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Sunday, June 24, 2012

New Testosterone Studies

Thanks to  Dr Michael Scally. Follow him at :

Posted: 24 Jun 2012 09:32 AM PDT

[SUN-75] Improvement in Sexual Function and Mood as a Function of Testosterone Level in Hypogonadal Men Receiving Testosterone Replacement Therapy

Xiao Ni, David Muram. Eli Lilly and Company, Indianapolis, IN; Eli Lilly and Company, Indianapolis, IN.

Based on limited available research (1-3) we undertook this post-hoc analysis to gain insights into symptomatic improvement in hypogonadal men on testosterone replacement therapy (TRT) based on whether they reached the pre-specified threshold of serum testosterone (T) 300 ng/dL.

This open-label trial (4) enrolled 155 testosterone-deficient (T<300ng/dL) men given an initial daily dose of T 60 mg in a topical solution applied to the axillae. Dose was adjusted on days 45 and 90 when necessary to maintain T within the physiological range (300-1050ng/dL). Sexual function and mood changes were assessed for 7 days preceding visits on days 1, 15, 60, and 120 by the Psychosexual Daily Questionnaire (PDQ) (5). Subjects were divided into two groups (T<300 and ≥300 ng/dL) based on their T level during therapy. Analysis of covariance (ANCOVA) with adjustments for baseline PDQ scores, age, and body mass index (BMI) was used to evaluate change in PDQ scores from baseline (pre-day1) to pre-day15 and pre-day 120 (with last-observation-carried-forward for dropouts) both within and between the groups.

As early as day 15, numerical improvement was observed in all PDQ scores (sexual desire, sexual activity, percent full erection, erection maintained, and positive and negative mood) for both groups. Within-group improvement was significant (p<0.05) for all parameters except positive mood in group T<300. Improvement in PDQ scores was maintained or increased on day 120. For example, sexual desire changes on day 15 for the T<300 and ≥300 ng/dL groups were 1.1 (0.2) and 0.8 (0.1) (least square means [standard error]) respectively; on day 120 values were 1.1 (0.3) and 1.5 (0.1) respectively. On day 120, changes in PDQ scores were numerically greater in the T≥300 ng/dL group than in the T<300 ng/dL group, although the between-group difference was not significant (p>0.05).

These data show that symptoms in hypogonadal men receiving TRT improved by day 15 and continued until day 120. Improvements were noted even in those with T<300 after treatment. The study is limited by lack of a placebo control group and a small number of patients with serum T levels <300ng/dL after treatment and also that the analysis groupings (T groups) are based on post-baseline efficacy results. Additional research is needed to better understand the thresholds at which patients and physicians can expect symptoms of hypogonadism to improve.

(1) Kelleher S et al., J Clin Endocrinol Metab 2004;89:3813.
(2) Zitzmann M et al., J Clin Endocrinol Metab 2006;91:4335.
(3) Saad F et al., Eur J Endocrinol 2011;165:675.
(4) Wang C etal., J Clin Endocrinol Metab 1996;81:3578.
(5) Lee KK et al., J Androl 2003;24:688.

Sources of Research Support: Eli Lilly and Company.

Disclosures: XN: Researcher, Eli Lilly & Company. DM: Clinical Researcher, Eli Lilly & Company.

Posted: 24 Jun 2012 09:31 AM PDT

[SAT-118] Restoring Testosterone to Normal Levels in Elderly Men Is Efficacious in Weight Reduction. A Follow-Up Study over 5 Years

Farid Saad, Ahmad Haider, Louis Gooren. Bayer Pharma AG, Berlin, Germany; Gulf Medical University School of Medicine, Ajman, United Arab Emirates; Private Practice, Bermerhaven, Germany; VUMC Amsterdam, Amsterdam, Netherlands.

Introduction: Obesity is associated with reduced testosterone, and low testosterone induces weight gain. This study analysed the effects of normalization of serum testosterone in mainly elderly, hypogonadal men.

Methods: Open-label, single-center, cumulative, prospective registry study of 255 men (aged 38 – 83 years, mean 60.6 ± 8.0 years), with testosterone levels between 1.7 –3.5 ng/mL (mean: 2.87 ± 0.4). Cut-off point for testosterone treatment was serum testosterone ≤ 3.5. ng/mL). 215 men were studied for at least 2 years, 182 for 3 years, 148 for 4 and 116 for at least 5 years. They received parenteral testosterone undecanoate 1000 mg/12 weeks after an initial interval of 6 weeks.

Results: After 5 years the following changes were observed: weight (kg) decreased from 106.22 ± 16.93 (minimum: 70, maximum: 139) to 90.07 ± 9.51 (min 74.00, max 115). The statistical significance was p<0.0001 vs baseline and vs the previous year over 5 years indicating a continuous weight loss over the full observation period. Waist circumference (cm) declined from 107.24 ± 9.14 (min 86, max 129) to 98.46 ± 7.39 (min 84, max 117) (p<0.0001 vs baseline and vs the previous year over 5 years). Body mass index (BMI, m/kg2) declined from 33.93 ± 5.54 (min 21.91, max 46.51) to 29.17 ± 3.09 (min 22.7; max 36.71) (p<0.0001 vs baseline and vs the previous year over 5 years). The mean per cent weight loss after 1 year was 4.12 ± 3.48%, after 2 years 7.47 ± 5.01%, after 3 years 9.01 ± 6.5%, after 4 years 11.26 ± 6.76% and after 5 years 13.21 ± 7.24%. At baseline, 96% of men had a waist circumference of ≥ 94 cm. This proportion decreased to 71% after 5 years.

