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Does Trenbolone or Resistance Exercise Reverse Hypogonadism Induced Bone and Muscle Loss?

Permanent Link: http://ufdc.ufl.edu/UFE0042779/00001

Material Information

Title: Does Trenbolone or Resistance Exercise Reverse Hypogonadism Induced Bone and Muscle Loss?
Physical Description: 1 online resource (135 p.)
Language: english
Creator: MCCOY,SEAN CONRAD
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: ANDROGENS -- HYPOGONADISM -- MUSCULAR -- TESTOSTERONE -- TRENBOLONE
Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre: Health and Human Performance thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Supraphysiologic testosterone (T) administration attenuates high turnover osteopenia in orchiectomized (ORX) rats; however T may increase prostate mass. Trenbolone enanthate (TREN) is a highly anabolic synthetic T analogue, which is purported to result in less prostate enlargement than T. The purpose of this experiment was to determine if T, TREN, and weighted ladder climbing (EX) prevent muscle and bone loss, effect prostate mass, fat mass, bone hormone and hemoglobin concentrations, and Pax7 positive satellite cells. Fifty, 10 month old male F344/Brown Norway rats were randomized into SHAM, ORX, ORX+EX, ORX+T, and ORX+TREN groups. The prostate, retroperitoneal fat pad, flexor hallicus longus (FHL), semimembranosus (SEMI), soleus, plantaris and levator ani bulbocavernosus (LABC) muscles were removed and weighed. The FHL and SEMI were examined for Pax7 expression. Femurs were analyzed by pQCT, while tibiae were evaluated for bone hormone concentrations by EIA. Differences were evaluated using a One Way ANOVA with a Tukey?s post hoc. No significant change was observed in the mass of the FHL, SEMI, soleus or plantaris. However, significant increases were seen in the LABC and hemoglobin concentrations of T and TREN treated animals while both animals reduced retroperitoneal fat pad mass. ORX reduced trabecular and cortical bone density compared with SHAMs. EX did not prevent ORX-induced bone loss. Conversely, TREN and T and maintained tBMC, tBMD, and cBMD at control values. Both ORX groups, prostate masses were reduced by 71% compared to SHAMs. TREN caused non-significant 20% reductions in prostate mass compared with SHAM; while T nearly doubled prostate mass (p<0.05). Conclusions: T and TREN prevent some indices of hypogonadism-induced muscle and bone loss, and decreased visceral adiposity. Progressive resistance exercise offered no protection to muscle and bone. An added benefit of TREN was the maintenance of prostate mass at SHAM levels.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by SEAN CONRAD MCCOY.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Borst, Stephen E.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-10-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042779:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042779/00001

Material Information

Title: Does Trenbolone or Resistance Exercise Reverse Hypogonadism Induced Bone and Muscle Loss?
Physical Description: 1 online resource (135 p.)
Language: english
Creator: MCCOY,SEAN CONRAD
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: ANDROGENS -- HYPOGONADISM -- MUSCULAR -- TESTOSTERONE -- TRENBOLONE
Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre: Health and Human Performance thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Supraphysiologic testosterone (T) administration attenuates high turnover osteopenia in orchiectomized (ORX) rats; however T may increase prostate mass. Trenbolone enanthate (TREN) is a highly anabolic synthetic T analogue, which is purported to result in less prostate enlargement than T. The purpose of this experiment was to determine if T, TREN, and weighted ladder climbing (EX) prevent muscle and bone loss, effect prostate mass, fat mass, bone hormone and hemoglobin concentrations, and Pax7 positive satellite cells. Fifty, 10 month old male F344/Brown Norway rats were randomized into SHAM, ORX, ORX+EX, ORX+T, and ORX+TREN groups. The prostate, retroperitoneal fat pad, flexor hallicus longus (FHL), semimembranosus (SEMI), soleus, plantaris and levator ani bulbocavernosus (LABC) muscles were removed and weighed. The FHL and SEMI were examined for Pax7 expression. Femurs were analyzed by pQCT, while tibiae were evaluated for bone hormone concentrations by EIA. Differences were evaluated using a One Way ANOVA with a Tukey?s post hoc. No significant change was observed in the mass of the FHL, SEMI, soleus or plantaris. However, significant increases were seen in the LABC and hemoglobin concentrations of T and TREN treated animals while both animals reduced retroperitoneal fat pad mass. ORX reduced trabecular and cortical bone density compared with SHAMs. EX did not prevent ORX-induced bone loss. Conversely, TREN and T and maintained tBMC, tBMD, and cBMD at control values. Both ORX groups, prostate masses were reduced by 71% compared to SHAMs. TREN caused non-significant 20% reductions in prostate mass compared with SHAM; while T nearly doubled prostate mass (p<0.05). Conclusions: T and TREN prevent some indices of hypogonadism-induced muscle and bone loss, and decreased visceral adiposity. Progressive resistance exercise offered no protection to muscle and bone. An added benefit of TREN was the maintenance of prostate mass at SHAM levels.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by SEAN CONRAD MCCOY.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Borst, Stephen E.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-10-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042779:00001


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1 DOES TRENBOLONE OR RESISTANCE EXERCISE REVERSE HYPOGONADISM INDUCED BONE AND MUSCLE LOSS? By SEAN C. MCCOY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIRE MENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011

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2 2011 Sean C. McCoy

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3 To my loving parents, my in laws my family and friends and Sandy and Liam

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4 ACKNOWLEDGMENTS During my non traditional path to my degree, n umerous individuals stepped in to carry me forward towards my career goals I would like to thank my mentor and committee chair Dr. Stephen Borst, without his mentorship, friendship, and assistance I would have never been able to continue on this path My committee members: Dr. Paul Borsa, Dr. Hordur Kristinsson, Dr. Sally Johnson and Dr. Mark Tillman for standing by me, guiding and supporting me through two separate and completely different dissertation proposals I would also like to thank Dr. Joshua Yarr ow for his friendship and support throughout my academic career Also, Dr. Barbara Smith and Dr. Ye Fan, and Dr. Chris Gregory whose expertise and patience made Pax7 and fiber typing run smoothly. I would like to thank Dr. Thomas Wronski and Jennifer Pinge l for their expertise on bone pQCT measurements. I would like to acknowledge Christine Conover for her assistance with surgical procedures, providing limitless support and motivation In addition, I wo uld like to thank Cesar Santill ana and Judyta Lipinska, members of the Borst lab, whose help and assistance was greatly appreciated I would also like to thank Dr. Bryan Conrad for his assistance with biomechanical analyses. I would like to extend additional gratitude to Dr. Borst from all the members of my fa mily Through his support we were able to remain in Gainesville and flourish. This work was supported in part by a VA Merit Award to Dr. Stephen Borst.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 14 Specific Aim, Hypothesis, Rationale 1. ................................ ................................ ... 20 Specific Aim, Hypothesis, Rationale 2. ................................ ................................ ... 22 Specific Aim, Hypothe sis, Rationale 3: ................................ ................................ ... 23 2 REVIEW OF LITERATURE ................................ ................................ .................... 25 Hypogonadism Induced Effects in Man ................................ ................................ ... 25 Testosterone Replacement Therapy for Hypogonadism ................................ ......... 25 Potential Therapeutic Use of Trenbolone Administration for Hypogonadism .......... 27 Hypogonadism Rat Model Selection ................................ ................................ ....... 30 Bone Effects in Orchiectomized (ORX) Rats ................................ .......................... 31 Bone and Serum Hormone Concentr ations in ORX rats ................................ ......... 33 Skeletal Muscle Effects in ORX rats ................................ ................................ ....... 33 Resistance Training for Sarcopenia and Osteoporosis ................................ ........... 34 Satellite Cells ................................ ................................ ................................ .......... 37 Definition ................................ ................................ ................................ .......... 37 Quiescence ................................ ................................ ................................ ...... 38 Self Renewal, Differentiation, and Activation ................................ .................... 39 Pax 7+ Responsiveness to Growth Factors ................................ ..................... 40 Androgen Effects on Satellite Cells ................................ ................................ ......... 41 3 METHODS ................................ ................................ ................................ .............. 43 Experimental Design ................................ ................................ ............................... 43 Drug Administration ................................ ................................ ................................ 44 Weighted Ladder Climbing ................................ ................................ ...................... 45 Hemoglobin and Serum Sex Hormone Measurements ................................ .......... 45 In Vivo Muscle Strength Testing (Grip Strength) ................................ .................... 46 Fiber Type and Fiber Cross Sectional Area Measurements ................................ ... 47 Hemotoxylin and Eosin Staining ................................ ................................ ............. 48 Satellite Cell (Pax 7) Staining ................................ ................................ ................. 49 Bone Sex Steroid Hormone Measurements ................................ ............................ 50 Prostate, Levator Ani Bulbocaveronsus Complex, Kidney, Adipose and Muscle Tissue Mass ................................ ................................ ................................ ........ 51

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6 Bone Mechanical Strength and Peripheral Quantitat ive Computed Tomography (pQCT) ................................ ................................ ................................ ................. 51 Statistical Analysis ................................ ................................ ................................ .. 52 4 RESULTS ................................ ................................ ................................ ............... 55 Body Weights at Sacrifice ................................ ................................ ....................... 55 Treatment Effects on Serum T ................................ ................................ ................ 55 Treatment Effects on Serum DHT ................................ ................................ ........... 56 Treatment Effects on Serum TREN ................................ ................................ ........ 57 Treatment Effects on Retroperitoneal Fat Mass ................................ ..................... 58 Treatment Eff ects on Hemoglobin at Sacrifice ................................ ........................ 58 Time Course of Treatment Effects on Hemoglobin ................................ ................. 59 Treatment Effects on the Levator Ani Bulbocav ernosus Complex (LABC) ............. 60 Treatment Effects on Prostate Mass ................................ ................................ ....... 60 Treatment Effects on Kidney Mass at Sacrifice ................................ ...................... 61 Treatment Effect on Hindlimb Muscles Mass ................................ .......................... 62 Grip Strength ................................ ................................ ................................ .......... 63 Progressive Resistance Training (Weighted Ladder Climbing) ............................... 63 Treatment Effects on Femoral and Tibial Bone Mass and Length .......................... 64 Serum Osteocalcin at Sacri fice ................................ ................................ ............... 65 Serum Trap 5b at Sacrifice ................................ ................................ ..................... 65 Femoral Neck ................................ ................................ ................................ ... 66 Femoral Midsh aft ................................ ................................ .............................. 67 Centralized and Internalized Nuclei of the FHL and Semimembransosus .............. 67 Pax 7 Positive Nuclei of the FHL and Semimembranosus ................................ ...... 68 Flexor Hallicus Longus ................................ ................................ ..................... 68 Semimembranosus ................................ ................................ .......................... 69 Treatment Effects on Cross Sectional Area of the Flexor Hallicus Longus ............. 69 Treatment Effects on Cross Sectional Area of the Semimembranosus .................. 70 Treat ment Effects on 5mm pQCT (Metaphysis) ................................ ...................... 70 Total Mineral Content, Total Density, Total Area, Trabecular Area, Cortical Content, Cortical Area ................................ ................................ ................... 70 Trabecular Content (CNT TRB ) ................................ ................................ ........... 71 Trabecular Density (TRAB DEN ) ................................ ................................ ......... 72 Cortical Density (CRT DEN ) ................................ ................................ ................ 72 Treatment Effects on 18 mm pQCT (Diaphysis) ................................ ..................... 73 Total Mineral Content, Total Density, Total Area ................................ .............. 73 Cortical Content, Cortical Density, Cortical Area ................................ .............. 73 Cortical Thickness, Periosteal and Endosteal Circumference .......................... 74 Intraske letal Hormone Concentrations ................................ ................................ .... 74 Intraskeletal Testosterone ................................ ................................ ................ 74 Intraskeletal DHT ................................ ................................ .............................. 74 5 DISCUSSION ................................ ................................ ................................ ....... 110 Overview of Principle Findings ................................ ................................ .............. 110

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7 Anabolic and Androgenic Effects of Testosterone and Trenbolon e Enanthate and Weighted Ladder Climbing on Muscle, Bone and Prostate ......................... 111 Background ................................ ................................ ................................ .... 111 Anabolic Effects on Skeletal Muscle ................................ ............................... 111 Anabolic Effects on Bone ................................ ................................ ............... 113 Intraskeletal Testosterone and DHT Concentrations ................................ ...... 114 Anabolic Effects on the Prostate ................................ ................................ .... 115 Anabolic Effects on Hemoglobin ................................ ................................ ..... 116 Effects of Testosterone and Trenbolone Enan thate and Weighted Ladder Climbing on Serum markers of T, DHT and TR ................................ ................. 117 Serum Testosterone at Sacrifice ................................ ................................ .... 117 Serum DHT at Sacrific e ................................ ................................ .................. 117 Serum TR at Sacrifice ................................ ................................ .................... 117 Effects of Testosterone and Trenbolone Enanthate and Weighted Ladder Climbing on Retroperitoneal F at Pad and Kidney Mass ................................ .... 118 Retroperitoneal Fat Pad Mass ................................ ................................ ........ 118 Kidney Mass at Sacrifice ................................ ................................ ................ 118 Effects of Testosterone and Trenbolone Enanthate and Weighted Ladder Climbing on Centralized and Internalized Myonuclei ................................ ......... 119 Effects of Testosterone and Trenbolone Enanthat e and Weighted Ladder Climbing on Pax7+ Nuclei in the Semimembranosus and Flexor Hallicus Longus ................................ ................................ ................................ ............... 119 Effects of ORX on Weighted Ladder Climbing ................................ ...................... 120 Effects of Weighted Ladder Climbing, Testosterone Enanthate, and Trenbolone Enanthate on Muscle Fiber Cross sectional Area of the Flexor Hallicus Longus ................................ ................................ ................................ ............... 120 Effects of Weighte d Ladder Climbing, Testosterone Enanthate, and Trenbolone Enanthate on Muscle Fiber Cross sectional Area of the Flexor Hallicus Longus ................................ ................................ ................................ ............... 121 Conclusion ................................ ................................ ................................ ............ 121 REFERENCES ................................ ................................ ................................ ............ 123 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 135

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8 LIST OF FIGURE S F igure P age 4 1 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on bodyweight at sacrifice. ................................ ........................ 76 4 2 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenb olone enanthate (TR) on serum testosterone levels at sacrifice. ................................ .. 76 4 3 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on serum DHT at sacrifice. ................................ ........................ 77 4 4 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on serum DHT at sacrifice. ................................ ........................ 77 4 5 Effects of ORX+V ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on retroperitoneal fat pad mass at sacrifice. .............................. 78 4 6 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone ena nthate (TR) on hemoglobin mass at sacrifice. ................................ .............. 78 4 7 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) time course of hemoglobin mass. ................................ .............. 79 4 8 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on levator ani bulbocavernosus muscle mass at sacrifice. ........ 79 4 9 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on prostate mass at sacrifice. ................................ .................... 80 4 10 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone e nanthate (TR) on kidney mass at sacrifice. ................................ ...................... 80 4 11 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on semimembranosus muscle mass at sacrifice. ...................... 81 4 12 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on plantaris muscle mass at sacrifice. ................................ ....... 81 4 13 Effe cts of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on soleus muscle mass at sacrifice. ................................ .......... 82 4 14 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on flexor hallicus longus muscle mass at sacrifice. ................... 82 4 15 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on forelimb grip strength at sacri fice. ................................ ......... 83

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9 4 16 Total weekly training load carried by ORX+EX animals on a 1.1m ladder inclined at 85 ................................ ................................ ................................ .... 83 4 17 Effects of ORX+V, ORX +EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on tibial mass at sacrifice. ................................ ......................... 84 4 18 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on tibia l length at sacrifice. ................................ ........................ 84 4 19 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on femoral mass at sacrifice. ................................ ..................... 85 4 20 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on femoral length at sacrifice. ................................ .................... 85 4 21 Effects of ORX+V, ORX+EX, testosterone enantha te (TE), or trenbolone enanthate (TR) on serum osteocalcin levels at sacrifice. ................................ ... 86 4 22 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on serum TRAP 5b leve ls at sacrifice.. ................................ ...... 86 4 23 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on femoral midshaft maximum load. ................................ .......... 87 4 24 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on femoral neck maximum load. ................................ ................ 87 4 25 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on internalized nuclei of the FHL. ................................ .............. 88 4 26 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on internalized nuclei of the s emimembranosus. ....................... 88 4 27 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on centralized nuclei of the FHL. ................................ ............... 89 4 28 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on centralized nuclei of the semimembranosus. ........................ 89 4 29 Effects of ORX+V, ORX+EX, testost erone enanthate (TE), or trenbolone enanthate (TR) on Pax 7+ nuclei expressed per 100 fibers of the flexor hallicus longus. ................................ ................................ ................................ ... 90 4 30 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or tr enbolone enanthate (TR) on Pax 7+ nuclei expressed per 100 fibers of the semimembranosus. ................................ ................................ ............................ 90

