIIB.Fc-treated group (+8g) than in the placebo group (-4g) (p<0.05). BMD also increased significantly more in the ActRIIB.Fc-treated macaques (+0.03g/cm2) than in the placebo-treated animals (+0g/cm2) (p<0.005). Serum osteocalcin was about two-fold higher in the ActRIIB.Fc-treated group than in the placebo group (p<0.05), but serum C-terminal telopeptide and testosterone levels did not differ significantly between groups. The expression levels of TNFalpha (p<0.05), GADD45 (p<0.005), and sclerostin (p<0.038) in the bone-marrow were significantly lower in the ActRIIB.Fc-treated group than in the placebo group.nnnCONCLUSIONnThe administration of ActRIIB.FC in SIV-infected juvenile macaques significantly increases BMC and BMD in association with reduced expression levels of markers of bone marrow inflammation.

Joint dysfunction and functional decline in middle age myostatin null mice

Since its discovery as a potent inhibitor for muscle development, myostatin has been actively pursued as a drug target for age- and disease-related muscle loss. However, potential adverse effects of long-term myostatin deficiency have not been thoroughly investigated. We report herein that male myostatin null mice (mstn(-/-)), in spite of their greater muscle mass compared to wild-type (wt) mice, displayed more significant functional decline from young (3-6months) to middle age (12-15months) than age-matched wt mice, measured as gripping strength and treadmill endurance. Mstn(-/-) mice displayed markedly restricted ankle mobility and degenerative changes of the ankle joints, including disorganization of bone, tendon and peri-articular connective tissue, as well as synovial thickening with inflammatory cell infiltration. Messenger RNA expression of several pro-osteogenic genes was higher in the Achilles tendon-bone insertion in mstn(-/-) mice than wt mice, even at the neonatal age. At middle age, higher plasma concentrations of growth factors characteristic of excessive bone remodeling were found in mstn(-/-) mice than wt controls. These data collectively indicate that myostatin may play an important role in maintaining ankle and wrist joint health, possibly through negative regulation of the pro-osteogenic WNT/BMP pathway.

Effects of an ActRIIB.Fc Ligand Trap on Cardiac Function in Simian Immunodeficiency Virus-Infected Male Rhesus Macaques

Abstract An important safety consideration in the use of antagonists of myostatin and activins is whether these drugs induce myocardial hypertrophy and impair cardiac function. The current study evaluated the effects of a soluble ActRIIB receptor Fc fusion protein (ActRIIB.Fc), a ligand trap for TGF-β/activin family members including myostatin, on myocardial mass and function in simian immunodeficiency virus (SIV)-infected juvenile rhesus macaques (Macaca mulatta). Fourteen pair-housed, juvenile male rhesus macaques were inoculated with SIVmac239; 4 weeks postinoculation, they were treated with weekly injections of 10 mg/kg ActRIIB.Fc or saline for 12 weeks. Myocardial mass and function were evaluated using two-dimensional echocardiography at baseline and after 12 weeks. The administration of ActRIIB.Fc was associated with a significantly greater increase in thickness of left ventricular posterior wall and interventricular septum both in diastole and systole. Cardiac output and cardiac index increased with time, more in animals treated with ActRIIB.Fc than in those treated with saline, but the difference was not statistically significant. The changes in ejection fraction, fractional shortening, and stroke volume did not differ significantly between groups. The changes in end-diastolic and end-systolic volumes did not differ between groups. In addition to a large reduction in IGF1 mRNA expression in the ActRIIB.Fc-treated animals, complex changes were detected in the myocardial expression of proteins related to calcium transport and storage. In conclusion, ActRIIB.Fc administration for 12 weeks was associated with increased myocardial mass but did not adversely affect myocardial function in juvenile SIV-infected rhesus macaques. Further studies are necessary to establish long-term cardiac safety.