Conclusions: Raising serum testosterone to normal produced loss of body weight, waist circumference and BMI. These improvements were progressive over the full 5 years of the study.
Sources of Research Support: Data entry and statistical analysis were supported by Bayer Pharma AG.

Disclosures: FS: Employee, Bayer Schering Pharma. AH: Principal Investigator, Bayer Schering Pharma. LG: Speaker, Bayer Schering Pharma.

Posted: 24 Jun 2012 09:29 AM PDT

[MON-51] Long-Term Testosterone Treatment in Hypogonadal Men Improves All Components of the Metabolic Syndrome

Aksam Yassin, Youssef El Douaihy, Ridwan Shabsigh, Aiman Yassin, Farid Saad. Segeberger Kliniken, Norderstedt, Germany; Dresden International University, Dresden, Germany; Gulf Medical University School of Medicine, Ajman, United Arab Emirates; Maimonides Medical Center, Brooklyn, NY; Klinikum Braunschweig, Braunschweig, Germany; Bayer Pharma AG, Berlin, Germany; Gulf Medical University School of Medicine, Ajman, United Arab Emirates.

Introduction: Metabolic syndrome as well as all its individual components are associated with testosterone deficiency. We hypothesize that long-term testosterone therapy improves the metabolic syndrome.

Materials and Methods: Since November 2004, 261 patients diagnosed with LOH in our center in Germany were entered into our registry study for long term testosterone replacement therapy. Men with a total testosterone of ≤3.5 ng/mL and symptoms of erectile dysfunction (IIEF-5< 21) met the inclusion criteria. Long acting testosterone undecanoate 1000 mg injections were administered at day 1, after 6 weeks and every 3 months thereafter. The mean follow up time was 4.25 years. Demographic data were collected at the baseline. Hormones, lipids, glucose and blood pressure were measured at every visit or every other visit. We used the International Diabetes Federation definition to define whether patients had the metabolic syndrome (MetS). MetS was considered in patients with central obesity and any two of the following: raised triglycerides, reduced HDL cholesterol, raised blood pressure, and raised fasting plasma glucose. Mean fasting glucose, triglycerides, and HDL were calculated at every T and linear regression was used to study their variation over time. Total testosterone and prevalence (%) of the MetS were plotted against time.

Results: we noticed a significant drop in the prevalence of the MetS from 56% to 30% after 57 months. There was a statistically significant drop of triglycerides, glucose, and the mean arterial pressure (p=0.00; β=-0.77, p=0.00; β=-0.67, and p=0.00; β=-0.78respectively) and an increase in HDL (p=0.05; β=0.53). In addition, we noticed a significant drop of 11 cm in the mean waist circumference.
Conclusion: All components of the MetS improved significantly and over the full observation period upon normalization of testosterone levels.

Sources of Research Support: Data entry was supported by Bayer Pharma AG.

Disclosures: AY: Speaker, Bayer Schering Pharma. RS: Speaker, Lilly USA, LLC. FS: Employee, Bayer Schering Pharma. Nothing to Disclose: YED, AY

Posted: 24 Jun 2012 09:26 AM PDT

[SUN-577] Identification of Four New Sex Hormone-Binding Globulin Variants with Abnormal Production or Function

Tsung-Sheng Wu, Caroline Underhill, Geoffrey Lewis Hammond. Child & Family Research Institute, Vancouver, Canada.

Background: Sex hormone-binding globulin (SHBG) binds sex steroids with high affinity and specificity. The levels of SHBG in blood determine the non-protein bound “free” fractions of sex steroids that are able to access target tissues and cells. Genetic polymorphisms of SHBG have been associated with altered SHBG levels or functions and linked to the risk of several diseases, including reproductive tissue cancers and type 2 diabetes. Recently, large scale genome and exome sequencing projects have revealed numerous single nucleotide polymorphisms (SNP) within the SHBG gene. Based on our knowledge of SHBG tertiary and quaternary structure, we selected nine new SNPs, which result in amino acid substitutions in the SHBG amino-terminal LG domain that mediates steroid-binding and dimerization, and examined their impact on SHBG production and/or function.

Methods: SHBG variants were expressed in Chinese hamster ovary cells for characterization by steroid-binding capacity and time-resolved immunofluorometric assays, which allow the steroid binding and dimerization properties of SHBG to be assessed.

Results: Four out of the nine naturally occurring SHBG variants studied have specific abnormalities. SHBG T48I and SHBG R123H have reduced steroid-binding affinity; SHBG R154W is dimerization deficient and has a lower binding affinity for estrogens but a normal affinity for androgens; SHBG G195E is not produced or secreted efficiently, probably due to misfolding during synthesis.

Conclusions: A method for the screening and functional analysis of SHBG genetic variants has been implemented to assess the impact of newly discovered genetic variants of SHBG on its production and/or function. This information will need to be assessed in relation to a wide range of diseases that have already been associated with SHBG polymorphisms. In addition, algorithms designed to calculate free testosterone or estradiol levels will need to take into consideration the effects of these new SHBG variants.

Posted: 24 Jun 2012 09:24 AM PDT

[SUN-575] The Role of Testosterone in the Control of Muscle Protein Balance

Lucian Nicolae Sandor, Daniel Lee, Gianluca Toraldo, Anqi Zhang, Ravi Jasuja, Shalender Bhasin, Carlo Serra. Boston Medical Center, Boston, MA.