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10 4 31 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on Type II a fibers of the flexor hallicus longus. .......................... 91 4 32 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on Type IIa fibers of the flexor hallicus longus. .......................... 92 4 33 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on femoral total bone content at 5mm. ................................ ...... 93 4 34 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on femoral total density at 5mm. ................................ ................ 93 4 35 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolon e enanthate (TR) on femoral trabecular content at 5mm ................................ ....... 94 4 36 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on trabecular density at 5mm. ................................ ................... 94 4 37 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on femoral total area at 5mm. ................................ .................... 95 4 38 Effects of ORX +V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on trabecular bone area at 5mm. ................................ ............... 95 4 39 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR ) on cortical bone content at 5mm. ................................ ............... 96 4 40 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on cortical bone density at 5mm. ................................ ............... 96 4 41 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on cortical bone area at 5mm. ................................ ................... 97 4 42 Effects of ORX+V, ORX+EX, test osterone enanthate (TE), or trenbolone enanthate (TR) on total bone content at 18mm. ................................ ................. 97 4 43 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on total bone d ensity at 18mm. ................................ .................. 98 4 44 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on total bone area at 18mm. ................................ ...................... 98 4 45 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on cortical bone content at 18mm. ................................ ............. 99 4 46 Effects of ORX+V, ORX+EX, testosterone enanthate ( TE), or trenbolone enanthate (TR) on cortical bone density at 18mm. ................................ ............. 99

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11 4 47 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on cortical bone area at 18mm. ................................ ............... 100 4 48 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on cortical bone thickness. ................................ ...................... 100 4 49 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on periosteal bone circumference. ................................ ........... 101 4 50 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbo lone enanthate (TR) on endosteal bone circumference. ................................ ........... 101 4 51 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on intraskeletal testosterone concentrations. .......................... 102 4 52 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on intraskeletal dihydrotestosterone concentrations. ............... 102 4 53 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on Pax 7 expression in the flexor hallicus longus ................... 103 4 54 Effects of ORX+V, ORX +EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on Pax 7 expression in the semimembranosus. ...................... 104 4 55 E ffects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enantha te (TR) on fiber type percentage and muscle cross sectional area of the flexor hallicus longus. ................................ ................................ ................. 105 4 56 E ffects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on fiber type percentage and muscle cross sectional area of the semimembranosus ................................ ................................ .................... 106 4 57 E ffects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on centralized and in ternalized nuclei in the flexor hallicus longus. ................................ ................................ ................................ .............. 107 4 58 E ffects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on centra lized and internalized nuclei in the semimembranosus. ................................ ................................ .......................... 108 4 59 Weighted ladder climbing on a 1m ladder inclined to 85 ................................ 109

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12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DOES TRENBOLONE OR RESISTANCE EXERCISE REVERSE HYPOGONADISM IN DUCED BONE AND MUSCLE LOSS? By Sean C. McCoy May 2011 Chair: Stephen Borst Major: Health and Human Performance Supraphysiologic testosterone (T) administration attenuates high turnover osteopenia in orchiectomized (ORX) rats; however T may increase pro state mass. Trenbolone enanthate (TREN) is a highly anabolic synthetic T analogue, which is purported to result in less prostate enlargement than T. The purpose of this experiment was to determine if T, TREN, and weighted ladder climbing (EX) prevent muscl e and bone loss effect prosta te mass fat mass, bone hormo ne and hemoglobin concentrations, and Pax7 positive satellite cells Fifty, 10 month old male F344/Brown Norway rats w ere randomized into SHAM OR X, ORX+EX, ORX+T, and ORX+TREN groups. The prostate retroperitoneal fat pad, flexor hallicus longus (FHL) semimembranosus (SEMI) soleus, plantaris and levator ani bulbocavernosus (LABC) muscles were removed and weighed The FHL and SEMI were examined for Pax7 expression Femurs were analyzed by pQCT, wh ile tibiae were evaluated for bone hormone concentrations by EIA. Differences were evaluated using a One Way ANOVA No significant change was observed in the mass of the FHL, SEMI, soleus or plantaris However, significant increases were seen in the LABC and hemoglobin concentrations of T and TREN treated animals while both animals reduced

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13 retroperitoneal fat pad mass ORX reduced trabecular and cortical bone density compa red with SHAMs. EX did not prevent ORX indu ced bone loss Conv ersely, TREN and T and maintained tBMC, tBMD and cBMD at control values. Both ORX groups prostate mass es were reduced by 71% compa red to SHAMs TREN caused non significant 20% reductions in prostate mass compared with SHAM; while T nearly doubled prostat e mass (p<0.05) Conclusions: T and TREN prevent some indices of hypogonadism induced muscle and bone loss, and decreased visceral adiposity Progressive resistance exercise offered no protection to muscle and bone. An added benefit of TREN was the mainten ance of prostate mass at SHAM levels

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14 CHAPTER 1 INTRODUCTION In males, aging results in a variety of physical decrements, including loss of skeletal muscle mass and function (i.e., sarcopenia), and reduced bone mineral density (BMD) (i.e., osteopenia), w hich combine to increase fall risk and consequently bone fracture risk ( 1 ) Guidelines (2010) sugge st evaluating patients for initial treatment of hypogonadism when serum testosterone concentrations are less than10.4 nmol/L and patients present with additional clinical manifestations Hypogonadism is one of s everal factors that underlie both sarcopenia and osteopenia ( 2 3 ) The incidence of hypogonadism increases with age, such that approximately 20% of the m ale population meets the clinical definition of hypogonadism by 60 years of age, while nearly 50% of those older than 80 years of age experience hypogonadism ( 4 ) H ypogonadism may occur as a result of primary (i.e., testicular level of failure) or secondary testicular failure (i.e., central defects of the hypothalamus or pituitary), or a combination of primary and secondary testicular failure (i.e., involving both the testis and pituitary) ( 5 ) However, treatment of older hypogonadal men with replacement doses of testosterone produces only modest improvements in skeletal muscle mass/strength and BMD ( 2 3 ) ; whereas, high dose testosterone administration produces robust improvements in both muscle mass ( 6 ) and BMD ( 7 ) in humans and animal models ( 8 11 ) However, testosterone administration is associated with a variety of side effects of which prostate enlargement and polycythemia are most prevalent ( 12 ) T h o rough routine clinical follow up and proactive monitoring of potential patient side effects can improve the benefit to risk ratio of testosterone replacement therapy The combination of side effects and u nknown

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15 optimal dosing regimens caused the Endocrine Society Clinical Practice Guidelines to recommend rigorous monitoring of hematocrit, LH and FSH for primary and secondary causes of hypogonadism, prostate specific antigen (PSA) levels, and gross prostate examinations for individuals receiving testosterone replacement therapy ( 5 ) The Institute of Medicine has requested that small and medium sized trials be conducted t o assess the safety and efficacy of testosterone replacement therapy for the treatment of a variety of conditions associated with hypogonadism, in cluding sexual dysfunction, cognitive impairment, depression, muscle weakness, and osteoporosis from Institute of Medicine 2003 position statement Additionally, examining alternative pharmacological therapies and non pharmacological interventions which may enhance skeletal muscle mass/strength and BMD, without causing prostate enlargement, or exacerbating polycyt hemia may improve treatment options for hypogonadism induced sarcopenia and osteopenia Osteoporosis affects nearly 200 million individuals worldwide making it the most prevalent metabolic bone disorder in the world ( 13 ) with falls contributing over $15 billion to US healthcare costs annually ( 14 ) Males represent approximately 25% of the total inci dence of osteoporosis and the estimated cost of osteoporotic bone fractures in men exceeds $4 billion per year ( 15 ) The lifetime incidence of hip fracture in white males resid ing in the US approximates 5%, while black males residing in the US have approximately a 50% lower lifetime incidence risk ( 16 ) Women experience a higher incid ence of osteoporotic fractures than men, although men experience a higher mortality, at least following hip fracture. In one UK based study, the likelihood of mortality following a hip fracture was eightfold higher in men compared to women ( 17 )

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16 Specifically, in the VA Healthcare System, men who experience a hip fracture have an approximate 32% mortality ra te within 12 months of fracture incidence ( 18 ) with higher mortality rates remaining higher than wom en for two years post fracture ( 17 ) demonstrating the severity of this condition Further, elderly indi viduals may also experience the physical and emotional burden of residual pain, short or long term disability, and possible deformities that limit quality of life, and perhaps a loss of skeletal muscle mass and strength following osteoporotic fractures ( 19 ) Sarcopenia (i.e., age related loss of skeletal muscle mass) is also associated with low BMD and occurrence of falls, suggesting that sarcopenia increases osteoporotic fracture risk in the elderly ( 20 ) It is estimated that complications from falls are the sixth leading cause of death ( 21 ) Sarcopenia is specifically defined as height adjusted muscle mass score two or more standard deviations below the mean of young adults ( 22 23 ) The definition of sarcopenia has recently been established as a multifactorial geriatric syndrome comprised of low muscle mass, impaired muscular strength, lifestyle, chronic disease, genetic susceptibility factors presenting as disordered mobility, disability, impaired quality of life, morbidity, and mortality ( 24 ) Hallmarks of sarcopenia include walking speeds of less than 1m/s and a mean lean body to fat mass ratio more than two standard deviatio ns greater than normative data for young adults ( 25 ) Current population estimates place 45% of the population aged 60 years and older in the category of mod erate to severe sarcopenia ( 26 ) The European Union Geriatric Medicine Society recently proposed classification of sarcopenia into primary (i.e., confined to age related changes), and secondary when loss of muscle mass and function is related to another disease or condition ( 27 ) Several factors may predispose

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17 individual s to sarcopenia such as: loss of functional capacity due to disease or disuse, nutrient malabsorption or deficiency, and organ failure ( 27 ) Against the backdro p of the advancing mean age the economic impact of sarcopenia is estimated at 19 billion dollars annually making it a prominent public health concern ( 26 ) Thus, treatments which are designed to prevent the loss of both skeletal muscle mass and function, and BMD may reduce the morbidity and mortality associated with hypogonadism. In addition, s everal common factors are implicated in the pathogenes is of both sarcopenia and osteopenia including reductions in endogenous sex steroid concentrations ( 28 ) skeletal muscle apoptosis and reduced physical activity ( 28 ) R ecent research has attempted to determine the safety and efficacy of interventions designed to prevent both sarcopenia and osteopenia Those interventions include treatment with testosterone ( 9 29 30 ) or selective an drogen receptor modulators (SARMs) ( 31 ) Ideally, SARMs would selectively bind and activate the androgen receptors and produce anabolic r esponses in tissues of inte rest (e.g., m uscle and bone), w hile avoiding androgenic activity in other tissues (e.g., prostate) ( 32 ) T renbolone is a synthetic analogue of testosterone The affinity of trenbolone for the human androgen receptor is 3 fold higher than that of testosterone and approximately equal to that of DHT ( 33 34 ) Trenbolone is capable of preventing hypogonadism induced muscle loss in animal models ( 35 39 ) and does not induce prostate enlar gement when administered in low doses ( 40 41 ) However, Freyberger a nd colleagues reported increased ventral prostate mass of castrated male rats when trenbolone was administered orally for ten days at a dosage of 40mg/kg whereas the

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18 1.5mg/kg dosage tended to maintain prostate levels similar to controls ( 42 ) They reported trenbolone administration at both low and high dosages reduced prostate masses by nearly 78% (low dosage) and 50% ( high dosage) compared to animals which received subcutaneous testosterone propionate at a d osage of 0.4mg/kg (41) Similarly Wilson and colleagues found that subcutaneous administration of trenbolone at doses below 200 micrograms /day for ten consecutive days did not significantly increase ventral prostate mass in immature male Sprague Dawley ra ts ( 34 ) Oral dosing with trenbolone resulted in approximately a 100 fold less induction of LABC mass compared to subcutaneous administration, similar to reduced b ioavailability of oral administered testosterone proprionate (139). Wilson and colleagues state that reductase enzyme ( 34 ) and that the levator ani reductase enzyme ( 34 ) Recently, Yarrow and colleagues reported significant increases of 35 40% hypertrophy in the levator ani bulbocavernosus complex compared to c ontrol animals, enhanced hemoglobin concentrations and reduced prostate size by 34% in the low dose (1.0mg/week) trenbolone administration ( 40 ) Currently, our laboratory is assessing the most balanced dosage r egimen to promote the desired anabolic/androgenic effects without inducing prostate enlargement ( 40 41 ) Preliminary mechanistic evidence from the H295R human adrenocortical carcinoma cell line indicates that trenbolone decrease endogenous T production similar to other administered androgens. Trenbolone may also increas e T metabolism through direct or indirect mechanims and potentially upregulates the aromatase gene CYP19 in vitro ( 43 ) Trenbolone concentrations d induction of estradiol in H295 R cell lines

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19 above values for testosterone and progresterone ( 43 ) This data may indicate a secondary effect of the trenbolone on steroidogenesis in the H2 95R bioassay but may not reflect in vivo effects of trenbolone on other tissues Yarrow and colleagues report that administration of trenbolone to rats increases serum, a n estradio l like or a trenbolone derived metabolite presenting as estradiol on commer cially available radioimmunoassay (unpublished laboratory results) For this reason, future work with GC MS or other advanced molecular techniques is necessary to determine the true identity of this metabolite. However, if trenbolone is truly a non aromati zable androgenic compound as suggested by Wilson and colleagues then trenbolone may offer protection agains t hypogonadism induced bone and skeletal muscle mass loss without estradiol mediated side effect (i.e., gynecomastia) ( 34 40 ) S pecifically androgenic and anti glucorticoid effects may be induced by trenbolone administration further work is necessary in determi ning how this compound effects both bone maintenance and development in males ( 29 44 47 ) E strogens are postu lated to inhibit osteoclast activity (i.e., bone resorption), whereas non aromotizable androgens (e.g., DHT) may induce bone formation and augment bone mass through enhanced anchorage of osteoblastic cells to the organic bone matrix ( 48 ) Thus, trenbolone may produce SARM like activity, at least in skeletal muscle and prostate tissue R ecent data suggest that trenbolone may cause positive skeletal responses i n orc hedectomized male rats ( 40 ) Our laboratory ass essed the dose dependent, anabolic/androgenic effects of trenbolone at dosages of 1.0mg/wk, 3.5mg/week or 7.0mg/week on skeletal muscle mass and bone mass prostate enlargement ( 40 ) These preliminary findings indicated that the low dose (1.0mg/wk) was most effica cious target dose of trenbolone administration for preserving

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20 bone and muscle mass while minimizing prostate enlargement in orchiectomized rodents ( 40 ) Progressive resistance training (PRT) (i.e., mechanical loading) is an established non pharmacological modality that augments skel etal muscle mass and function, and BMD ( 49 50 ) and may also reduce fracture and fall risk ( 51 ) at least in eugonadal elderly men Few studies have specifically evaluated the skeletal muscle and bone responses to PRT in hypogonadal men or using hypogonadal animal models; h owever, several studies have shown that PRT combined with testosterone replacement resulted in significant improvement in leg lean muscle mass and strength compared to either PRT or testosterone alone demonstrating that sex hormones influence the skeletal muscle responses to PRT ( 52 56 ) Therefore, future research designed to evaluate the safety and efficacy of alternative therapies, such as trenbolone administrati on or PRT, using hypogonadal models may improve treatment options for both sarcopenia and osteopenia Specific Aim Hypothesis, Rationale 1. Specific Aim : To determine the effect of trenbolone enanthate, progressive resistance exercise, and testosterone e nanthate administration on skeletal muscle, bone, kidney, retroperitoneal fat pad mass, hemoglobin concentration, and prostate growth in a mature orchiectomized male rat model Hypothesis : In mature orchiectomized male rats, 42 days of trenbolone enanthate administration will attenuate the hypogonadism induced skeletal muscle and bone loss associated with orchiectomy, maintain kidney mass, reduce retroperitoneal fat pad mass, and elevate circulating hemoglobin concentrations without inducing prostate enlarg ement. Rationale: Supraphysiological testosterone administration completely prevents the loss of skeletal

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21 muscle mass and BMD in hypogonadal rodents, but results in significant prostate reduction of testosterone to DHT Trenbolone a synthetic analogue of testosterone, reduces orchiectomy induced skeletal muscle loss in rodents ( 34 40 ) but i reductase enzyme ( 34 41 ) and thus should not induce prostate enlargement Conversely, supraphysiological administration of testosterone enanthate for 28 days has been shown to augment the anthropometric (i.e., length and mass) and biomechanical characteristics of the femur in both dev eloping male and female rodents ( 11 ) However, in this study 10 month old rats are mature and present with closed epiphysis making length increases impossible. In a graded dose response study by Yarrow and colleagues, low dose trenbolone (i.e., 1.0mg/week) reduced retroperitoneal fat pad mass by 23% compared to orchiectomized animals They reported that low dose trenbolone also augmented LABC mass by 35%, and preven ted reductions in kidney mass and hemoglobin concentration without enlarging the prostate. Additionally, testosterone administration preserved cancellous bone volume and trabecular number in male rats following ORX ( 10 11 57 58 ) However, a separate study examin ing the effects of oral administration of 50mg/day testosterone propionate administration in hypogonadal male mice reported decreases in cortical area and width ( 59 ) Unlike estradiol where reducti ons in cortical bone were fully compensated for by an increased trabecular structured network, testosterone administration resulted in only a partial compensation of this network ( 59 ) Thus, some c ontroversy exists regarding the efficacy of testosterone administration in preventing hypogonadism induced bone loss. The Fischer 344 male rat model aged 3 months shows a modest decline in the plantaris muscle (8%) ( 60 ) at eight week s following ORX.

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22 However, during a four week experimental period by Yarrow and colleagues observed that plantaris muscle and femoral and tibial bone mass were not significantly reduced following ORX ( 11 ) Therefore the current study was extended to six weeks to evaluate a longer period of ORX on muscle and bone mass. Unlike testosterone replacement therapy, PRT is not reported to increa se prostate size, but has produced significant effects in muscle ( 61 62 ) and bone turnover markers ( 63 ) Our proposed model of PRT in the rat (weighted ladder climbing) increases the flexor hallicus muscle mass of eugonadal rodents by 23% in eight weeks ( 61 62 ) and increases the external load on bone Increased external load through weight bearing, increased body weight or resistive exercise has shown positive effects on bone mass and strength, howeve r these specific effects have not been reported In addition, PRT is not associated with one of the most reported side effects of androgen therapy prostate enlargement To accomplish this aim we will quantify the degree trenbolone enanthate, PRT, testoster one enanthate (positive control), or vehicle (negative control) alter skeletal muscle mass (wet weight) retroperitoneal fat pad mass, bone mass and morphometry (as evaluated by femoral pQCT measurements), bone biomechanical strength, hemoglobin concentrat ion, and mass of the kidney and prostate mass (wet weight) in mature ORX male rats Specific Aim Hypothesis, Rationale 2 Specific Aim: To characterize the effects of trenbolone enanthate administration testosterone enanthate administration or progressiv e resistance exercise (i.e., weighted ladder cl imbing) on the serum, and bone concentrations of testosterone, trenbolone, and DHT in an orchiectomized mature male rat model. Hypothesis: O rchiectomized male rats, rats receiving trenbolone or progressive res istance training will display lower

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23 bone, and serum hormone conc entrations of Testosterone and DHT compared to control, whereas supraphysiological testosterone administration will exhibit the highest levels of bone (T,DHT) and serum hormones (T, DHT) Rati onale: Previous work by Yarrow and colleagues ( 11 ) has found that ORX reduces serum concentrations of both testosterone and elevates intraskeletal DHT and maintain s bone concentrations of testosterone; while testosterone administration of ORX rodents significantly elevates both serum and bone testosterone and DHT concentrations To date, no published data exists on the effects of trenbolone or PRT on bone hormone co ncentrations To accomplish this aim, we will quantify the degree to which trenbolone enanthate, PRT, testosterone enanthate (positive control), or vehicle (negative control) therapy alters the serum, and bone sex hormone concentrations in mature ORX male rats. Specific Aim Hypothesis, Rationale 3 : Specific Aim: To characterize the effects of trenbolone ena n thate administration testosterone enanthate administration or progressive resistance exercise (i.e., weighted ladder climbing) on Pax 7 satellite cell activation, fiber type distribution, and fiber cross sectional area in the semimembranosus and flexor hallicus longus. Hypothesis : Administration of trenbolone enanthate and testosterone enanthate will increase Pax 7 activated satellite cell populations, increase Type II fiber cross sectional area, and maintain Type II fiber distribution in both muscles of the semimembranosus and flexor hallicus longus Progressive resistance tr aining will increase Pax 7 activated satellite cell numbers in the flexor halli cus longus only. Rationale : Administration of androgens has been shown to increase satellite cell number in humans and animals ( 35 64 ) Progressive resistance training leads to muscular hypertrophy in exercised muscles of both humans and rodents ( 56 61 65 ) However, limited peer reviewed data exists

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24 establishing the ability of progressive resistance training to induce satellite cell activation during a superimposed hypogonadal state in either the rodent or human model.

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25 CHAPTER 2 REVIEW OF LITERATURE Hypogonadism Induced Effects in Man Male h ypogonadism (i.e., serum T <300ng/dL) manifest s as sexual dysfunction, reduced energy, depression, cognitive deficits, anemia ( 53 ) and may result in reductions in lean body mass and bone mineral density, and increased adiposity ( 66 67 ) Together these changes limit functional independence and increase the risk of fracture ( 68 ) In males, the incidence of osteoporosis related fractures is increasing H owever, osteoporosis remains under diagnosed and under treated area of clinical medicine in men ( 59 ) Strikingly, hip fractures result in far greater mortality in men than in women The one year period post fracture mortality risk (32%) for male veterans versus 18% in females ( 18 ) Sarcopenia is an age related decline in skeletal muscle mass and strength leading to decreased functional capacity and predisposition to chronic metabolic disease ( 69 ) The l oss of skeletal muscle results in a reduced metabolic rate, a reduction in the primary storage depot for glucose amino acids, and for lipid oxidation ( 70 ) Reductions in muscle mass also limit locomotor ability ultimately reducing ability to maintain cardiorespiratory endurance. Testosterone Replacement Therapy for Hypogonadism Testosterone is the prim ary androgen in circulation and is closely bound to sex hormone binding globulin and albumin (95%) whereas free te stosterone circulates at around 2% of total testosterone ( 71 ) The Institute of Medicine recently requested for small clinical trials to be conducted to elucidate the potential anabolic and androgenic effects of testosterone re placement therapy aimed at reducing, reversing or preventing

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26 the catabolic effects of hypogonadism in males published Clinical Practice Guidelines, states that a male presenting with a serum total testosterone level of 300 ng/dL is likely to be hypogonadal ( 28 ) A clinical test with a level between 200 400ng/dL should have the test repeated along with a measurement of free testoster one ( 28 ) Clinical measurement of luteinizing hormone ( LH ) and follicle stimulating hormone ( FSH ) are also recommended to determine whether primary testicular fai lure is the causative factor or a secondary disturbance of the hypothalamus pituitary gonadal axis ( 28 72 ) Tw enty percent of men over the age of 60 have serum testosterone levels below 11.3 nmol/L ( 325ng/dL) the normal range for normal eugonadal men ( 73 ) Bhasin and col leagues first proved the clinical effectiveness of testosterone in a trial of seven hypogonadal men receiving a 100 mg*week 1 of testosterone enanth ate by intramuscular injection for ten weeks ( 74 ) Individuals participating in this trial underwent a 12 week pre experimental period of withdrawal from previous androgen or gonadotropin therapy with no weight training or heavy endurance training four weeks prior to st udy initiation Significant effects were seen in accrual of lean body mass (5.0 0.8 kg) and increase d muscle strength (22%) following ten weeks of 100 mg *week 1 of testosterone enanthate ( 74 ) Similarly, Wang and others administered a dosage of 5m g/ 3 times a day, sublingually in non exercising hypogonadal men and found positive changes in fat free mass leg strength, and markers of bone resorption after six mon ths of treatment ( 75 ) Administration of testosterone (100 mg/day) in a transdermal gel (which has a low rate of absorption) placed on selected skin sites raised ser um testosterone concentrations from approximately 6 nmol/L, up to 29 nmol/L 24 hours

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27 after application at one site ( 76 ) Additional evidence exists in humans that s upraphysiologic doses of 600 mg*week 1 of testosterone for t wenty weeks resulted in significant improvements in lean body mass, muscle cross sectional area, and muscle strength without exercise in eugonadal young mal e s made hypogonadal through the administ ration of lupron or older hypogonadal m ales ( 6 ) In a recent study, by Page and colleagues ( 77 78 ) improvements in timed functional tests, handgrip strength, bone mineral density, and lean body mass were reported for hypogonadal elderly men receiving 200 mg intramuscular testost erone bi weekly Additionally, t estosterone administration has demonstrated positive effects on body composition in older hypogonadal males In hypogonadal men aged 65 years or older receiving 200 mg* week 1 Page and colleagues ( 77 78 ) found increases of more than 3.0kg of lean body mass and a 5% reduction in total body fat after 36 months In another 36 month long st udy, Snyder and colleagues ( 79 ) investigated a testosterone replacement therapy utilizing a scrot al patch delivering 6 mg/day of t estosterone This dosage level an d route of administration resulted in significant increases in lean body mass of 1.9 kg and a decreased total fat mass of 3.0 kg following the intervention. Bone mineral density improvements were found for both study populations ranging and 2 10% change in regional BMD sites of the hip and lumbar spine, respectively ( 7 79 80 ) Potential Therapeutic Use of Trenbolone Administration for Hypogonadism Trenbolone Hydroxyestra 4,9,11 trien 3 one) is a synthetic analog of testosterone that is generally administered in an acetate or enanthate form Historically, trenbol one has been confined to experimentation in laboratory animals and livestock ( 81 ) to en hance muscle growth, and market characteristics of slaughter animals ( 82 ) Administration of trenbolone (120mg +24mg estradiol) to finishing steers (i.e., castrated

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28 m ale bovines) tended to increase t ype II a myosin heavy chain (MHC) mRNA concentrations after 30 days in the semimembranosus muscle ( 83 ) while sarcopenia tends to dimini sh type II fiber concentrations ( 3 ) In a study by Gonzalez an d colleagues, crossbred cull cows had increases in Type I muscle fiber cross sectional area and diameter were larger when trenbolone acetate was co adminstered with a beta agonist ractopamine HCL ( 84 ) Its application in humans as a method to prevent or attenuate hypogonadism induced osteopenia and sarcopenia has not been explored Currently, human application has been hindered because the specific mechanism of g rowth promotion by trenbolone is not completely elucidated However, a recent review by Yarrow and colleagues discusses the tissue selectivity and potential clinical applications of trenbolone administration in humans ( 41 ) however the dosage and safety profile of trenbolone in humans has not been established to date Pottier and colleagues reported that trenbolone is hydrolyzed to 17 beta t renbolone ( 17B TbOH, 17 B hydroxy estra 4 ,9,11, trien 3 one) following ab sorption ( 85 ) I n a classic st udy by Danhaive and Rousseau it is stated that trenbolone exerts its anabolic action through androgenic and potent anti glucocorticoid effects ( 86 ) Meyer and colleagues state that trenbolone binds to the androgen receptor with similar affinity to DHT (i.e., 3 times greater than testosterone) ( 87 ) Interestingly, trenbolone also binds to the progesterone receptor with the same affinity as progesterone however the role of the progesterone receptor in tissue anabolism or catabolism warra nt further study ( 87 ) The binding affinities of trenbolone for both the and rogen and progesterone receptor coupled with the potent anti gluccocorticoid effects are different than testosterone and may result in differing tissue effects in muscle and bone. However, a general conse nsus

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29 s effects on tissue accretion has not been reached. In a two week study, young female Sprague Dawley rats (60 120g) gained 26% more weight in muscle and skeletal tissue during trenbolone administration at 80 micrograms of trenbolone /100g body weight comp ared to control animals ( 35 ) In additi on, there was a higher efficiency of feed conversion to lean body mass b y the trenbolone treated intact female rats ( 35 ) There was no significant increase in skeletal muscle (wet weight) of the gastrocnemius, peroneus, and tibialis anterior, however total DNA content was significantly increase d in the peroneus and tibialis anterior ( 35 ) Interestingly, the semimembranosus a large muscle of the hindlimb saw significant increases in both skeletal muscle wet weight (24%) and total DNA/muscle (38%) following two weeks of trenbolone administration in intact, young female rats ( 35 ) Without confirmation by histological samplin g, the potential growth observed cannot be attributed solely to accretion of skeletal muscle tissue and may have been through changes in lipid deposition or connective tissue. Vernon and Buttery reported that trenbolone administration exert s a more profou nd effect on minimizing protein degradation compared to placebo allowing for the accretion lean body mass ( 88 ) Similarly, total carcass nitrogen content of the trenbolone trea ted animals was increased while tot al body fat content tended to decrease ( 89 90 ) An additional possible mechanism hypothesized by Thompson and colleagues is that trenbolone administration may increase the responsiveness of satellite cells to FGF and IGF 1 ( 35 ) However, the direct role that androgens play in

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30 regulating muscle and plasma IGF 1 and associated binding proteins remains to be determined. Hypogonadism Rat Model Selection S everal orchiectomized rodent models have been utilized to mimic the effects of human hypogonadism including orchiectomized Brown Norway, Wistar and Fischer 344 (F344) rats, among others Older Brown Norway experience minor reductions in skeletal muscle mas s ( 91 ) while undergoing spontaneous prostate enlargement similar to aging men Similarly, Borst and colleagues evaluated the responsiveness of bone resor ption, prostate mass, and muscle mass in a group of 13 month old Brown Norway rats receiving a 5 alpha reductase inhibitor without blocking the anabolic effects of T on muscle and bone ( 8 ) However, p revious work by Borst and colleagues reported that orchiectomized Brown Norway experience only minor decrements in skeletal muscle hindlimb mass, and do not experience an i ncreased in fat mass or a significant alterat ion in serum bone resorption markers following orchiectomy ( 8 ) Therefore, Borst and colleagues evaluated three rat models (i.e., Brown Norway, Wistar and Fischer 34 4) to identify the most robust strain for measuring the catabolic consequences of hypogonadism and found that F 344 rats were the only strain where orchiectomy induced a cascade of effects similar to that observed in hypogonadal men, including reductions in body and muscle mass, high turnover osteopenia (i.e., increased bone resorption and a reflux increase in bone formation), and increased adiposity ( 60 ) Recent data by Hershberger and colleagues, establi shed that the levator ani bulbocavernosus muscle complex are androgen responsive skeletal muscle and tissues in the rat ( 40 ) with a greater androgen receptor density compared to human skeletal muscle ( 42 92 ) Therefore, we evaluated F344 /Brown Norway rodent s to examine the robustness of this

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31 model in mimicking the effects of human hypogonadism on skeletal muscle, bone, fat, hemoglobin, an d prostate tissue Bone Effects in Orchiectomized ( ORX ) Rats Previous work by Borst and colleagues determined that rapid changes in bone resorptive markers were present for both Fischer 344 and Brown Norway 3 month old r ats following orchiectomy ( 60 ) Both species experienced an increase in urinary excretion of urinary DpD/creatinine 28 days an d 56 days following ORX surge ry Bone resorption indices of urinary DpD/creatinine in Fisher 344 rats increased by 87% in the first m onth, with makers of bone resorption still increased at 60% into month two, whereas Brown Norway rats experienced a 50% increase in urinary DpD/creatinine excretion in month one and a 99% increase at month two of the study ( 60 ) Loss or withdrawal of endogenous testosterone results in catabolic effects in muscle and bone ( 60 ) that become apparent within two weeks of ORX and are stea dily increased compared to control animals, over the course of 9 months ( 93 ) However administration of 1.0 mg of testosterone per day for 56 days suppressed the urinary excretion of Dpd/creatinine in ORX rats (20). Substantial osteoporotic changes occur in cancellous bone of the axial and appendicular skeleton of ORX a ged 13 month old orchiectomized male Fischer 344 rats ( 8 ) In the vertebrae, t he majority of cancellous bone loss consisted of reductions in t rabecular number and thinning of the trabeculae, whereas tibial cancellous bone loss in the tibial consists only of the loss of trabe cular number without a con commitant decrease in trabecular thickness ( 93 ) ORX also reduces periosteal appositional growth in cortical bone, an effect that is reversible by testosterone or dihydrotestosterone administration ( 58 94 ) Within two months following orchiectomy, a rapid and large loss of cortical bone mass occurs in mature male Fischer

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32 344 rats and is associated with increased bone remodeling and an expansion of the marrow cavity under conditions of androgen deficiency ( 95 ) However, subcutaneous administration of testosterone undecanoate 6mg/kg*week 1 prevented cortical bone loss in ORX animals ( 95 ) Expectedly, E rben and colleagues reported reductions in serum concentrations of estradiol following ORX in rats ( 93 ) although re ductions in serum estradiol were not observed in male F344 rats following ORX in another study ( 11 ) Erben and others suggest ed that estradiol in serum and bone ma y attenuate changes in high turnover osteopenia following androgen withdrawal ( 93 ) In a human study by Riggs and colleagues, the authors stated that estradiol is a prominent hormone regulating skeleta l metabolism in elderly men (average age 68 years), and in fact, is the dominant hormone governing bone resorption ( 96 ) They reported that resorption mark ers were reduced by 70% following estradiol administration in men deficient in T and E ( 96 ) Whereas T may have a more limited, but important role in preve nting apoptosis in mature osteoblastic cells ( 96 ) In addition, Reim and colleagues suggest that circulating levels of estradiol may function in the suppre ssion of endocortical bone resorption in aged male rats ( 95 ) These studies indicate that the loss of naturally produced estradiol may be implicated in skeletal loss, and the presence of estradiol may maintain the balance between for mation and resorption in the adult skeleton. Currently the mechanisms governing bone resorption during androgen replacement therapy are still being refined At least one study has reported that subcutaneous t estosterone undecanoate administration of 8 mg /kg *week 1 to ORX rats restores circulating levels of estradiol ( 93 ) However, a recent study by Yarrow and colleagues reported that intramuscular