Background: Skeletal muscle is the main protein and energy storage of the body. Aging and pathological conditions perturb protein turnover and the muscle response to anabolic stimuli. Gains in muscle mass occur through either increases in muscle protein synthesis or declines in muscle protein breakdown. Several lines of evidence indicate that testosterone increases muscle mass by stimulating either protein synthesis and reducing protein degradation. In addition, androgens prevent muscle atrophy and weakness caused by glucocorticoids in humans and rodents. These data suggest that androgens inhibit proteolysis, and enhance specific steps of protein synthesis and/or protect from degradation key factors of the translational machinery.

Methods: To assess the effect of testosterone on muscle protein synthesis and degradation we used the Hershberger assay in rodents. 2-month old male mice were castrated and treated with vehicle or testosterone propionate. Mice were killed after 4 and 7 days of testosterone treatment. Sham-operated, untreated mice and 7-day castrated mice served as control.

Results: The androgen-dependent levator ani muscle underwent rapid atrophy after castration, showed reduced level of androgen receptor and FoxOs phosphorylation, as well as increased MAFbx and MuRF1 gene expression. Testosterone administration rescued these changes. Castration induced the expression of key autophagy genes, such as Bnip3, LC3B, p62 and Cathepsin-L, indicating an active recruitment of autophagy components during levator ani muscle atrophy. Castrated mice showed also the increased gene expression of Fis1, a component of the mitochondrial fission machinery activated during autophagy. Castration was associated to reduced activation and activity of mTOR, as indicated by the level of its phosphorylation and by that of 4E-BP1. Testosterone rescued these effects and enhanced the phosphorylation of eIF4G and of the ribosomal protein S6rp, a target of activated p70S6k1. Castrated mice showed reduced protein level of eIF3f, of eIF2Bε, the enzymatic subunit of the eIF2B complex, and of the elongation factor eEF2. Testosterone rescued all these changes.

Conclusion: Testosterone supplementation re-establishes an optimal protein balance in atrophic muscle, and regulates the protein degradation pathways as well as the early steps of mRNA translation. These data indicate potential new therapeutic targets in androgen-based therapies for muscle wasting.

Posted: 24 Jun 2012 09:20 AM PDT

[OR28-1] Association between Serum Testosterone Levels and Vascular Calcification in Community-Dwelling Men: The Framingham Heart Study

Thomas G Travison, Shehzad Basaria, Shalender Bhasin, Anh-Hoa Nguyen, Joseph Massaro, Warren J Manning, Ramachandran S Vasan, Christopher J O'Donnell. Boston University School of Medicine, Boston, MA; Boston University School of Public Health, Boston, MA; Harvard Medical School, Boston, MA; National Heart, Lung and Blood Institute, Framingham, MA.

Background: Observational associations between male circulating testosterone (T) levels and cardiovascular disease and mortality have been widely reported, but the relationship between T and atherosclerosis is poorly understood. Here we report an analysis of association between serum testosterone and vascular calcification in adult men.

Methods: Data were available on 1678 participants belonging to Generation 2 (examination 7) and Generation 3 (examination 1) of the Framingham Heart Study cohort, and were obtained in clinical study visits conducted between 1998 and 2008. Early-morning serum total testosterone (TT) was measured by liquid chromatography-tandem mass spectrometry, and free testosterone was calculated (cFT) using mass action equations. Sex hormone-binding globulin (SHBG) was measured using immunofluorometric assay. Thoracic aortic calcification (TAC), abdominal aortic calcification (AAC), and coronary artery calcification (CAC) were measured by computed tomography scan. Covariables considered included age, body mass index (BMI), smoking, fasting lipids, and comorbid conditions. Estimates of association were obtained by Tobit regression, which acknowledges the influence of floor effects on outcomes.

Results: Subjects had mean (SD) age 49 (10) y with range 31-80 y; mean (SD) BMI was 28 (5) kg/m2. Mean (SD) TT, cFT, and SHBG were 616 (224) ng/dl, 111 (45) pg/ml, and 46 (23) nmol/l, respectively. TT and cFT exhibited highly significant positive, and SHBG negative, association with vascular calcification at all sites (p<0.001). After adjusting for age, SHBG displayed no association with vascular calcification. Scaled to the mean sample CAC, a 100-ng/dl between-subject increase in TT was associated with a mean (SE) age-adjusted decrease in CAC of -8.6% (4.5%), p=0.04. Similarly, a 10 pg/ml positive difference in cFT was associated with a mean (SE) CAC decrease of -5.1% (2.5%), p=0.04. Trends of similar magnitude were observed for AAC and TAC. However, multivariable model adjustment for other cardiovascular risk factors (BMI, history of smoking, lipids and comorbidities) reduced estimated associations between testosterone and vascular calcification scores at all sites to statistical nonsignificance.

Conclusion: Low circulating testosterone levels in men exhibit an association with increased vascular calcification that is not explained by age alone, but which may be accounted for by their co-occurrence with established cardiovascular risk factors.

Posted: 24 Jun 2012 09:17 AM PDT

[MON-42] Cardiovascular Safety and Testosterone Replacement Therapy in Male Hypogonadism Including Men with Type 2 Diabetes and Cardiovascular Disease

Jonathan Charles Brooke, David Stuart McLaren, Deborah J Walter, Vakkat Muraleedharan, Thomas Hugh Jones. Barnsley Hopsital, NHSFT, Barnsley, UK; University of Sheffield, Sheffield, UK.