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33 administration of supraphysiologic testosterone enanth ate (7.0 mg/week) to 3 month old ORX F344 rats did not elevate the serum or bone estradiol concentrations, but did result in significant elevations in serum and bone testosterone and DHT concentrations ( 11 ) S upraphysiolgic testosterone enanthate increased tibia l bone mass by 10% in the tibia and by 23% in the femur in young rodents ( 144) Similarly, bone mechanical strength was improved by 12 19% in both sexes of gonadectomized rodents administered testosterone ( 11 ) Currently, the significance of bone androgen and estrogen concentrations role in bone strength and bone tur nover is not understood. Bone and Serum Hormone Concentrations in ORX rats Recently, Yarrow and colleagues reported the first ever measurements of the bone sex hormone concentrations for intact, ORX and testosterone treated animals ( 11 ) In intact F344 rat tibial concentrations of testosterone were reported at 2.5ng/g, whereas ORX induce d a near ly 50% decline in bone testosterone concentrations Conversely, suprap hysiologic testosterone enanthate administration to ORX male rats resulted in a 9.5 fold increase in tibial testosterone compared with control animals and a 12.5 fold greater testosterone concentration that what was found in serum ( 11 ) Tibial DHT concentrations were 91% greater in male rats receiving testosterone enanthate compared to controls whereas serum levels of DHT were 21 fold higher than intact controls ( 11 ) However, neither ORX nor testosterone enanthate administration demonstrated altered bone estradiol concentrations in male rats ( 11 ) Skeletal Muscle Effects in ORX rats Orc hi ectomy induces rapid loss of skeletal muscle mass in some hindlimb muscles within two months following surgery ( 60 ) Coupled with reductions in muscle mass, ORX induces losses of glycogen stores and impair s protein synthesis ( 97 ) Intramuscular

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34 testosterone enanthate administration (7.0mg/week) has been shown to increase rat plantaris wet weight compared to ORX rats following 28 days administration ( 11 ) Hourde and c olleagues reported that while three months of androgen treatment in orchiectomized rats did not cause hypertrophy of the soleus muscle, force and endurance were increased by 69% and 35% respectively ( 98 ) Resistance Training for Sarcopenia and Osteoporosis Resistance traini ng is well known to produce favorable increases in skeletal muscle mass/strength ( 99 ) functional abilities ( 99 ) and bone mineral density (BMD) ( 100 101 ) without the deleterious side effects associated with testosterone replacement therapy In fact, the American College of Sports Medicine statement recommends that resistance exercis e should be performed by all healthy older adults in order to prevent the reductions in muscle function and BMD that occur primarily between 50 80 years of age ( 3 ) Further, in a recent review, Hunter and colleagues report that resistance training is the most favorable intervention for ameliorating the deleterious ef fects of sarcopenia ( 102 ) Despite the established benefits of resistance exercise on muscle mass and BMD, few studies have evaluated the effects of resistance training in hypogonadal older men or hypogona dal animal models The resistance exercise induced augmentations of muscle mass are believed to be influenced by the acute changes in the systemic and localized (i.e., within muscle) sex hormone and growth factor concentrations that occur following exerci se ( 103 107 ) Specifically, following heavy resistance exercise, the serum concentrations of growth hormone and testosterone are elevated for approximately 60 minutes in healthy young and older men ( 108 ) However, r ecent work by Vingren and colleagues (2008) reported

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35 that the concentrations of testosterone within mus cle were not elevated in humans following heavy resistance training ( 109 ) which is in contrast to reports in rat models undergoing swimming ( 110 ) and treadmill running ( 111 ) ; although the modalities of swimming and treadmill running in the rat are not parallel stimuli to resistan ce trainin g in the human Hornberger and colleagues have developed a weighted ladder climbing exercise protocol to model the overload stimulus typically seen in progressive resistance training programs in humans ( 62 ) An important feature of ladder climbing as a model for skeletal muscle hypertrophy is the volitional nature of the activity Rats typically take to th e ladder without induction of the noxious stimuli (i.e., forced running, over heating, and electrical shock) that occurs with treadmill exercise Presumably, ladder climbing initiates a lower stress response when compared to treadmill running I ndeed, Lee and colleagues have utilized the weighted ladder climbin g model and observed increases of 23% in the flexor hallicus longus muscle mass following eight weeks of training ( 61 ) By the end of this eight week training period, rats were able to carry nearly 1,400g attached to their tail representing a load nearly 3 times their body weight ( 61 ) Indeed, in this study increases in skeletal muscle wet we ight are not a direct reflection in accrural of muscle cross sectional area or improvements in myofibrillar content In the study by Lee and colleagues the resistanc e trained group had lower levels of myofibrillar protein content when expressed as mg/g of flexor hallicus longus muscle tissue compared to control animals ( 61 ) One possible mechanism for this discrepancy suggested by Lee and colleagues is that the eccentric phase of ladder climbing induces larger degrees of muscle damage and possibly resultant fibrosis and damage to the surrounding

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36 sar colemma ( 61 ) The eccentric mode was suggested by Lee and colleagues, as the animals engage the ladder rung with their toes, followed by an initial lengthening contra ction of the flexor hallicus longus and flexor digitorum longus ( 61 ) The second phase consists of concentric contraction of the plantar flexors and knee extenors while loaded ( 61 ) Increased damage may lead to increased swelling as evidenced by decreased total muscle protein content in the FHL reported by Lee and colleagues ( 61 ) Another potential mechanism for the increased strength displayed with weighted ladder climbing could be attributable to learning, or growth in other muscle groups not evaluated by the researchers. Although, weighted ladder climbing is a potent stimulus for increasing skeletal muscle hypertrophy; systemic and l ocal sex hormone and growth factor responses to weighted ladder climbing have not been evaluated O steopor osis is metabolic disease of bone tissue affecting bone microarchictecture, fracture risk, and bone mass ( 112 ) Osteoporotic fractures are estimated to occur at a rate of 1.5 million per year in individuals over the age of 50 years ( 100 ) Scane and colleagues reported that aging males experience a reduction in trabecular bone content of 15 45%, and a loss of cortical bone ranging from 5 15% ( 113 ) Resistance training is a recommended modality to combat the deleterious consequences of osteoporosis although the Centers for Disease Control and Prevention reported that only 12% of individuals aged 65 74 and 10% of individuals over 75 years of age engaged in muscular strength and endurance activities at least two days a week. Recent studies by Vincent and Braith have found increases in bone miner al density (2%) in older men and women engaged in a 24 week high intensity progressive resistance exercise study, whereas lower intensity exercises may not be as effective

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37 ( 65 ) L ittle research has examined the role of resistance training in the prevention of osteoporosis in males specifically hypogonadal males A majority of longitudinal studies have focused on women as the critical population at risk for osteopo rosis ( 100 ) However, a population at more immediate post fracture r isk is elderly males as one in three elderly males dies within twelve months of a hip fracture whereas females have more overall fracture rates ( 18 ) Theref ore, non pharmacological interventions such as resistance training and pharmacological strategies for the attenuation of hypogonadism induced sarcopenia and osteopenia in males are necessary. Satellite Cells Definition Satellite cells are globally defined as a heterogenous population of mononuclear myogenic precursors that contribute to the regulat ion, maintenance and function of essential for muscle cell hypertrophy, and repair; satellite cells are histologically defined by their location beneath the basa l lamina of the muscle fiber Satellite cells have the potential to proliferate to fuse with the myofiber or form new myofibers giving rise to multi nucleated myotubes Dhawan and colleague state that satellite cells can be termed muscle stem cells of post natal skeletal muscle ( 140) In a similar light, other authors refer to adult skeletal muscle stem cells as satellite cells ( 141 ). By the def inition of Hawke and colleagues, once satellite cells begin expressing myogenic markers they are termed myoblasts ( 142 ). Collins and colleagues (2005) report that 3,000 myonuclei can originate from a single satellite cell, highlighting the enormous potential for satellite cells to address skeletal muscle myopathies (143) As the field of studying satellite cell activat ion, quiescence, and signaling pathways evolve so will the current definitions.

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38 Quiescence Satellite cells exist in a quiescent state until recruited for repair, hypertrophy, renewal or differentiation The homeostatic balance of quiescence and activation is necessary to maintain satellite cell function ( 144 ) I n response to disease or injury satellite cells of the Pax 7+ lineage may enter the cell cycle to initiate myofiber repair while a sub population returns to quiescence During the proliferation ph ase myoblasts expand their cytoplasmic nuclei ratio and initiate fusion with existing fibers or initiate de novo myofiber synthesis ( 142 ) Recent research suggests that Sprouty1, a receptor tyrosine kinase signaling inhibitor, is highly expressed by satel lite cells in the quiescent state ( 144 ) and is down regulated during the proliferative phase of muscle regeneration ( 145 ) Shea and colleagues suggest that the expression of Sprouty1 occurs in a sub population of satellite cells and is essential for their return to the quiescent state ( 145 ). Subsequently, Sprouty1 appears critical for regulating quiescence but is not an obligatory component of muscle differentiation. In contrast, Abou Khalil and colleagues, suggest that quiescence may be regulated by the in teraction of satellite cells with the vascular bed, and the simultaneous promotion of endothelial growth, muscle regeneration and angiogenic factors ( 1 4 ) Angiopoieit i n 1/Tie 2 (e.g., an angiogenesis promoting factor/tyrosine kinase endothelial receptor) i s described as maintaining vascular integrity; where Ang iopoietin 1 /Tie 2 increases the nu m ber of satellite cells in the quiescent state Whereas, blocking Ang 1/Tie 2 initiates satellite cells to re enter the cell cycle ( 146 ). The role Ang 1/Tie 2 is theo rized to regulate angiogenesis in response the satellite cell pool, ensuring that muscle growth does not exceed angiogenesis. The Ang 1/Tie 2 regulation of satellite cell pools entry and emergence from the quiescent state requires further elucidation to ad vance the field of muscle regeneration.

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39 Self Renewal, Differentiation, and Activation In a classic study by Reznik, satellite cells were characterized as having the potential to be mobilize d from their quiescent state, expand, generate myoblasts and t erm inally differentiate to fuse with surrounding fibers for growth or repair ( 114 ) In the rodent, satellite cells represent approximately four percent of the total myonuclei, and decrease to two percent of the myonuclei in senescence ( 146) Occurrence of myotrauma through exercise or injury results in the activation, proliferation, and migration of satellite cells to the injured fiber Upon arriving at t he area of trauma satellite cells which will then fuse with the damage d myofiber (hypertrophic response) or fuse together to produce additional myofibers (hyperplas t ic response) The regenerated fiber will then appear with central nuclei these newly added nuclei will then migrate to the periphery. Satellite cells not directly committed to muscle repair or growth are involved in satellite cell self renewal ( 142 ). Moss and colleagues confirmed that a sub population of satellite cells exists that do not termi nally differentiate but which function to renew the satellite cell pool ( 115 ) R ecently, Notch signaling and the canonical Wnt (ligand for Frizzled family of receptors) pathway signaling are implicated in the myogenic properties of muscle regeneration, growth, and activation of satellite cells In response to muscle injury, Notch sign aling components (Delta 1, Notch 1, and active Notch ) are upregulated and are necessary to activate satellite cells into a highly proliferative state ( 147 ) Wnt functions in recovery from muscle injury and has a prominent role in myoblast dif ferentiation a nd myotube fusion (140) Under conditions of injury or exercise, satellite cells initiate proliferation and exhibit a myoblast cell fate under the regulation and control of Myf 5, MyoD, integrin, and desmin ( 149 ). T he specific mechanism s by

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40 which Wnt and Notch regulate or co regulate cell fates dur ing postnatal myogenesis remain to be determined. During the postnatal period, myonuclei remain in a mitotically inactive state quiescence in the absence of appropriate stimuli ( 116 ) Pax 7 is expressed universally in satellite cells in rats, other mammals, salamanders, and zebrafish ( 117 120 ) Deletion of the Pax 7 gene in knock out models cause s depletion of satellite cells by causing embryonic progenitors to apoptose or adopt alternative non myogenic cell fates ( 121 123 ) Exp resssion of Pax 7+ in satellite cell s allows for their self renewal and survivability, while removal of Pax 7+ leads to decline in their number as determined in Pax 7 null mice ( 122 124 ) Therefore, measuring and monitoring the expression of Pax 7 will allow for the detection of early and late phase therapeutic interventions aimed at increasing skeletal muscle in at risk populations. Pax 7+ Responsiveness to Growt h Factors T ransforming growth factor depending upon the phase of the cell cycle Transforming growth factor implicated in reducing migration in C2C12 skeletal muscle satellite cells, while reducing the ability of IGF 1 t o increase migration. However, d uring the regenerative process, the expression of transforming growth factor ligand are required for proliferation and differentiation ( 150 ). The insulin like growth factor family i s a known inducer of satellite cell proliferat ion and differentiation (151 ). IGF 1 may support the satellite cell pool by down regulating caspace/apoptotic pathways and activating the serine threonine protein kinase Akt ( 152 ) Following exercise, the induc tion of IGF 1 signaling also promotes the fusion of satellite cells to the myofiber, and committment of the satellite cell to participate in hypertrophy ( 142 )

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41 Androgen Effects on Satellite Cells In a recent study by Sinha Hikim, it was suggested that tes tosterone may partially regulate the cycle of cell proliferation, and commitment of daughter cells through the activation of Notch ( 153 ) Sinha Hikim and colleagues reported that supraphysiologic al doses of testosterone enanthate (300 vs.600 mg/week) resul ted in a dose dependent increase in satellite cell number size, and motility in healthy men ( 125 ) However, a lower dose of testosterone ( 125mg/week ) whic h remains anabolic did not increase satellite cell number ( 125 ) Androgens may directly affect the proliferation of satellite cells through binding with a ndrogen receptors present on muscle satellite cells ( 126 ) Alternatively, and rogens may indirectly stimulate satellite cell activation through the presence of muscle IGF 1, although the relative contributions of the se two mechanisms are unknown. Similarly, in cultured satellite cells nuclei and myotubes from an in vitro porcine mod el administered testosterone; Doumitt and colleagues reported increased androgen receptor numbers were present on satellite cells after incubation with testosterone ( 127 ) Lastly, Sinha Hikim suggest that testosterone may function through regulating satel lite cell replication, inhibition of satellite cell apoptosis or dedication of stem cells into a myogenic lineage ( 125 ) The combinat ion of trenbolone and e stradiol when implanted in the semimembranosus muscle of yearling steers, causes a significant increase in satellite cell number ( 39 ) In addition, trenbolone an d estradiol increased daily carcass protein accretion by 82% over the course of 40 days of administration ( 39 ) Kamanga Sollo and others have reported that trenbo lone also increases IGF 1 mRNA in bovine satellite cell cultures as a possible mechanism for muscle cell hypertrophy ( 128 129 ) Thompson

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42 and colleagues reported that trenbolone administration increases muscle size and DNA content when administered to mice and that trenbolone increases satellite cell sensitivity to IGF I and to fibroblast growth fa ctor (FGF) in a culture system ( 35 ) Ho wever, the mechanisms by which trenbolone or testosterone regulate directly or indirectly induce satellite cell proliferation and differentiation remain to be elucidated.

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43 CHAPTER 3 METHODS Experimental Design All experimental procedures conformed to the ILAR Guide to the Care and Use of Experimental Animals and were approved by the Institutional Animal Care and Use Committees at t he Malcolm Randall VA Medical Center Fifty F isher 344/Brown Norway F1 (N=50) intact male rats, were obtained from Charles Riv er Laboratories (Wilmington, MA ), and were housed in separate cages at 20 C under a 12 hour light cycle All rats underwent a minimum one week acclimatization phase in the laboratory prior to any procedural intervention Rats were allowed to move freely a nd were fed standard rat chow and water F ood intake was measured on a weekly basis. Borst and Conover have established F344 and Brown Norway male rat s as model s for studying the anabolic/ androgenic pathways and catabolic state associated with hypogonadism ( 60 ) For thi s study, male F344/Brown Norway F1 rats, aged 10 months, were matched for initial body weight and randomized i nto the following groups (n = 10 /group ; Total=50 ) and treated for 42 days as fol lows : 1) Sham + vehicle (Sham) 2) ORX + vehicle (ORX), 3) O RX + testosterone (ORX+T), 4) ORX + trenbolone (ORX+TREN), 5) ORX + progressive resistance exercise (ORX+EX) Following randomization animals underwent Sham vs. ORX surgery and while receiving the following treatments for 42 days: testosterone enanthate (7.0m g/week), trenbolone enanthate (1 .0mg/week), vehicle (sesame oil ) or exercise (i.e., weighted ladder climbing performed 3 days/week +vehicle ) treatments for 42 days Blood was sampled every two weeks via t he tail tip ambulation method ( 130 ) prior to and during

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44 drug administration or progressive resistance training exercise and by a single intrac ardiac puncture at sacrifice ( 131 ) in order to evaluate serum hormon e concentrations and to verify drug delivery. During all surgical procedures, animals were kept on a circulating heat pad to maintain body tempera ture, remained under isoflurane anesthesia (5% induction, 1.5 2.5% maintenance), and receive d buprenophine analgesia to reduce pain Following surgery, a nutritional supplement (Jell O cube with added protein and fat) ( NIH protocol diet ) was provided f or o ne week in order to minimize weight loss resulting from surgery The rats were sacrificed at day 42, via an intraperitoneal injec tion of 120mg/kg pentobarbital blood, bone, muscle, and prostate tissues were removed for group comparisons There were a total of ten animals per group, as determined by a p ower analysis, for a total of 50 rats. Drug Administration The slow releasing enanthate ester s of both testosterone ( 1 1 ) and trenbolone (unpubli shed laboratory results) result in a sustained supraphysiological drug serum concentrations for at least seven days following intramuscular injection Testosterone enanthate (Savient Pharmaceutical East Brunswick, NJ), trenbol one enanthate (Steraloi ds, Newport, RI), and vehicle were dissolved in sesame oil and administered (0.1mL) once every seven days, under isoflurane anesthesia, into the quadriceps musculature Injections were alternated between legs in order to reduce possi ble discomfort of repeated injections Additionally, previous studies from our laboratory have reported that once weekly supraphysiological (7.0 mg/week) testoste rone enanthate administration successfully prevents skeletal muscle mass/strength and BMD loss in growing ORX rodents (aged 3 months) ( 8 11 30 )

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45 Weighted L adder Climbing Weighted ladder climbing is an established form of progressive resistance training (PRT) in rodents that results in overload of the flexor hallicus longus musculature ( 62 ) During training, rats repeatedly climbed a 1 meter ladder inclined 85 while carry ing progressively heavier (in 25 g increments) lead weights The weights were placed in a nylon bag and hung from a Velcro strap that was secured by a protective foam pad on Prior to weighted training, rats underwent a 3 day acclimatization period, carrying their body weight up the ladder If the rat paused during the climb, it was induced to complete the climb with a brief pulse of compressed air On the fourth training day, rats carried a series of progressively increasing loads in order to establish the baseline maximum Over the 42 day experimental period, exercising rats performed the PRT protocol consisting of five completed cli mbs per session (3 sessions/week), during which time the weight were progressively increased following successful completion of the training protocol Each week the load was increased between 10 30% over the load carried the previous week If the rat was u nable to complete greater than 3 sessions at the new workload, the load was reduced by 5 10% on the subsequent session Next, weights were reduced to most recently utilized load to ensure 5 weighted climbs were completed Each climb was separated by two mi nutes. Testing was completed when the required number of repetitions were completed or when a brief Hemoglobin and Serum Sex Hormone Measurements Whole blood samples were acquired from the tail at ba seline and week 2, week 4, and week 6 under isoflurane anesthesia and assayed in duplicate using the Hgb Pro ( ITC, Edison, NJ) photometer, which has an intra assay CV of less than 2.41 % Briefly,

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46 approximately 20 l of whole blood was placed on Hgb Pro test strips membrane Once dispersed in the membrane the red blood cells contact a lysing agent and release their hemoglobin The photometer analyzed the hemoglobin concentration at a wavelength of 522nm The remaining blood samples were centrifuged at 3000g for 12 minutes and serum aliquots were separated and stored at 80C for later analysis All serum hormone measurements were determined in duplicate within the same plate Testosterone was 0.04ng/ml with an intra assay CV of 5.3% ( Alpco Diagnostics, Windham, NH ) DHT was of 6.0 pg/ml with and intra assay CV <11% (Alpco Diagnostics, Windham, NH) Trenbolone Corporation, Lexington, KY) with a sensitivity of 0.1ng/ml and an intraassay CV of 3.76%, according to methods previously devised in our laboratory (unpublished laboratory results) Specifically, trenbolone (Sigma Aldrich) as dissolved in 100% ethanol (1:1) and subsequently serially diluted in hormone free EIA buffer (Neogen Corporation, Lexin gton, KY) to produce a quantitative standard curve. In Vivo Muscle Strength Testing (Grip Strength) The Ring Grip Performance Test (Columbus Instruments, Columbus, OH,USA) is an established in vivo muscle strength test, which measures the maximal gripping strength of the forelimb (i.e., digital flexors) muscles ( 132 ) The test measures the peak tension generated from the forelimbs of the rodent during testing For this test, animals were placed in a horizontal position an d allowed to grasp the wire mesh Subsequently, the researcher grasps the animal by the tail and force is applied in an opposing direction

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47 ken The average of three successful trials is recorded and utilized to calculate specific force similar to procedures reported by Borst and colleagues ( 30 ) Fiber Type and Fiber Cross Sectional Area Measurements The right flexor hallicus longus, and semim embranosus muscles were excised, pinned at resting length, coated with OTC, and frozen on a slurry of isopentane, cooled with liquid nitrogen and stored at 80C pr ior to analysis width were taken from the mid belly of each muscle. Initially, plated muscle cross section slides were first permeabilized with 0.5% Triton X100 in phosphate buffered saline (PBS) and subsequently rinsed wit h a series of PBS washes. The samples were incubated with primary antibodies for laminin (Lab Vision, Fremont, CA), type I myosin heavy chain (A4.840), and type IIa myosin heavy chain (SC 71). Secondary antibody treatments were completed with rhodamine, Al exa Fluor 350 and Alexa Fluor 488 (Invitrogen, Carlsbad, CA). Cover slips were mounted with Vectashield fluorescent mounting medium (Vector Labs, Burlingame, CA). Samples were visualized using fluorescence microscopy (10x magnification) with N21, GFP, and A4 cube filters (Leica DM LB, Solms, Germany), and then imaged with a digital camera T he CSA, fiber type, and the area fraction (A A ) occupied by each fiber type were recorded from a minimum of 250 fibers Encoded images were calculated for fiber CSA usin g Scion Image (NIH) software. Fibers that fluoresced blue were assigned as Type I (slow fibers), those fluorescing green were assigned to Type IIa fibers, whereas the remainder of non fluourescing fibers were assigned to Type IIb/x for each muscle The A4. 840 and SC 71 antibo dies were obtained from the Developmental

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48 Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242 Hemotoxylin and Eosin Staining Serial cros s sections of the semimbranosus and flexor hallicus longus (10 width were taken from the mid belly of each muscle Muscle sections were placed on a glass slide and subjected to a progressive hydration protocol beginning with an initial exposure to 1 00% ethanol (ETOH) solution for one minute followed by immersi on in a 70% ETOH bath for one minute A final rinse in deionized water for two minutes completed the first phase of the protocol Muscle sections were immersed in hematoxylin for one minute, followed by a rinsing in deionized and tap water for three minute s to reduce non specific tissue staining Briefly, differentiation of hematoxylin staining was accomplished by immersing muscle sections seconds followed by a 15 second rinse in deionized water N ext, muscle samples were partiall y dehydrated in a 70 % ETOH solution for one minute followed by placement in Eosin stain for two minutes Muscle sections were progressively dehydrated by submersion in a 95% and 100% ETOH ba th for two minutes Last samples were clarified in xylene for fo ur minutes Samples were allowed to air dry and were mounted with a glass cover slip using Crystal M ount ing M edium (Genetex, Irvine, CA, USA). Muscle cross sections were visualized with brightfield microscopy (Leica DM LB, Solms Germany) at 20x magnificati on. Ten randomly selected images were analyzed from each right flexor hallicus longus and semimembranosus for a minimum of 200 fibers analyzed An investigator blinded to the treatment group analyzed and recorded the presence on centralized or internalized nuclei present in the corresponding number of fibers per field of view Individual images were randomly selected from each group and

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49 analyzed in the following directional pattern: top left, top right, bottom right, bottom left until the required numbers o f muscle fibers were analyzed Centralized nuclei were defined as nuclei residing in a position equidistant from two or more sides of the myofiber Internalized nuclei were classified as nuclei residing greater than 20 pixels from the inner membrane of the fiber Data are MEAN SE, expressed per 100 fibers Satellite C ell (Pax 7) Staining The right flexor hallicus longus and semimembranosus muscle were pinned at resting length covered with OTC medium and frozen in isopentane chilled by liquid nitrogen Sa tellite cell staining for the presence of Pax 7 was conducted on 10 muscle sections previously frozen at 80 C Muscl e sections were rinsed with 100 mL of 1x PBS Muscle sections were fixed with a methanol:a cetone (1:1) for 3 5 min at room temperature F ixed muscle sections were rinsed in 1x PBS (3x) with a rinse duration of 5 minutes M uscle sections were blocked with a Superblock (Pierce Biotechnology, Rockford, IL) in 1x PBS for 60 min at room temperature A gain the fixed section s were rinsed with P BS a total of three times with a 5 minute rinse duration. Next the primary antibody (Pax7 ( 1:50 ) ) and rabbit laminin ( 1:200 ) were incubated with the muscle section for 1 2 hours at room temperature After the incubation time, rinse the section with 1x PBS 4 5 times with a five minute rinse duration Then, a dd the secondary antibody (A lexa fluor 488 ( 1:300 ) ); Rhodami n (Gand R ( 1:500 ) ) and incubate for one hour in diminished light. R inse the muscle section with 1x PBS 5 times with a five minute rinse duration un der diminished light Cover slips were mounted over fixed muscle sections with Vectashield fluorescent mounting medium with DAPI (Vector Labs, Burlingame, CA) and sealed with nail polish.

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50 Representative muscle samples from the flexor hallicus longus and se mimembranosus for Pax 7 positive nuclei were visualized using fluorescence microscopy (Leica DM LB, Solms Germany) at 20x A miminum of ten fields of view were recorded for each muscle sample The images were loaded into Scion Image and separated into thei r respective layers of N21, GFP, and A4 Representative populations of Pax7 nuclei per expressed per 100 fibers, and as the percentage of Pax7 positive per myonuclei population from a sample of 200 fibers. Pax7 positive nuclei were identified by yellow gre en fluorescent nuclei located between the basal lamina and the extra cellular membrane, overlayed by DAPI positive tissue The investigator was blinded to the group assignments during analysis Individual images were randomly selected from each group and a nalyzed in the following directional pattern: top left, top right, bottom right, bottom left until the required numbers of muscle fibers were analyzed Bone Sex Steroid Hormone Measurements The right tibia e were harvested and the sex hormones (i. e., testos terone, DHT ) and trenbolone were extracted according to the methods of Yarrow and others ( 11 ) First, the tibia was cut into small pieces, pulverized with a liquid nitrogen cooled Spex Certiprep freezer mill (Edison, NJ USA ) for two minutes and stored at 80C until homogenization Bone powder was homogenized in 20 volumes of 4C Krebs Ringer phosphate buffer that consisted of 116mM NaCl, 10 mM of phosphate, 4.5mM K CL, 2.5mM MgCl 2 1.2mM CaCl 2, 5% glycerol (pH 6.9), 2mM EDTA and 4mM DTT (Sigma Aldrich, St. Loui s, MO) The bone homogenate was disrupted via a high speed polytron (15s) and probe sonic ation (30s) The homogenate was then diluted with 1:2 chloroform metha nol (2:1 v:v), vortexed for 45 seconds and centrifuged at 1500 rpm for 10 minutes in order to s eparate the organic aqueo us layers The upper aqueous layer is

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51 re diluted in 2 ml chloroform, vortexed for 45 seconds and re centrifuged at 1500rpm for 10min Ne xt, the bottom organic layers from both extraction steps were combined, dried under a gentle stream of nitrogen, and equilibrated for 48 hours at room tempeature solutions prior to analyses All reconstituted samples were assayed in duplicate on a Previous work by Yarrow and colleagues demonstrated greater than 90% recovery for all hormones a nd minimal interference ( 11 ) Prostate, Levator Ani Bulbocaveronsus C omplex, Kidney, Adipose and Muscle Tissue Mass Prostates were cleaned of adipose tissue, sectio ned and weighed in order to compare the effects of the sex hormone t estosterone trenbolone, and resistance training on prostate mass The LABC was excised, weighed and cleaned of adipose tissue to examine the effects of the sex hormone testosterone, dehyd rotestosterone, trenbolone, and resistance training on tissue mass The left kidney and retroperitoneal fat pad were removed to determine the effects of the sex hormone tes tosterone, trenbolone, and resistance training on mass The soleus, plantaris, flexo r hallicus longus, and semimembranosus muscles were removed and weighed to evaluate changes in mass associated with administration of testosterone, trenbolone or weighted ladder climbing on mass. Bone Mechanical Strength and Peripheral Quantitative Compute d Tomography (pQCT) After excision, the right and left femora were weighed and femoral length was measured using a digital caliper (Mitutoyo Aurora, IL) Femora was immediately wrapped in saline soaked gauze to prevent dehydration, and stored at 20C in order to

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52 maintain the mechanical properties of the bone ( 133 ) Prior to mechanical testing, femora were thawed to room temperature and were kept wrapped in sal ine soaked gauze except during measurements. The midshaft of the left femur was subjected to a medial/lateral three point bending test, using an MTS material testing machine (MTS Systems Co., Erden Prairie, MN) as described by Leppanen and others ( 134 ) while the neck of the right femur was subjected to an axial load biomechanical test Before mechanical testing, a preload (10 N/0.1 mm/s) was applied at 1.0 mm/s until failure. From the load deformation curve, the fo llowing parameters were determined for the femoral shaft: breaking load, yield load Bone mechanical strength is expressed both as force (measured in Newtons) as previously described by Yarrow and colleagues ( 11 ) Prior to pQCT assessment, the saline soaked gauze was removed from the femora and it was placed into the polycarbonate holding device The femora wa s placed in position within the holding device and aligne d with the specific landmarks for the condyles A test scan was performed to verify the correct positioning of the femur within the holder device, prior to the fina l scan being performed Cross sections of the femoral diaphysis and meta physis were scanned by p eripheral quantit ative computerized tomography (p QCT ) The left femoral diaphysis and metaphysis were scanned with a Stratec XCT Research M Instrument (Norland Medical Systems,Fort Atkinson, WI). Scans were performed at a dis tance of 5mm (metaphysis) a nd 18 mm (diaphysis) proximal to the distal end of the femur for total, trabecular, and cortical bone area (mm), content (mg/mm), and density (mg/cm) Statistical Analysis A One Way ANOVA using SPSS (version 18 ) were utilized to examine differences betwee n treatments on normally distributed data for the variables of muscle mass and

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53 strength, bone mass, strength and cross sectional area, serum sex hormones, bone hormone concentrations and prostate mass All results were reported as Mean SE Prior to anlay sis, outcome variables were tested for normality If the recorded data for the primary outcome variables were unable to be transformed and meet the conditions of a normal distribution a non parametric test were performed Non parametric data was assessed b y either the Kruskal Wallis or Mann Whitney test for final analyses A power analysis based on previous work from our laboratory ( 11 ) comparing group means betwee n testosterone administration, and ORX+Vehicle animals during a 28 day intervention utilizing pooled standard deviations, powered at 80%, with a type I error of 0.05% was conducted for the following primary outcome variables: femoral load, femoral mass, t ibial mass, plantaris muscle mass, and prostate mass Similarly, a power analysis was constructed for progressive resistance training (i.e., weighted ladder climbing) based on the work Lee and colleagues ( 61 ) evaluating hypertrophy in the flexor hallicus longus The muscle hypertrophic response to trenbolone was based on the findings of Thompson and colleagues based on a lower dose of trenbolone resulting in an 8% increase in gastrocnemius mass and a 24% increase in the mass of the semimembranosus ( 35 ) The estimated minimal group sizes necessary for to achieve statistical and clinicall y relevant data from the intervention groups (ORX+T, ORX+Tren, ORX+PRT) are reported as follows (Group size/% Expected change: femoral load (n=13/10%), femoral mass (n=6/10%), tibial mass (n=6/10%), plantaris mass (with T) (n=9/15%), gastrocnemius mass (with TREN) (n=11/8%), semimembranosus (with TREN) (n =10/15%), prostate growth n=10/10%) In addition, the extension of the ORX treatment period by two weeks from the

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54 preliminary data utilized for the power analysis is expected to induce a greater osteope anic and sarcopenic effect Therefore, group sizes were set at N=10/group to ensure adequate power >80%, for the expected effect size of treatment and an alpha of p<0.05 for the primary outcome variables.