Testosterone replacement therapy (TRT) has recently been shown to have beneficial effects on CVD risk factors including insulin resistance, dyslipidaemia, obesity, hypertension and pro-atherogenic states. Nevertheless, the use of TRT remains controversial in men with cardiovascular disease.

A long-term retrospective audit of 401 (47.4% Type 2 diabetes (T2D), 34.0% CVD) hypogonadal men (age 58.7±14.3 years; mean baseline testosterone 7.16±2.59 nmol/l) receiving physiological TRT (84% testosterone gels) for up to 27 years (mean 5.41 years) in normal clinical practice was carried out to evaluate TRT's safety. BMI, waist circumference, blood pressure (BP), hemoglobin (Hb), hematocrit (HCT), lipid profile, liver function, testosterone, estradiol and PSA levels were monitored at 3, 6 and 12 months and yearly thereafter. Data from the most recent visits represent the primary endpoint. Hospital admissions, major adverse cardiovascular events (MACEs), mortalities and prostate-related outcomes were recorded.

TRT was associated with a reduction in total cholesterol (-0.28 mmol/l, p=0.004), non- fasting triglycerides (-0.244 mmol/l, p=0.04) and liver transaminases (ALT and AST) at the primary endpoint. HDL cholesterol fell by 0. /l (p=0.044). Hb increased by 0.72g/dl (p=0.002), HCT by 0.035 (p=0.011), PSA by 0.26g/l (p=0.008) and estradiol 128.45±44.58 at baseline vs 157±66.89 pmol/l. At the end of the study PSA levels were comparable to those of eugonadal males and were not grossly elevated. No significant changes in BMI, waist circumference or BP were observed. Safety outcomes: Polycythaemia (HCT >0.52) developed in 22 patients. 3 new cases of BPH and 2 prostate carcinomas were diagnosed, but this is no more than expected in this population which included aging males. 4 patients were diagnosed with T2D during the study, and 24 MACEs (4 MI, 8 angina, 5 TIAs, 2 CVAs, 4 CABG, 1 CCF) were identified. Over the course of the study there were 54 hospital admissions and 2 deaths which are no higher than expected in this morbid population.

This audit confirms the cardioprotective role of physiological doses of testosterone and demonstrates that in clinical practice TRT has beneficial effects on cardiovascular risk factors including circulating lipid and cholesterol levels. This is the largest and most comprehensive study of TRT safety to date, representing over 1642 patient years of TRT. TRT was not associated with an increase in prostate carcinoma, MACEs or mortality.

Posted: 24 Jun 2012 09:14 AM PDT

[MON-41] Effect of Testosterone Replacement on Response to Sildenafil Citrate in Men with Erectile Dysfunction: A Randomized, Placebo-Controlled Trial

Matthew Jonathan Spitzer, Shehzad Basaria, Maithili N Davda, Thomas G Travison, Amanda Paley, Beth Kaplan, Norman A Mazer, Philip E Knapp, Samson Hanka, Jagadish Ulloor, Anqi Zhang, Katie Orwoll, Richard Eder, Lauren Collins, Nurahmed Mohammed, Shalender Bhasin. Boston University School of Medicine and Boston Medical Center, Boston, MA; Boston University School of Public Health, Boston, MA; Hoffman LaRoche, Basel, Switzerland.

Background: Erectile dysfunction (ED) and low testosterone levels frequently co-occur in men. It is unclear whether the addition of testosterone to a phosphodiesterase-5-inhibitor improves erectile response.

Methods: In this randomized, double-blind, placebo-controlled trial, men age 40 to 70 years old with Erectile Function Domain (EFD) scores of ≤25 on the International Index of Erectile Function (IIEF), total testosterone <330 ng/dL (measured by LC-MS/MS) and/or free testosterone <50 pg/mL, were randomized to either 10-g testosterone or placebo gel for 14-weeks after they had received sildenafil for a period of 3-7 weeks. The primary outcome was change in EFD score. Secondary outcomes included changes in other domains of the IIEF and Male Sexual Health Questionnaire (MSHQ), frequency of total and successful sexual encounters, ED-Quality-of-Life (ED-QOL), marital-intimacy, vitality, and affectivity-balance. Sample mean changes and 95% confidence intervals (CI) were computed, and a Student's T-test was used to assess for differences by treatment arm.

Results: Testosterone and placebo groups were similar at baseline. Mean serum total testosterone at screening was 248 ng/dL and 254 ng/dL, which increased to 649 ng/dL and 338 ng/dL on sildenafil plus testosterone and sildenafil plus placebo, respectively. Administration of sildenafil alone was associated with substantial increases in EFD score (mean change for both arms 7.7; 95%CI 6.5 to 8.8) as well as in other domains of IIEF, MSHQ, frequency of total and successful sexual encounters, and ED-QOL. However, after randomization, the change in EFD score did not differ significantly between testosterone plus sildenafil (mean change 1.7) and placebo plus sildenafil (mean change 0.4; difference 1.3; 95%CI -0.8 to 3.5; P=0.23) arms. Similarly, changes with testosterone vs. placebo gel in other domains of the IIEF, frequency of total and successful sexual encounters, ED-QOL, marital-intimacy, vitality, and affectivity-balance did not differ between the two groups. Sensitivity analyses of men with baseline testosterone <250ng/dL did not reveal significant differences in EFD scores between arms.