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55 CHAPTER 4 RESULTS Body W eights at Sacrifice Admi nistration of testosterone enanthate significantly lowered overall body mass compared to control animals At sacrifice, the body weights were 443.5 17.1 g (SHAM), 419.9 12.5 g (ORX), 404.0 5.17 g (ORX+EX), 404.8 10.9 g (ORX+T), and 412.7 7.64 g ( ORX+TR) (see Figure 4 1) Orchiectomy resulted in a non significant reduction in body weight of 5.3% in the ORX (p>0.05 ) group and significan tly decreased bodyweight by 8.9% ORX+EX com pared with SHAM at sacrifice (p< 0.05). ORX+TR treated animals body weigh ts were 6.9% below SHAM animals, however this reduction was not statistically significant (p>0.05) Whereas, ORX+T resul ted in a significant reduction 8.7% in body weight comp ared to SHAM animals (p<0.05). Removed testes and epididymal fat weights were sim ilar between surgical groups 15.5 0.3 g (ORX), 14.8 0.3 g (ORX+EX), 15.1 0.1 g (ORX+T), 14.9 0.2 g (ORX+TR) (p>0.05), removed weights represented approximately 3% of pre surgical body mass All time course comparisons are based on post surgical bo dyweight Following six weeks of treatment, ORX+TR animals were not significantly different than ORX+V, ORX+EX, or ORX+T animals (p>0.05). Similarly, ORX+T animals were not statistically different from ORX+V, ORX+EX, or ORX+TR (p>0.05). Treatment Effects o n Serum T Intramuscular injections of testosterone enanthate at 7.0 mg week 1 resulted in supraphysiologic concentrations (6 fold) above SHAM Notably, intramuscular injections of trenbolone enanthate at 1.0 mg week 1 maintained testosterone levels simila r to the other castrated groups. Following six weeks of treatment, serum testosterone levels

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56 were 2.46 0.51 ng/mL (SHAM), 0.12 0.05 ng/mL (ORX+V), 0.06 0.03 ng/mL (ORX+EX), 14.8 2.02 ng/mL (ORX+T), and 0.20 0.15 ng/mL (ORX+TR) (see Figure 4 2) O RX+V animals serum testosterone levels were significantly lower 95% than SHAM six weeks following surgery (p<0.05) Similarly, ORX+EX animals were significantly lower 98% than SHAM (p<0.05) Also, ORX+TR ani mals were significantly lower 92% than SHAM (p<0. 05) In contrast, the superphysiologic administration of testosterone enanthate i n the ORX+T resulted in (6 fold ) increase of serum testosterone levels compared to SHAM (p<0.01) ORX+TR treated animals were signifi cantly lower 99% than ORX+T treated animal s (p<0.05). Serum levels of T were not significantly different between ORX+TR, ORX+V, and ORX+EX animals (p>0.05) However, serum levels of T in the ORX+T treated group were significantly greater than OVX+V ( 10 fold) and OVX+EX (20 fold ) treated animals (P <0.01) Treatment Effects on Serum DHT Intramuscular injections of testosterone enanthate at 7.0 mg week 1 resulted in supraphysiologic concentrations (~9 times control) of serum dihydrotestosterone in orchiectomized male rats. Following six weeks of tre atment, serum DHT levels were 461.1 70.6 pg/mL (SHAM), 112.7 14.0 pg/mL (ORX+V), 99.0 13.2 pg/mL (ORX+EX), 4427.0 383.4 pg/mL (ORX+T), and 62.7 11.6 p g/mL (ORX+TR) (see Figure 4 3) At sacrifice, OVX+V ser um DHT levels were reduced by 75.6% compa red to SHAM (p<0.01) OVX+EX animals had a similar red uction in serum DHT levels of 78.5% (p<0.01) ORX+TR treated an imals had significantly lower 86.4% serum DHT levels compared to SHAM (p<0.01) Indeed, treatment with T significantly inc reased serum DHT levels by (9 fold ) compared to SHAM conditions (p<0.01) OVX+TR animals had significantly lower seru m DHT levels than OVX+V 44.4% and OVX+EX 36.7%

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57 (p<0.05) Also, OVX+TR had lower 99% serum DHT levels compared to ORX+T treated animals (p<0.01) Whereas, se rum DHT in the group undergoing testosterone suppl ementation (ORX+T) were (4 fold ) greater than ORX+V and were similarly incre ased compared to ORX+EX (4 fold ) (p<0.01) Serum DHT levels were not significantly different in ORX+EX animals compared to ORX+V ( p>0.05) Treatment Effects on Serum TREN Intramuscular injections of trenbolone enanthate at 1.0 mg week 1 resulted in elevated serum trenbolone concentrations in orchiectomized male rats Following six weeks of treatment, serum TREN levels were 0.72 0 .21 ng/mL (SHAM), 0.64 0.16 ng/mL (ORX+V), 0.51 0.08 ng/mL (ORX+EX), 1.11 0.27 ng/mL (ORX+T), and 4.77 0.42 ng/mL (ORX+TR) (see Figure 4 4) Administration of synthetic TREN resulted in significantly elevated levels of serum tren bolone compared to SHAM (6 fold), ORX+V (6 fold), ORX+EX (8 fold), and ORX+T (3 fold ) treated animals (p<0.05) Serum TREN levels were not significantly different in ORX+EX animals compared to ORX+V (p>0.05) Since, trenbolone is an administered synthetic analog of testoster one, no trenbolone should be present in the SHAM, ORX+V, ORX+EX, or ORX+T groups. A small percentage (3%) of cross reactivity is present between trenbolone and testosterone at a physiologic concentration of 3.2 ng/mL as reported by the manufacturer (Neogen Corporation, Lexington, KY) The SHAM group had serum T values with a mean of 2.5 ng/mL However superphysiologic administration of testosterone in these animals resulted in mean serum T values nearly 4.6 times the stated concentration used by the manufa cturer to determine cross reactivity which may account for the apparent presence of trenbolone in these groups. In contrast, the cross reactivity of trenbolone is negligible when assayed on the testosterone assay.

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58 Treatment Effects on Retroperitoneal Fat M ass Androgen administration significantly reduced retroperitoneal fat pad mass, whereas progressive resistance in the hypogonadal male rat model tended to increase fat pad mass. At sacrifice, retroperitoneal fat pad masses were 3.26 0.33 g (SHAM), 4.06 0.20 g (ORX+V), 3.77 0.27g (ORX+EX), 2.25 0.08g (ORX+T), and 2.84 0.12 g (ORX+TR) (see Figure 4 5) ORX+V animals experienced a 24 .7 % increase in fat pad mass six weeks following surgery compared to SHAM animals, however this was not statistically significant (p =0.052) Similarly, ORX+EX animals increased retroperitoneal fat pad mass by 15.9% compared to SHAM, however this was not statistically significant(p>0.05) Indeed, significant decreases in retroperitoneal fat pad mass were evident for ORX+TR EN 12.7 % and ORX+T 31% compared to SHAM animals ( p < 0.05 ) In a similar fashion androgen treatment with TREN sig nificantly decreased fat pad mass 30% compared to the ORX+V treatment group ( p<0.01). TREN treatment decrease d fat pad mass 25% co mpared to ORX +EX (p<0.01) The ORX+T treatment significantly reduced retroper itoneal fat pad mass by 44.7% compared to the ORX+V group (p<0.01) A similar reduction in retroperitoneal fat pad mass by the ORX+T group 40.5% was observed when compared to the ORX+EX group (p<0.05) ORX+T animals experienced significant retroperitoneal fat loss compared ORX+TR animals (p<0.01) Weighted ladder climbing had no effect in altering retroperitoneal fat pad mass compared to ORX+V animals (p>0.05). Treatment Effects on Hemoglobin at Sacrifice Androgen administration of testosterone and trenbolone enanthate significantly elevated circulating hemoglobin levels by the conclusion of the study. In contrast, progressive resistance exercise in the hypogonadal male rat model tended to mai ntain

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59 circulating hemoglobin at control levels At sacrifice, hemoglobin values were 15.28 0.44 g /dL (SHAM), 15.18 0.25 g /dL (ORX+V), 15.16 0.26 g /dL (ORX+EX), 16.79 0.16 g /dL (ORX+T), and 16.98 0.33 g /dL (ORX+TR) (see Figure 4 6) Following six weeks of orchiectomy no significant changes in hemoglobin concentrations were present for either vehicle injections ORX+V or ORX+EX compared to SHAM (p>0.05) Whereas, ORX+TR treated animals had a si gnificant elevation of 11.1% in circulating hemoglobin l evels compared to SHAM (p<0.01). Similarly, ORX+T resulted in a significant elevation 9.9% in circulating hemoglobin levels compared to SHAM (p<0.01). Androgen administration of ORX+TR and ORX+T had similar responses for hemoglobin concentration (p>0.05). ORX+T administration resulted in significant elevations of circulating hemoglobin c ompared to ORX+V 10.6% and ORX+EX 10.8%, (p<0.01) respectively. Weighted ladder climbing (ORX+EX) had no significant effect on circulating hemoglobin compared to vehicle inj ections (ORX+V) (p>0.05). Time Course of Treatment Effects on Hemoglobin Androgen administration of testosterone and trenbolone enanthate significantly elevated circulating hemoglobin levels by week four of the study, with non significant elevations from week four to six. Hemoglobin (Hb) levels were not significantly different for any treatment groups when assessed at week 0 and week 2 of the experimental period (p>0.05) (see Figure 4 7) At week 4, significant elevat ions were evident for ORX+TREN 9% and ORX+ T 8% treatment groups compar ed to SHAM and both ORX groups 11% vs 1 0% respectively ( p<0.01). However, there were no significant differences between the androgen treatment (ORX+TR and ORX+T) groups at week 4 (p>0.05) In addition, e levations in hemogl obin levels were present in both androgen administration groups compared to SHAM and both ORX groups at week 6 (p<0.0 1 )

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60 Weighted ladder climbing exerted no measurable effect on Hb levels over SHAM or ORX+V treatments (p>0.05). Treatment Effects on the Lev ator Ani Bulbocavernosus Complex (LABC) Androgen administration of testosterone and trenbolone enanthate to the highly androgen sensitive levator ani bulbocavernosus muscle resulted in significant hypertrophy. In contrast, orchiectomy and orchiectomy coup led with exercise resulted in significant reductions in the LABC. At the conclusion of the experiment LABC masses were 1.07 0.04 g (SHAM), 0.68 0.03 g (ORX+V), 0.64 0.02 g (ORX+EX), 1.54 0.03 g (ORX+T), and 1.54 0.04 g (ORX+TR) (see Figure 4 8) ORX+V and ORX+EX animals had a significant reduction in levator a ni bulbocavernosus (LABC) mass 3 6.2 % and 40%, respectively compared to SHAM animals (p<0.01) However, ORX+TR and ORX+T animals resulted in significant myotrophic growth of the LABC compar ed to SHAM 4 4.5 % and + 44 .1% respectively Moreover, ORX+TR and ORX+T treated animals experienced a similar increase 126% in LABC mass compared to both ORX+V and ORX+EX conditions (p<0.01). ORX+EX had a non significant 6% reduction in LABC masses compared to ORX+V and treated groups (p>0.05) Similarly, androgen administered groups ORX+TR and ORX+ T had similar effects on LABC mass (p>0.05) Treatment Effects on Prostate Mass Androgen administration of testosterone enanthate significantly increased the m ass of the prostate compared to control and orchiectomized groups. Trenbolone enathate exhibited a non significant reduction in prostate mass compared to control conditions. Androgen ablation through orchiectomy and orchiectomy coupled with exercise result ed in significant reductions in prostate mass compared to controls. Prostate masses were the following: 0.231 0.02 g (SHAM), 0.066 0.02 g (ORX+V),

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61 0.66 0.01 g (ORX+EX), 0.513 0.04 g (ORX+T), and 0.185 0.02 g (ORX+TR) (see Figure 4 9) ORX+V and ORX+EX animals prostate masses were significantly smaller 71.5% and 71.6%, respectively, at six weeks following surgery compared to SHAM (p<0.01). A non significant reduction of 20% in prostate mass was observed for ORX+TR animals compare d to SHAM (p=0.12 ). In contrast androgen treated ORX+T animals had prostate mass was significantly increased by 122% compared to SHAM (p<0.01). Androgen treatment with TREN resulted in prostate masses 180% vs. +181%, compared to the ORX+V and ORX+EX, respectively (p<0.01). Androgen treatment with T, significantly increased prostate mass (6 fold) compared to the ORX+V and ORX+EX groups, respectively (p<0.01). ORX+TR had significantly smaller prostate masses 64% compared to ORX+T (p<0.01). Weighted ladder climbing had no impa ct significant impact on prostate mass compared to the ORX+V treatment group (p>0.05). Treatment Effects on Kidney Mass at Sac rifice Androgen administration of testosterone and trenbolone enanthate maintained kidney mass at control levels. In contrast, o rchiectomy and orchiectomy coupled with exercise resulted in significant reductions in kidney mass K idney masses were 1.12 0.04g (SHAM), 0.97 0.02 g (ORX+V), 0.96 0.02 g (ORX+EX), 1.20 0.04 g (ORX+T), and 1.15 0.02 g (ORX+TR) (see Figure 4 10) After 6 weeks of ORX conditions, ORX+V kidney masses were significantly decreased 12.5% compared to SHAM (p<0.05) ORX+EX animals kidneys were significantly decreased 14.2% compared to SHAM conditions (p<0.05). Although not statistically significant both a ndrogen treated groups ORX+T and ORX+TR group increased kidney mass by 7.5% vs. +3.3% compared to the SHAM group (p<0.10 ). ORX+TR significa ntly increased kidney mass 18.1% compared to ORX+V (p<0.01). Similarly, ORX+T treatmen t

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62 increased kidney mass 22.9% a bove ORX+V conditions (p<0.01). Both ORX+TR a nd ORX+T increased kidney mass 20.4% vs. 25.2% compared to the ORX+EX group, respectively (p<0.01). There was no difference in kidney mass response between ORX+TR and ORX+T animals (p>0.05). Treatment Effect on Hindlimb Muscles Mass There was no significant effect of androgen administration of testosterone enanthate, trenbolone enanthate, orchiectomy or orchiectomy coupled with exercise on hindlimb muscle mass At sacrifice, semimembranosus values were 0.39 0.01g (SHAM), 0.38 0.01 g (ORX+V), 0.38 0.01 g (ORX+EX), 0.38 0.01 g (ORX+T), and 0.40 0.01 g (ORX+TR) (see Figure 4 11) Plantaris masses were 0.44 0.02 g (SHAM), 0.40 0.02 g (ORX+V), 0.42 0.01 g (ORX+EX), 0.40 0.01 g (ORX+T), and 0.44 0 .02 g (ORX+TR) (see Figure 4 12) Soleus masses were 0.18 0.01 g (SHAM), 0.16 0.01 g (ORX+V), 0.16 0.01 g (ORX+EX), 0.16 0.01g (ORX+T), and 0.17 0.01 g (ORX+TR) (see Figure 4 13) Flexor hallicus longus masses were 0.55 0.03 g (SHAM), 0.52 0.0 3 g (ORX+V), 0.50 0.04 g (ORX+EX), 0.53 0.02 g (ORX+T), and 0.52 0.03 g (ORX+TR) (see Figure 4 14) ORX+V treatment did not alter muscle wet weight of the flexor hallicus longus, semimembranosus, soleus, or plantaris compared to SHAM or ORX+EX condi tions (p>0.05) Androgen treatment (ORX+TREN and ORX+T) did not alter muscle wet weight of the flexor hallicus longus, semimembranosus, soleus, or plantaris compared to ORX, ORX+EX, or SHAM (p>0.05) Interestingly, weighted ladder climbing (ORX+EX) did not increase wet muscle weights compared to any treatment conditions (p>0.05).

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63 Grip Strength Neither androgen administration nor progressive resistance training significantly improved grip strength values in the hypogonadal male rats. The grip strength values were 1.72 0.07 kg (SHAM), 1.83 0.11 kg (ORX), 1.76 0.08 kg (ORX+EX), 1.94 0.08 kg (ORX+T), and 1.83 0.08 kg (ORX+TR) (see Figure 4 15) Group comparisons of forelimb strength found no difference between SHAM, ORX+V, ORX+EX, ORX+TREN or ORX+T (p> 0.05). Progressive Resistance Training (Weighted Ladder Climbing) Orchiectomized male rats improved the total load (body weight+tail weight summed across climbs) carried over the course of the study. One week following ORX surgery, animals in the weighted ladder climbing group ascended the ladder with an average weekly load of 0.41 0.01 kg (Week 1), 1.47 0.13 kg (Week 2), 1.88 0.18 kg (Week 3), 2.51 0.37 kg (Week 4) and 3.00 0.40 kg (Week 5) (see Figure 4 16) Load values increased significant ly 6 31% from Week 1 to Week 5 of the training intervention (p<0.01). Training loads significantly increased 285% between Week 1 and Week 2 (p<0.05). However, training loads did not significantly increase between Week 2 and Week 3 (p>0.0 5) and tended to increas e 33% between Week 2 and Week 4 (p=0.054) Training load was significantly increased between Week 1 and Week 3 of the study (p<0.05) Although, Week 2 training loa d was significantly increased 104% by Week 5 of the study (p<0.01) Similarly, training load did not significantly increase between Week 3 and Week 4 (p>0.05), however Week 3 training loa d was significantly increased 60% by Week 5 (p<0.05) Lastly, training load did not significantly increase between Week 4 and Week 5 of the intervention (p>0.05).

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64 During week one of the study animals ascended the ladder carrying their bodyweight. In week two, the animals carried an average of 72.4 12.2 g attached to their tail during their repeated ladder climbs. Weight increased during week three, with the anima ls 98.5 12.7 g, and further increased the weight in week four to 137.0 14.2 g. Lastly, tail weight increased by week five to 175.0 22.0 g Treatment Effects on Femoral and Tibial Bone Mass and Length There was no significant effect of administratio n of testosterone enanthate, trenbolone enanthate, orchiectomy or orchiectomy coupled with exercise on femur or tibial mass or length. Femur weights were 1.01 0.03 g (SHAM), 1.07 0.02 g (ORX), 1.05 0.02 g (ORX+EX), 1.08 0.04 g (ORX+T), and 1.13 0.04 g (ORX+TR). Femur lengths were 40.3 0.36 mm (SHAM), 40.1 0.23 mm (ORX), 39.9 0.25 mm (ORX+EX), 40.0 0.31 mm (ORX+T), and 40.5 0.29 mm (ORX+TR). Tibia weights were 0.83 0.03 g (SHAM), 0.83 0.02 g (ORX), 0.82 0.02 g (ORX+EX), 0.85 0.03 g (ORX+T), and 0.86 0.02 g (ORX+TR). Tibia lengths were 43.1 0.45 mm (SHAM), 42.8 0.33 mm (ORX), 42.8 0.30 mm (ORX+EX), 42.6 0.24 mm (ORX+T), and 43.4 0.27 mm (ORX+TR) (see Figure 4 16 through 4 20) ORX+V conditions did not significantly dec rease femoral bone mass or length (p>0.05). Androgen treatment (ORX+TREN and ORX+T) did not significantly increase femoral or tibial bone mass or length compared to other treatment conditions (SHAM, ORX+V, ORX+EX) ( p >0.05) No differences existed between t he response of ORX+TREN or ORX+T for femoral or tibial bone mass or length (p>0.05). Exercise training (ORX+EX) did not induce significant changes in femoral or tibial mass or length compared to any treatment following six weeks of the intervention (p>0.05 ).

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65 Serum Osteocalcin at Sacrifice A dministration of testosterone and trenbolone enanthate resulted in significant reduction in circulating serum osteocalcin levels. In contrast, orchicectomy resulted in increased levels of circulating serum osteocalcin, w hereas orchiectomy coupled with exercise maintained serum osteocalcin at a level similar to controls. At sacrifice serum osteocalcin values were 167.4 11.4 ng/ml (SHAM), 255.5 11.1 ng/ml (ORX), 199.6 15.5 ng/ml (ORX+EX), 82.1 7.13 ng/ml (ORX+T), an d 101.1 9.0 ng/ml (ORX+TR) (see Figure 4 21) Following six weeks of treatment, SHAM an imals had significantly 52.7% lower levels of serum osteocalcin than ORX+V treated animals (p<0.01) ORX+EX, serum osteocalcin levels were 19% higher than SHAM animals although these values did not reach statistical significance (p>0.05) ORX+TREN animals were lower 39.6% than SHAM (p<0.05). A lso, ORX+T animals were lower 50.9% than SHAM (p<0.05) ORX+TR animals were lower 60.4% than OVX+V animals (p<0.05) Similarly, O RX+TR animals were also lower 4 9.4% than OVX+EX (p<0.05) The ORX+T trea tment was significantly lower 67.9% compared to the ORX+V group (p<0.05) There was no significant difference between ORX+TREN or ORX+T treatment for serum osteocalcin (p>0.05). Serum osteocalcin levels were sig nificantly lower 21.9% in ORX+EX animals compared to ORX+V (p<0.05) Serum Trap 5b at Sacrifice Androgen administration of testosterone and trenbolone enanthate resulted in significant reduction in Trap 5b levels. While orchiec tomy did not significantly elevate Trap 5b above SHAM Trap 5 b levels in ORX+V animals was significantly higher than in androgen treated animals. At sacrifice serum Trap 5b values were 5.22 0.38 U/L (SHAM), 5.94 0.68 U/L (ORX), 5.89 0.78 U/L (ORX+E X), 3.01 0.17 U/L (ORX+T),

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66 and 3. 29 0.30 U/L (ORX+TR) (see Figure 4 22) At s ix weeks following surgery serum Trap 5b levels in SHAM animals were not significantly different than ORX+V, and ORX+EX animals (p>0.05) In SHAM animals Trab 5b levels were 37.0% highe r than in ORX+TR animals (p<0.01) Simil arly, SHAM animals had 42.3% higher serum Trap 5b levels compared to ORX+T treated animals (p<0.01). ORX+TR ani mals were significantly 40% lower compared to ORX+V and ORX+EX groups (p<0.01) ORX+T animals had s ignificantly lower Trap 5b serum levels compared to both ORX+V and ORX+EX, (p<0.01) ORX+ TR and ORX+T had similar effects on lowering serum Trap 5b values compared to the other treatment groups (p>0.05). Treatment Effects on Bone Biomechanical Charact eristics Femoral Neck A dministration of trenbolone resulted in increased maximum load values compared to ORX+EX animals, and non significantly increased maximum load va lues above ORX+V animals Supraphysiologic testosterone enanthate tended to increase ma ximum load of the femoral neck compared to ORX+EX and was not significantly stronger than ORX+V animals At sacrifice femoral neck maximum load values in Newtons (N) were : 206.2 3.13 (SHAM), 196.3 3.72 (ORX +V ), 178.9 3.12 (ORX+EX), 212.9 2.13 (O RX +T), and 22 7.8 1.74 (ORX+TR ) (see Figure 23 24) S ix weeks following surgery serum femoral neck maximum load in SHAM animals were not significantly different than an y treatment group (p>0.05) ORX+TR animals had significantly strong er 22% femoral necks than ORX+EX (p<0.01), but only tended to increase 14% femoral neck maximum load values compared to ORX+V (p=0.1 1) Also, ORX+T tended to have higher maximum load values for the fe moral neck compared to

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67 ORX+EX 16% (p=0.08), but were not significantly differ ent than ORX+V (p>0.05) There was no significant difference between ORX+T and ORX+TR treated animals (p>0.05). Femoral Midshaft Androgen administration and progressive resistance training had no significant effect on femoral midshaft maximum load. At sacr ifice femoral neck maximum load values in Newtons (N) were : 172.5 3.04 (SHAM), 161.1 3.51 (ORX+V), 164.0 1.77 (ORX+EX), 164.9 2.85 (O RX+T), and 164.0 2.34 (ORX+TR ) A slight non significant reduction in femoral midshaft maximum load was present f or all ORX groups ranging between 4 7% (p>0.05). Centralized and Internalized N uclei of the FHL and Semimembransosus The initial procedures froze the flexor hallicus and semimembranosus muscle tissue (n=6 7/group) directly in liquid nitrogen cooled isope ntane The direct immersion of O.C.T coated muscle tissue into liquid isopentane resulted in freeze artifact preventing the acquisition of acceptable histology samples Procedures were adapted for the remaining animals (n=3 4/group) so that muscle tissue w as frozen on an isopentane slurry Although the number of animals initially proposed in the power analysis were unavailable for immunohistochemical analyses a smaller sample was available for analysis The small sample size points to potential trends for internalized nuclei in the ORX EX, ORX+T and ORX+TR groups in the flexor hallicus longus, but not the semiembranosus At sacrifice internalized nuclei counts (expressed per 100 fibers) of the FHL were as follows: 5.75 1.8 nuclei (SHAM), 4.50 2.0 nuclei (ORX), 12.0 3.9 nuclei (ORX+EX), 7.83 2.3 nuclei (ORX+T), and 10.0 3.3 nuclei (ORX+TR) At sacrifice centralized nuclei counts (expressed per 100 fibers) of the FHL, 1.25 0.63 nuclei (SHAM), 1.50 1.5 nuclei (ORX), 1.83 1.4 nuclei (ORX+EX), 2.1 7 0.60 nuclei

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68 (ORX+T), and 0.83 0.83 nuclei (ORX+TR) (see Figure 4 25 and 27) Following six weeks of treatment, no significant group differences were detected for the presence of centralized nuclei or internalized nuclei in the flexor hallicus muscle g roup of these anima ls (p >0.05). At sacrifice internalized nuclei counts (expressed per 100 fibers) of the SEMI were as follows: 6.25 1.7 nuclei (SHAM), 5.83 1.3 nuclei (ORX), 10.2 5.4 nuclei (ORX+EX), 4.00 0.58 nuclei (ORX+T), and 8.00 0.29 nucle i (ORX+TR) At sacrifice, centralized nuclei counts (expressed per 100 fibers) of the SEMI, 0.88 0.31 nuclei (SHAM), 2.50 0.76 nuclei (ORX), 2.5 1.3 nuclei (ORX+EX), 0.17 0.17 nuclei (ORX+T), and 1.5 0.76 nuclei (ORX+TR) (see Figure 26 and 28) Fo llowing six weeks of treatment, no significant group differences were detected for the presence of centralized nuclei or internalized nuclei in the semimembransosus muscle group of these animals (p >0.05). Pax 7 Positive Nuclei of the FHL and Semimembranosu s Because of freeze artifacts only 3 4 samples/group were suitable for analysis Treatment with androgens had no significant effects on the presence of Pax 7 positive nuclei in the flexor hallicus longus Similarly, progressive resistance exercise did not increase Pax 7 positive nuclei populations in either muscle, and may have negatively impacted Pax 7 expression in the flexor hallicus longus Flexor Hallicus Longus Treatment with androgens had no significant effects on the presence of Pax 7 positive nuc lei in the flexor hallicus longus. Similarly, progressive resistance exercise did not effect Pax 7 positive nuclei populations in the primary mover of weighted ladder climbing the flexor hallicus longus. At sacrifice Pax 7 positive nuclei (expressed per 10 0

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69 fibers) of the FHL were as follows: 14.3 1.0 nuclei (SHAM), 11.3 3.8 nuclei (ORX), 7.8 2.4 nuclei (ORX+EX), 13.5 2.0 nuclei (ORX+T), and 11.5 5.5 nuclei (ORX+TR) (see Figure 4 29) At sacrifice Pax 7 positive nuclei counts (expressed as percent age of Pax 7 positive nuclei/ total myonuclei counts per 100 fibers) of the SEMI, 3.7 0.43 % (SHAM), 2.3 0.60 % (ORX), 3.3 0.89% (ORX+EX), 4.1 0.95 % (ORX+T), and 2.6 0.92% (ORX+TR). No significant differences were detected between treatment grou ps for Pax7 positive nuclei of the FHL expressed per 100 fibers or as a percentage of total myonuclei per 100 fibers (p>0.05). Semimembranosus Treatment with androgens had no significant effects on the presence of Pax 7 positive nuclei in the semimembransu s Similarly, progressive resistance exercise did not effect Pax 7 positive nuclei populations. At sacrifice Pax 7 positive nuclei (expressed per 100 fibers) of the SEMI were as follows: 20.4 6.2 nuclei (SHAM), 15.8 2.9 nuclei (ORX), 21.0 6.4 nuclei (ORX+EX), 44.2 17.0 nuclei (ORX+T), and 42.0 8.9 nuclei (ORX+TR) (see Figure 4 30) At sacrifice Pax 7 positive nuclei counts (expressed as percentage of Pax 7 positive nuclei/ total myonuclei counts per 100 fibers) of the SEMI, 1.9 0.38 % (SHAM), 3. 9 0.72 % (ORX), 5.3 1.6 % (ORX+EX), 4.1 0.95 % (ORX+T), and 3.7 0.64 % (ORX+TR). No significant differences were detected between treatment groups for Pax7 positive nuclei of the SEMI expressed per 100 fibers or as a percentage of total myonuclei p er 100 fibers (p>0.05). Treatment Effects on Cross Sectional Area of the Flexor Hallicus Longus Although the number of animals initially proposed in the power analysis were unavailable for immunohistochemical analyses; a small sample size identified potent ial trends of increased cross sectional area of Type IIa in ORX+EX animals participating in

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70 weighted ladder climbing.Type IIbx fiber cross sectional appear to be preserved in ORX+T for the flexor hallicus longus, but appears to decline in ORX+EX treated a nimals (see Figure 4 31) Treatment Effects on Cross Sectional Area of the Semimembranosus Although the number of animals initially proposed in the power analysis were unavailable for immunohistochemical analyses; a small sample size identified potential trends in Type IIa fiber preservation in ORX+T and ORX+TR, whereas Type IIa fiber cross sectional area tends to decrease in ORX+EX animals All treatement groups tend to provide partial protection to Type IIbx fiber cross sectional area compared to ORX (se e Figure 4 32) Treatment Effects on 5mm pQCT (Metaphysis) Total Mineral Content, Total Density, Total Area, Trabecular Area, Cortical Content, Cortical Area Group comparisons revealed no significant differences for the 5mm pQCT outcome variables of total mineral content, total density, total area, trabecular area, cortical content, and cortical area. Significant group differences were detected for the following variables: trabecular content, trabecular density, and cortical density. Total bone content val ues were 14.5 0.60 m g /mm (SHAM), 13.2 0.30 m g /mm (ORX), 13.6 0.52 m g /mm (ORX+EX), 13.5 0.37 m g /mm (ORX+T), and 13.9 0.04 m g /mm (ORX+TR) (see Figure 4 33) Total femoral density values were 668.1 10.74 m g /mm 2 (SHAM), 631.5 7.56 m g /mm 2 (ORX), 6 62.4 12.0 m g /mm 2 (ORX+EX), 672.2 12.5 m g /mm 2 (ORX+T), and 669.1 11.1 m g /mm 2 (ORX+TR) (see Figure 4 34). Total area values at the 5mm landmark of the femur were 21.8 0.80 mm 2 (SHAM), 21.0 0.52 mm 2 (ORX), 20.6 0.72 mm 2 (ORX+EX), 20.0 0.32 mm 2 ( ORX+T), and 20.8 0.43

PAGE 71

71 mm 2 (ORX+TR). Trabecular area values at the 5mm landmark of the femur were 6.51 0.24 mm 2 (SHAM), 6.29 0.16 mm 2 (ORX), 6.17 0.21 mm 2 (ORX+EX), 6.00 0.10 mm 2 (ORX+T), and 6.22 0.13 mm 2 (ORX+TR) (see Figure 4 35 through 38) Whereas, cortical bone content values at the 5mm landmark of the femur 9.60 0.55 m g (SHAM), 8.50 0.23 m g (ORX), 9.26 0.48 m g (ORX+EX), 8.93 0.38 m g (ORX+T), and 9.13 0.43 m g (ORX+TR) (see Figure 4 39) Trabecular Content (CNT TRB ) Trabecular cont ent values at the 5mm landmark of the femur were 1.86 0.09 m g /mm (SHAM), 1.41 0.06 m g /mm (ORX+V), 1.49 0.09 m g /mm (ORX+EX), 1.71 0.09 m g /mm (ORX+T), and 1.77 0.09 m g /mm (ORX+TR) (see Figure 4 35 through 38). Six weeks following surgery femurs ev aluated from ORX+V and ORX+EX animals had lower CNT TRB 31.9% and 24.8% respectively, than SHAM animals (p<0.05) ORX+TR and ORX+T maintained CNT TRB similar to SHAM conditions (p>0.05) O RX+TR femurs maintained 25.5% greater CNT TRB than ORX+V femurs (p<0.05 ) Notably, ORX+TR animals maintained CNT TRB at an 18.8% higher level than ORX+EX animals, however this was not statistically significant (p=0.10) A similar pattern was seen for CNT TRB with ORX+T animals retaining 15.6% greater content compared to ORX+V (p<0.05) Wher eas, ORX+T maintained a 14.8% greater CNT TRB when compared to ORX+EX, however this was not statistically significant (p=0.09) Weighted ladder climbing did not conserve CNT TRB in excess of ORX+V conditions (p>0.05). There were no significant differences between ORX+T and ORX+TREN for CNT TRB (p>0.05).