Conclusions: Combined administration of testosterone plus sildenafil was not superior to sildenafil plus placebo in improving erectile function in men with ED and low testosterone levels.

Posted: 24 Jun 2012 09:12 AM PDT

[SUN-74] Effects of Baseline Testosterone Levels on Symptom Improvement in Hypogonadal Men Receiving Testosterone Replacement Therapy

Xiao Ni, David Muram. Eli Lilly and Company, Indianapolis, IN; Eli Lilly and Company, Indianapolis, IN.

It is possible that symptom improvement may be affected by baseline testosterone (T) level in men receiving testosterone replacement therapy (TRT). We performed a post-hoc analysis to evaluate the effects of baseline T level on changes in sexual function and mood in hypogonadal men receiving 2% testosterone in a topical solution applied daily to the axillae.

An open-label trial (1) was conducted in 155 androgen-deficient men started on daily 60 mg T. Dose was adjusted on days 45 and 90 based on average serum T on days 15 and 60, respectively, as necessary to maintain T in the physiologic range (300-1050ng/dL). Sexual desire, sexual activity, positive mood, negative mood, percent full erection and erection maintained were assessed by the Psychosexual Daily Questionnaire (PDQ) (2) for 7 days preceding visits at days 1, 15, 60 and 120. Subjects were divided into four subgroups depending on baseline (pre-day 1) total serum testosterone (TT)<100, 100<200, 200<300, and ≥300 ng/dL. For each PDQ parameter, change from baseline to days 15, 60 and 120 was analyzed using repeated measures analysis of covariance (ANCOVA) adjusted for TT group, age, body mass index (BMI) and visit, with baseline TT subgroup effect assessed by the type III test of fixed effects.

All PDQ domains showed significant (p<0.05) improvement for subjects in all TT subgroups. In general, improvement was maintained or increased during the 120-day treatment. When subgroups were compared, T levels at baseline did not appear to affect the magnitude of improvement; p>0.05 for all PDQ domains. Subjects in the lowest TT group (TT<100ng/dL) seemed to have the greatest numerical improvement in sexual desire, sexual activity, percent full erection, and erection maintained after adjusting for baseline age, BMI and visit. For example, the least squares (LS) means of the TT groups (main effects, averaged across other factors) for sexual desire were 1.70, 1.15, 1.13 and 0.97 for the TT<100, <200, 200<300, and ≥300ng/dL groups respectively. This trend was not observed in the positive and negative mood domains.

The study is limited by lack of a placebo control group and a relatively small number of patients with very low serum T levels at baseline. While adjustments were made for age and BMI, other confounders may have affected patients' response to therapy. Additional research is needed to better understand the effects of TRT on symptom improvement in men with hypogonadism.

(1) Wang C et al., J Clin Endocrinol Metab 1996;81:3578.
(2) Lee KK et al., J Androl 2003;24:688.

Sources of Research Support: Eli Lilly and Company.

Disclosures: XN: Researcher, Eli Lilly & Company. DM: Clinical Researcher, Eli Lilly & Company.

Posted: 24 Jun 2012 09:10 AM PDT

[SUN-LB18] Short-Term Testosterone Treatment Suppresses Skeletal Muscle NIK Expression in Older Men

Astrid M Horstman, Sanjeev Choudhary, William J Durham, E Lichar Dillon, Ronald G Tilton, Randall J Urban, Melinda Sheffield-Moore. University of Texas Medical Branch, Galveston, TX.

A number of events are known to trigger muscle loss with aging including inflammation, inactivity, and loss of anabolic hormones such as testosterone. It is unclear, however, what role NF-κB inducing kinase (NIK), an upstream regulatory MAP kinase in the NF-κB activation pathway, plays in mediating age-related muscle loss. Our data show that a link exists between increased NIK and decreased Notch levels in aging skeletal muscle. Moreover, testosterone has been shown to exert anti-catabolic properties in the skeletal muscle of older men.

Thus, we hypothesized that testosterone may be involved in regulating NIK levels, as NIK has been shown to be significantly elevated in aging and diseases where chronic inflammation is present. We investigated whether short-term administration (7 days) of testosterone was capable of counteracting aberrantly regulated NIK signaling in older men with serum testosterone concentrations in the low normal range (≤500 ng/dl), suggestive of a possible NIK-testosterone link in counteracting muscle loss with aging.

Blood was collected and biopsies were obtained from the mixed vastus lateralis muscle before and 7 days after either a single dose (intramuscular injection) of testosterone enanthate (100 mg on Day 1) or initiation of daily topical testosterone gel (two 5g packets of 1%AndroGel®/day). Muscle samples were homogenized and protein lysates were fractionated on 10% SDS-PAGE and immunoblotted for NIK using specific antibodies. GAPDH served as a loading control. Serum testosterone was measured on an Immulite 2000.

Skeletal muscle NIK levels significantly decreased following 7 days of testosterone treatment. Further, we found a significant negative association (r=-0.841, p<0.036) between baseline serum testosterone and skeletal muscle NIK protein levels. Moreover, a negative correlation was found between the change in serum testosterone levels and the change in NIK protein expression after one- week treatment (r=-0.412), although this correlation did not reach statistical significance due to the small sample size.

These data clearly demonstrate that as little as seven days of testosterone treatment can suppress skeletal muscle NIK levels in older men, suggesting that testosterone may exert its anti-catabolic properties in skeletal muscle via its regulation of NIK.

Posted: 24 Jun 2012 09:05 AM PDT

[SAT-39] Testosterone Measurement by Mass Spectrometry — A Tale of Three Internal Standards

Laura Jane Owen, Brian George Keevil. UHSM, Manchester, UK.

Introduction - Testosterone measurement by liquid chromatography tandem mass spectrometry (LC-MS/MS) is well accepted as the preferred technique for the analysis of serum testosterone in both males and particularly females. However variation is seen between LC-MS/MS assays and is most likely to be due to method differences between laboratories. One area of inconsistency amongst routine LC-MS/MS assays is the choice of internal standard. We investigated the effects of three internal standards on the results obtained.
Methods - Stable isotopes of testosterone were prepared in methanol. Testosterone with two deuterium (D2), five deuterium (D5) and three carbon thirteen enrichment (C13) were separately assessed. Sera, calibrators and quality controls (100 µL) were extracted using 0.5 mL of ether following the addition of 10 µL of internal standard. All aliquots were prepared in triplicate, one for each type of internal standard. After mixing the ether was transferred to the wells of a 96-deep well block then evaporated to dryness. Extracts were reconstituted with 100 µL of 50% mobile phases and analysed using a Waters Acquity LC and Quattro Premier tandem mass spectrometer. This method had previously shown to have excellent agreement with a reference method using the D2 internal standard.
Results - Lower results were obtained when using D5 testosterone when compared to D2 testosterone. The Passing-Bablock regression equation was; testosterone (D5) nmol/L = 0.86 x testosterone (D2) + 0.04. The C13 internal standard also gave lower results but was closer than the D2 target than the D5 internal standard. The Passing-Bablock regression analysis was; testosterone (C13) nmol/L = 0.90 x testosterone (D2) + 0.02.
Discussion - The choice of internal standard alone can have a significant affect on the results obtained by LC-MS/MS assays for testosterone using this chromatography. The effects of the combination of chromatography and internal standard choice should be investigated during method development.

Posted: 24 Jun 2012 08:26 AM PDT

[OR12-2] Disruption of Glucocorticoid Receptor Signaling in Kisspeptin Neurons Accelerates the Recovery of Reproductive Function in the Post-Traumatic Stress Period

Oulu Wang, Anne Lanjuin, Caroline Ho, Juliana Basko, Catherine Dulac, Joseph Majzoub. Children's Hospital Boston, Boston, MA; Harvard Medical School, Boston, MA; Harvard University, Howard Hughes Medical Institute, Cambridge, MA.

Stressors can generate adaptive stress responses that prepare for the return to homeostasis, but also maladaptive responses, including the inhibition of reproductive function even after cessation of the stressor. How acute stress causes these maladaptive, post-traumatic effects is not well understood.

Kisspeptin (KISS1) is required for the activation of the hypothalamic-pituitary-gonadal (HPG) reproductive axis in humans and mice (1-3). We hypothesized that acute stress in adult mice transiently inhibits kisspeptin neurons and the downstream HPG axis, and that restoration of kisspeptin signaling is necessary to reactivate the axis, similar to its role during puberty. This inhibition of kisspeptin expression could be caused by the concomitant stress-induced elevation of glucocorticoids, with the subsequent fall in glucocorticoids allowing the restoration of kisspeptin expression required for the recovery of the reproductive axis after stress.

To test this hypothesis, we first examined the response of hypothalamic Kiss1 mRNA expression to different stressors in male mice. After restraint, plasma corticosterone was increased, testosterone was decreased, and Kiss1 expression was decreased. After food deprivation, corticosterone was increased, testosterone was decreased, and Kiss1 expression was decreased. After cold exposure, corticosterone was unchanged, and Kiss1 expression was unchanged, even though testosterone was decreased. All changes were significant.

The consistent relationship between a rise in corticosterone and fall in Kiss1 expression suggested that elevated corticosterone caused stress-induced inhibition of Kiss1 expression. To test this directly, we injected mice with a stress dose of corticosterone and observed that testosterone and Kiss1 expression were decreased.

To evaluate the role of glucocorticoid signaling in kisspeptin neurons, we generated mice lacking glucocorticoid receptors specifically in kisspeptin-containing neurons. In these animals, Kiss1 expression was no longer inhibited during restraint stress, even though corticosterone was increased. Both testosterone and copulatory behaviors showed significantly accelerated recovery in the post-traumatic stress period. Blockade of glucocorticoid receptor signaling in kisspeptin neurons during stress accelerates the recovery of reproductive function during the post-traumatic stress period, and this finding may have therapeutic implications in humans with post-traumatic stress disorders.

(1) Seminara et al., 2003.
(2) De Roux et al., 2003.
(3) Lapatto et al., 2007.

Posted: 24 Jun 2012 07:17 AM PDT

[SUN-693] Continuous Kisspeptin Infusion Restores Pulsatile LH Secretion in Patients with Neurokinin B Signaling Deficiencies

Jacques Young, Jyothis T George, Javier A Tello, Bruno Francou, Jerome Bouligand, Anne Guiochon-Mantel, Sylvie Brailly-Tabard, Richard A Anderson, Robert P Millar. Université Paris-Sud, Paris, France; (INSERM U693) Institut National de la Santé et de la Recherche Médicale, Paris, France; Hôpital Bicêtre, Paris, France; University of Edinburgh, Edinburgh, UK; University of Edinburgh, Edinburgh, UK; Hôpital Bicêtre, Paris, France; University of Pretoria, Pretoria, South Africa; Medical Research Council/University of Cape Town, Cape Town, South Africa.

Pulsatile gonadotropin releasing hormone (GnRH) is a requirement for normal reproductive function. Abnormalities in pulse frequency result in reproductive dysfunction. Kisspeptin and neurokinin B (NKB) are neuropeptides secreted by the same neuronal population in the ventral hypothalamus, which are crucial central regulators of GnRH and thus gonadotropin secretion. Patients with mutations resulting in loss of signaling by either of these neuroendocrine peptides fail to advance through puberty but the mechanisms mediating this remain unresolved. We show that continuous kisspeptin infusion restores gonadotropin and pulse frequency and amplitude in patients with loss-of-function mutations in neurokinin B (TAC3) or its receptor, (TAC3R). Thus indicates that kisspeptin on its own is sufficient to stimulate pulsatile GnRH secretion. Moreover, our findings suggest that NKB action is proximal to kisspeptin in the reproductive neuroendocrine cascade regulating GnRH secretion, and may act as an autocrine modulator of kisspeptin secretion. The ability of continuous kisspeptin infusion to induce pulsatile gonadotropin secretion further indicates that GnRH neurons are able to set up pulsatile secretion in the absence of pulsatile exogenous kisspeptin.
Sources of Research Support: Medical Research Council(UK) _ Experimental Medicine Grant.

Nothing to Disclose: JY, JTG, JAT, BF, JB, AG-M, SB-T, RAA, RPM

Posted: 24 Jun 2012 07:13 AM PDT

[OR28-6] Obstructive Sleep Apnea Is Not an Independent Determinant of Plasma Testosterone

Gary Wittert, Sean Martin, Robert Adams, Amy Reynolds, Zumin Shi, Anne Taylor, Andre Araujo, Andrew Vakulin. University of Adelaide, Adelaide, Australia; University of South Australia, Adelaide, Australia; New England Research Institutes, Watertown, MA; Repatriation General Hospital, Adelaide, Australia.

It is generally considered that obstructive sleep apnoea (OSA) lowers testosterone in men, although the data supporting this are relatively limited. We determined the relationship between the presence and severity of sleep disordered breathing and plasma testosterone in a comprehensively characterized community based cohort of men aged over 40 yrs (MAILES) from whom fasting morning plasma samples were drawn in 2009-2010 (n=1869). Plasma total testosterone (T) was measured by liquid chromatography mass spectrometry (LC-MS/MS).

In 2011, home polysomnography was done in 810 randomly selected men from the cohort using an 8 channel Embletta X100 device. The Apnoea Hypopnea Index (AHI), and absence or presence and severity (mild: AHI 10-20; moderate AHI 21-30; severe: AHI ≥ 30) of OSA were classified according to the International Classification of Sleep Disorders (ICDS-2) from the American Academy of Sleep. After excluding men with pathological conditions or taking medications affecting testosterone, with missing values or using CPAP, 654 men aged 41-85 remained.

The effect of OSA severity, or AHI, on T were analysed using generalized linear models controlling for potential confounders (age, BMI, smoking, marital status, presence of depression (self-report), HbA1c and SHBG).

The mean (±SD) characteristics of the men were: age 59 (10) yrs, T 16.5 (5.4) nmol/L, SHBG 33.1 (13.4) nmol/L, BMI 28.4 (4.2) kg/m2, AHI 14.1 (14). OSA was present in 53.7%, (mild 28.6%, moderate 13.6, and severe 11.5%). A significant inverse relationship between AHI and T (Beta -.118, P=0.002), remained after adjustment for age, smoking, marital status, presence of depression, and HbA1c (Beta-.109, P=0.007), and SHBG (Beta -0.077, P=0.017), but not after additional adjustment for BMI (Beta -0.022, P=0.504). The results using OSA category rather than AHI were similar.

These data suggest that obesity, or sleep related factors rather than OSA per se, determine T. This accords with the graded effect of weight loss, but limited effect of CPAP to increase T and highlights the importance of managing obesity effectively, particularly in the context of OSA.

Sources of Research Support: This study was supported by the National Health and Medical Research Council of Australia (NH&MRC grant 627227).

Disclosures: RA: Speaker, GlaxoSmithKline.
Nothing to Disclose: GW, SM, AR, ZS, AT, AA, AV

Posted: 24 Jun 2012 06:57 AM PDT

[MON-34] Longitudinal Changes in Testosterone over 5 Years in Community-Dwelling Men

Andre B Araujo, Zumin Shi, Sean A Martin, Gary A Wittert. New England Research Institutes, Inc, Watertown, MA; University of Adelaide, Adelaide, Australia.

There are few population-based studies reporting longitudinal changes in testosterone (T) levels, and limited information on risk factors for longitudinal change in T. We examined changes in serum T levels assessed by liquid chromatography mass spectrometry (LC-MS/MS) among a cohort of 1,588 men aged 35-80y at baseline with available data at 2 clinic visits separated by 5 years. Excluded from analyses were men on medications (N=102) or with medical conditions (N=45) known to affect hormones, and men with pathological laboratory values (N=59), leaving 1,382 for analysis. Multivariate linear regression was used to assess the association between baseline predictors as well as change in predictors and annualized change in T. All models were minimally adjusted for baseline age.

At baseline, mean age was 54±11y, 21% were unmarried, 19% were current smokers, 30% had BMI ≥ 30 kg/m2, 48% had waist ≥ 100 cm, and 8% were depressed. Mean baseline T was 16.2±1.4 nmol/L, with 3%<8 nmol/L, 36% 8-15 nmol/L, and 61%>15 nmol/L. Mean T at follow-up was 15.6±1.4 nmol/L, representing a non-significant T change of -0.13 nmol/L/y (95% CI: -0.62, 0.41) or an approximate decline of -0.80%/y.

Baseline variables significantly associated with annualized T changes were: being unmarried (-0.18 nmol/L/y vs. married, p<.01), central obesity (+0.13 nmol/L/y vs. non-central obesity, p<.01), BMI ≥ 30 kg/m2(+0.22 nmol/L vs. <25 kg/m2, p<.01), and current smoking (-0.17 nmol/L/y vs. non-smoker, p<.01).

The effects of smoking status was driven by decreasing T in men who quit smoking (-0.17 nmol/L/y vs. non-smoker at both time points, p<.01). The effects of central obesity and high BMI were driven by changes in status between baseline and follow-up, with for instance, a T change of -0.25 nmol/L/y in men who became centrally obese (waist ≥ 100cm) and a T change of +0.20 nmol/L/y in men who became non-centrally obese, both compared to men who were not centrally obese at both time points (both p<.01). In addition, men who were depressed at both time points had a significantly greater T decline (-0.28 nmol/L/y vs. men without depression at both time points, p<.01).

In conclusion, this study strongly suggests that
(i) T decline is not an inevitable part of aging and
(ii) variability in T changes are largely explained by smoking behavior and intercurrent changes in health status, particularly obesity and depression.

Sources of Research Support: This study was supported by the National Health and Medical Research Council of Australia (NHMRC grant 627227).

Declining Testosterone Levels in Men Not Part of Normal Aging

Posted: 24 Jun 2012 06:45 AM PDT

[S39-2] When Is Testosterone an Appropriate Treatment for Erectile Dysfunction?

Jacques Buvat. CETPARP, Lille, France.

Erectile dysfunction (ED) is prevalent in young hypogonadal men. Some randomized, placebo-controlled trials (RCPTs) demonstrated that testosterone therapy (Tth) normalizes sexual function in such men. This has led to the belief that hypogonadism is an easily curable cause of ED.

Serum testosterone (T) is < 10.4 nmol/L in 15% of middle-aged and older men presenting with ED. Observational studies of the effects of Tth on erectile function of such men show limited success rate (< 40%). Several meta-analyses of the RPCTs of Tth also analyzed the effects of this therapy on erectile function of men of any age. A first one, not restricted to men with sexual complaints, concluded to an improvement of erections in men with baseline T < 12 nmol/L. Two further meta-analyses, restricted to men with ED or desire problems, did not confirm the benefit of Tth for erectile function, except in young hypogonadal men.

Several reasons may account for such differences in the effects of Tth on erectile function of young and older hypogonadal men with ED. Above all vascular comorbidities are prevalent in older men with ED and late onset hypogonadism (LOH).These may prevent improvement of erections with Tth alone, and may require combination therapy with T and phosphodiesterase type V inhibitor (PDE5I) for optimal results.

Moreover, ED-associated LOH is often mild or moderate, and may not be the real cause of ED, since 2 population-based studies found that in older men low T is associated with ED, but only below a threshold of 8 nmol/L. In certain cases, the low T associated with ED might be a consequence of reduced sexual activity, as suggested by an increase in T following improvement of erectile function after non-hormonal treatment of ED.

Lastly, evidence of requirement of a minimum T level for a complete efficacy of PDE5I therapy recently emerged. The corresponding thresholds seem close to 10.4 nmol/L for total T, 2.1 nmol/L for bioavailable T, and 180 pml/L for calculated Free T.

In conclusion, the lower the patient's age and T level, the higher the chance of success of Tth alone in a hypogonadal patient with ED. Low T is probably a plausible cause of ED only below 8 nmol/L, it but may reduce sexual desire at less decreased levels. After the age of 50, the frequent association of endothelial and vascular factors often requires PDE5I therapy, that should be combined with Tth in case of T < 10.4 nmol/L, due to the need for a minimum T level for a full PDE5I efficacy.

(1) Buvat J, Maggi M, Gooren L, Guay A, Kaufman J, Morgentaler A, Schulman C, Tan H.M, Torres L.O, Yassin A, Zitzmann M. Endocrine Aspects of Male Sexual Dysfunctions. J Sex Med 2010;7:1627-1656.
(2) Buvat J, Montorsi F, Maggi M, Porst H, Kaipia A, Colson MH, Cuzin B, Moncada I, Martin-Morales A, Yassin A, Meuleman E, Eardley I, Dean JD, Shabsigh R. Hypogonadal men nonresponders to the PDE5 inhibitor tadalafil benefit from normalization of testosterone levels with a 1% hydroalcoholic testosterone gel in the treatment of erectile dysfunction (TADTEST study). J Sex Med 2011;8:284-293.

Posted: 24 Jun 2012 06:04 AM PDT

Jones DS, Podolsky SH, Greene JA. The Burden of Disease and the Changing Task of Medicine. New England Journal of Medicine 2012;366(25):2333-8.

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