PAGE 72

72 Trabecular Density (TRAB DEN ) Trabecular density values at the 5mm landmark of the femur were 285.6 8.74 m g /mm 2 (SHAM), 225.4 8.92 m g /mm 2 (ORX+V), 242.0 10.6 m g /mm 2 (ORX+EX), 285.1 13.0 m g / mm 2 (ORX+T), and 283.4 11.2 m g /mm 2 (ORX+TR) (see Figure 4 35 through 38). At the end of the study, femurs evaluated from ORX+V and ORX+EX animals had lower TRAB DEN 26.7% and 18.0% respectively, than SHAM animals (p<0.05) ORX+TR and ORX+T maintained TRA B DEN similar to SHAM conditions (p>0.05) O RX+TR femurs maintained 25.7% greater TRAB DEN than ORX+V femurs (p<0.05). Similarly, ORX+TR animals maintained TRAB DEN 17.1% higher than ORX+EX animals (p<0.05) Assessment of TRAB DEN found ORX+T animals retaining 26.4% greater density compared to ORX+V (p<0.05) Wher eas, ORX+T maintained a 17.8% greater TRAB DEN when compared to ORX+EX (p<0.05) Weighted ladder climbing did not protect against losses in TRAB DEN compared to ORX+V conditions (p>0.05). There were no s ignificant differences between ORX+T and ORX+TREN for TRAB DEN (p>0.05). Cortical Density (CRT DEN ) Cortical density values at the 5mm landmark of the femur 960 6.70 g (SHAM), 995.8 7.92 m g /mm 2 (ORX), 999.6 12.2 m g /mm 2 (ORX+EX), 972.7 6.20 m g /mm 2 (O RX+T), and 974.8 6.67 m g /mm 2 (ORX+TR) (see figure 4 40) At the conclusion of the study, femurs evaluated from ORX+V and ORX+EX animals had higher CRT DEN 3.77% and +4.1% respectively, than SHAM animals (p<0.01) ORX+TR and ORX+T maintained CRT DEN simil ar to SHAM conditions (p>0.05) Assessment of CRT DEN found ORX+T an imals with a tendency towards lower density 2.7% compared to ORX+EX (p=0.08). Weighted ladder climbing did not improve CRT DEN compared to

PAGE 73

73 ORX+V conditions (p>0.05). There were no difference s between ORX+T and ORX+TREN for CRT DEN (p>0.05). Treatment Effects on 18 mm pQCT (Diaphysis) No significant main effects were present at the 18mm femoral site for total mineral content, total density, total area, cortical content, cortical density, corti cal area, cortical thickness, periosteal circumference and endosteal circumference (p>0.05) (see Figure 4 42 through 50) Total Mineral Content, Total Density, Total Area Total bone content values at the 18mm site were 11.8 0.18 m g /mm (SHAM), 11.3 0.24 m g /mm (ORX), 11.1 0.23 m g /mm (ORX+EX), 11.4 0.32 m g /mm (ORX+T), and 11.0 0.25 m g /mm (ORX+TR) Total femoral density values at the 18 mm site were 985.9 11.46 m g /mm 2 (SHAM), 992.5 11.64 m g /mm 2 (ORX), 981.2 13.08 m g /mm 2 (ORX+EX), 1010.0 14.40 m g /mm 2 (ORX+T), and 993.48 11.61 m g /mm 2 (ORX+TR) Total bone area values at the 18 mm landmark of the femur were 12.0 0.19 mm 2 (SHAM), 11.4 0.32 mm 2 (ORX), 11.4 0.29 mm 2 (ORX+EX), 11.3 0.27 mm 2 (ORX+T), and 11.1 0.25 mm 2 (ORX+TR) Cortical Con tent, Cortical Density, Cortical Area Cortical bone content values at the 18 mm landmark of the femur 11.5 0.17 m g /mm (SHAM), 11.0 0.22 m g /mm (ORX), 10.9 0.22 m g /mm (ORX+EX), 11.2 0.30 m g /mm (ORX+T), and 9.13 0.43 m g /mm (ORX+TR). Cortical bone de nsity values at the 18 mm landmark of the femur 1390.6 5.09 m g /mm 2 (SHAM), 1388.4 5.40 m g /mm 2 (ORX), 1395.5 5.10 m g /mm 2 (ORX+EX), 1396.6 4.20 m g /mm 2 (ORX+T), and 1398.5 5.39 m g /mm 2 (ORX+TR).Cortical bone area values at the 18

PAGE 74

74 mm landmark of the f emur 8.26 0.12 mm 2 (SHAM), 7.91 0.17 mm 2 (ORX), 7.78 0.16 mm 2 (ORX+EX), 7.99 0.21 mm 2 (ORX+T), and 7.71 0.18 mm 2 (ORX+TR). Cortical Thickness, Periosteal and Endosteal Circumference Cortical bone thickness values at the 18 mm site were 0.87 0. 01 mm (SHAM), 0.85 0.01 mm (ORX), 0.83 0.01 mm (ORX+EX), 0.87 0.02 mm (ORX+T), and 0.85 0.01 mm (ORX+TR).Periosteal circumference values at the 18 mm landmark were 12.26 0.10 mm (SHAM), 11.96 0.17 mm (ORX), 11.94 0.15 mm (ORX+EX), 11.92 0.1 5 mm (ORX+T), and 11.81 0.13 mm (ORX+TR). Endosteal circumference values at the 18mm landmark site were 6.82 0.12 mm (SHAM), 6.60 0.16 mm (ORX), 6.70 0.16 mm (ORX+EX), 6.46 0.12 mm (ORX+T), and 6.52 0.11 mm (ORX+TR) Intraskeletal Hormone Conc entrations Intraskeletal Testosterone Tibial intraskeletal testosterone concentrations were 3.81 1.07 ng/g (SHAM), 2.59 0.72 ng/g (ORX), 2.59 0.79 ng/g (ORX+EX), 11.4 2.51 ng/g (ORX+T), and 2.80 0.78 (ORX+TR) (see Figure 4 51) At the conclusion of the study, there were no significant differences between SHAM, ORX, ORX+EX and ORX+TR ORX+T animals intraskeletal testosterone concentrations were 4 fold greater than all ORX conditions and 3 fold greater than SHAM conditions ORX+T animals were signif icantly greater than all other treatment groups (p<0.01). Intraskeletal DHT Tibial intraskeletal dihydrotestosterone concentrations were 4013.2 204.0 pg/g (SHAM), 3419.3 290.1 pg/g (ORX), 3504.4 214.9 pg/g (ORX+EX), 12413.1 1177.5 pg/g (ORX+T), 368 1.0 164.1 pg/g (ORX+TR) (see Figure 4 52) At the conclusion of the study, there were no significant differences between SHAM, ORX, ORX+EX and

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75 ORX+TR ORX+T animals intraskeletal DHT concentrations were 3.3 fold greater than all ORX conditions and 3 fold greater than SHAM conditions ORX+T animals were significantly greater than all other treatment groups (p<0.01).

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76 FIGURE 4 1 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on bodyweight at sacrifice. V alues are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 2 Effects of ORX+V, ORX+EX, te stosterone enanthate (TE), or trenbolone enanthate (TR) on serum testosterone levels at sacrifice. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. O RX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). a a a a ,b ,c a ,d

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77 FIGURE 4 3 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on serum DHT at sacrifice. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 4 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on serum DHT at sacrifice. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). a a a ,b ,c a Background a*,b*,c*,d* *,

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78 FIGURE 4 5 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on retroperitoneal fat pad mass at sacrifice. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 6. Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on hemoglobin mass at sacrifice. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). a ,b # c # ,e # a*,b* a*,b*,c* a*,b*c*

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79 FIGURE 4 7 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone ena nthate (TR) time course of hemoglobin mass Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR ). FIGURE 4 8 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on levator ani bulbocavernosus muscle mass at sacrifice. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labele d groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). a # a # a # ,b # ,c # a # ,b # ,c # a*,b*,c* a*,b*,c*

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80 FIGURE 4 9 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on prostate mass at sacrifice. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 10 Effects of ORX+V, ORX+EX, testo sterone enanthate (TE), or trenbolone enanthate (TR) on kidney mass at sacrifice. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). a # a # a # ,b # ,c # ,e # b # ,c # ,d # a a b # ,c # b # ,c #

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81 FIGURE 4 11 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on semimembranosus muscle mass at sacrifice. Values are Means SE, n = 9 10/group Letters a e indicate difference s from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 12 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on plantar is muscle mass at sacrifice. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR).

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82 FIGURE 4 13 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on soleus muscle mass at sacrifice. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 ( a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 14 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on flexor hallicus longus muscle mass at sacrifice. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR).

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83 FIGURE 4 15 Effects of ORX+V, ORX+EX, testosterone enanthate (T E), or trenbolone enanthate (TR) on forelimb grip strength at sacrifice. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = v s. ORX + T, e = vs. ORX + TR). FIGURE 4 16 Total weekly training load carried by ORX+EX animals on a 1.1m ladder inclined at 85 Values are Means SE, n = 10 Significant at # p < 0.05 or p < 0.01 (a = vs. Week 1, b = vs. Week 2, c = vs Week 3+ EX, d = vs. Week 4, e = vs. Week 5. a* a* a* ,b* a*,b*,c*

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84 FIGURE 4 17 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on tibial mass at sacrifice. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively lab eled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 18 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on tibial length at sacrifice. Valu es are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR).

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85 FIGURE 4 19 Effects of ORX+V, ORX+EX, test osterone enanthate (TE), or trenbolone enanthate (TR) on femoral mass at sacrifice. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 20 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on femoral length at sacrifice. Values are Means SE, n = 9 10/group Letters a e indicate differences from respe ctively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR).

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86 FIGURE 4 21 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on serum osteocalcin le vels at sacrifice. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 22 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on serum TRAP 5b levels at sacrifice. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). a # b* a b *,c* a b *,c* a # ,b # ,c # a # ,b # ,c #

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87 FIGURE 4 23 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on femoral midshaft maximum load. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 24 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenb olone enanthate (TR) on femoral neck maximum load. Values are Means SE, n = 8 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. OR X + TR). c* c

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88 FIGURE 4 25 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on internalized nuclei of the FHL. Values are Means SE, n = 8 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs. ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 26 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on internalized nuclei of the semimembranosus. Values are Means SE, n = 8 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs. ORX + EX, d = vs. ORX + T, e = vs. ORX + TR).

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89 FIGURE 4 27. Effects of ORX+V, ORX+EX, test osterone enanthate (TE), or trenbolone enanthate (TR) on centralized nuclei of the FHL. Values are Means SE, n = 8 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs. ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 28 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on centralized nuclei of the semimembranosus. Values are Means SE, n = 8 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs. ORX + EX, d = vs. ORX + T, e = vs. ORX + TR).

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90 FIGURE 4 29 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enantha te (TR) on Pax 7+ nuclei expressed per 100 fibers of the flexor hallicus longus Values are Means SE, n = 4/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 30 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on Pax 7+ nuclei expressed per 100 fibers of the semimembranosus Values are Means SE, n = 4/group Letters a e indicat e differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR).

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91 FIGURE 4 31 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate ( TR) on Type IIa fibers of the flexor hallicus longus Values a re Means SE, n = 1 4/group. (A=Type IIa FIBERS); (B =Type IIbx) A. B.

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92 FIGURE 4 32 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on Type IIa fib ers of the flexor hallicus longus Values a re Means SE, n = 1 4/group. (A=Type IIa FIBERS); (B =Type IIbx) B. A.

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93 FIGURE 4 33 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on femoral total bone content at 5mm. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 34 Effects of ORX+V, ORX+EX, testosteron e enanthate (TE), or trenbolone enanthate (TR) on femoral total density at 5mm. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX d = vs. ORX + T, e = vs. ORX + TR).

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94 FIGURE 4 35 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on femoral trabecular content at 5mm. Values are Means SE, n = 9 10/group Letters a e indicate differences from re spectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 36 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on trabecular densit y at 5mm. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). a* a* b* b* a a b ,c* b ,c*

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95 FIGURE 4 37 Effects of ORX+V ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on femoral total area at 5mm. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. OR X, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 38 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on trabecular bone area at 5mm. Values are Means SE, n = 9 10/group Letters a e indicate differenc es from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR).

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96 FIGURE 4 39 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on cortical bone content at 5mm. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 40 Effe cts of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on cortical bone density at 5mm. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. S HAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). a # a #

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97 FIGURE 4 41 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on cortical bone area at 5mm. Values are Means SE, n = 9 10/group Letters a e indi cate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 42 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (T R) on total bone content at 18mm. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR).

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98 FIGUR E 4 43 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on total bone density at 18mm. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 44 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on total bone area at 18mm. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR).

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99 FIGURE 4 45 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enant hate (TR) on cortical bone content at 18mm. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR) FIGURE 4 46 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on cortical bone density at 18mm. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 o r p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR).

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100 FIGURE 4 47 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on cortical bone area at 18mm. Values are Means SE, n = 9 10 /group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 48 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on cortical bone thickness. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs ORX + TR).

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101 FIGURE 4 49 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on periosteal bone circumference. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 50 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on endosteal bone circumference. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR).

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102 FIGURE 4 51 Effects of ORX+V, ORX+EX, testosterone enan thate (TE), or trenbolone enanthate (TR) on intraskeletal testosterone concentrations. Values are Means SE, n = 9 10/group Letters a e indicate differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.O RX + EX, d = vs. ORX + T, e = vs. ORX + TR). FIGURE 4 52 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on intraskeletal dihydrotestosterone concentrations. Values are Means SE, n = 9 10/group Letters a e indicat e differences from respectively labeled groups at # p < 0.05 or p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs.ORX + EX, d = vs. ORX + T, e = vs. ORX + TR). a b* c* e* a b* c* e*

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103 FIGURE 4 53 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate ( TR) on Pax 7 expression in the flexor hallicus longus A) = SHAM, B)=ORX, C)=ORX+EX, D )=ORX+T, E)=ORX+TR. White Arrows Denote Pax 7+ Cells.

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104 FIGURE 4 54 Effects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on Pax 7 expression in the semimembranosus. A) = SHAM, B)=ORX, C)=ORX+EX, D )=ORX+T, E)=ORX+TR. White Arrows Denote Pax 7+ Cells.

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105 FIGURE 4 55 E ffects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on fiber type percentage and muscl e cross sectional area of the flexor hallicus longus A) = SHAM, B)=ORX, C )=ORX+EX, D)=ORX+T, E)=ORX+TR Type 1 Fibers=Blue; Type Iia Fibers=Green; Type Iix/B=Black

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106 FIGURE 4 56 E ffects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enan thate (TR) on fiber type percentage and muscle cross sectional area of the semimembranosus A) = SHAM, B)=ORX, C)=ORX+EX, D)=ORX+T, E)=ORX+TR Type 1 Fibers=Blue; Type IIa Fibers=Green; Type IIx/B=Black

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107 FIGURE 4 57 E ffects of ORX+V, ORX+EX, te stosterone enanthate (TE), or trenbolone enanthate (TR) on centralized and internalized nuclei following hematoxylin and eosin staining in the flexor hallicus longus A) = SHAM, B)=ORX, C)=ORX+EX, D)=ORX+T, E)=ORX+TR. White Block Arrow=Internalized; Black Arrow=Centralized

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108 FIGURE 4 58 E ffects of ORX+V, ORX+EX, testosterone enanthate (TE), or trenbolone enanthate (TR) on centralized and internalized nuclei following hematoxylin and eosin staining in the semimembranosus A) = SHAM, B)=ORX, C)=O RX+EX, D)=ORX+T, E)= ORX+TR. White Block Arrow=Internalized; Black Arrow=Centralized

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109 FIGURE 4 59 Weighted ladder climbing on a 1m ladder inclined to 85

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110 CHAPTER 5 DISCUSSION Overview of Principle Findings In this experiment we studied the anabolic effects of weighted ladder climbing, testosterone enanthate and trenbolone enanthate on serum hormone levels of T and DHT, muscular hypertrophy and histology bone, adiposity, and hemoglobin in orchiectomized 10 month old Fisher Brown Norway rats. Testoste rone enanthate and trenbolone enanthate were effective at improving LABC muscle mass, reducing adiposity and improving hemoglobin levels in these animals. Specifically testosterone enanthate and trenbolone enanthate reduced bone turnover through their eff ects on markers of bone osteoclast activity (i.e., indirect resorption measure) and formation through lowered serum levels of Trap5b and osteocalcin. Similarly, ORX+T and ORX+TR animals prevented ORX induced deficits in trabecular bone content, trabecular bone density, and cortical bone density at the femoral metaphysis Treatment with testosterone enanthate and resulted in elevated levels of intraskeletal testosterone and DHT. Opposing effects of the individual androgens were seen in the prostate as testos terone resulted in large increases in prostate mass, whereas trenbolone administration tended to decrease prostate mass While testosterone elevated intraskeletal T and DHT; elevated levels of trenbolone were not detectable with current methodologies. Howe ver, the trenbolone group increased maximal load of the femoral neck while testosterone did not suggesting that trenbolone exerts a bone protective effect similar to testosterone In the androgen responsive kidney tissue both androgen treated groups ele vated kidney mass compared to the ORX treatment groups. Whereas,

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111 progressive resistance exercise training without the presence of testosterone failed to induce positive changes in muscle mass, adiposity, Pax 7, and hemoglobin. Anabolic and Androgenic Effec ts of Testosterone and Trenbolone Enanthate and Weighted Ladder Climbing on Muscle, Bone and Prostate Background In the aging male population, dysregulation of supportive anabolic pathways in muscle and bone may lead to decreased independence, increased ri sk of falls, fracture risk, and higher rates of morbidity and mortality ( 18 52 68 ) The role of testosterone in the preservation of bone and muscle has been reported in both human ( 6 9 32 64 74 125 135 137 ) and animal studies ( 8 11 30 40 ) However, administration of testosterone does cause prostate enlargement, polycythemia and other adverse events, even at replacement dosages ( 9 138 ) In the current study, we provided our orchiectomized animals with a weekly supraphysiologic dose of testosterone which elevated serum testosterone ~9 fold over SHAM. In an effort to minimize prostate growth an additional group of animals were also treated with trenbolone enanthate a synthetic analog of testosterone with reported SARM like effects ( 40 ) A recent review by Yarrow and colleagues, reports 17beta hyd roxyestra 4,9,11 trien 3 one ( trenbolone ) has a full spectrum on anabolic effects in muscle and bone, but shows reduced androgenic and estrogenic activity originating from reduced 5 alpha reductase and aromatase conversion to DHT and estradiol ( 41 ) respectively. Anabolic Effects on Skeletal Muscle Once weekly testosterone enanthate or trenbolone enanthate injection yielded higher levels of serum hormones in circulati on At the conclusion of the study, T treated animals, serum T was elev ated (9 fold), whereas circulating D HT increased to levels

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112 (15 fold ) above SHAM. In TR treated animals levels of circulating T and DHT fell to the levels of ORX treated animals. In T tr eated animals, the h igher circulating levels of T and DHT may be implicated in the large increases seen in the LABC muscle mass. Both T and DHT are capable of binding to the androgen receptor and may increase muscular hypertrophy through direct or indirect mechanisms. However, anabolic effects on the soleus, plantaris, semimembranosus, and flexor hallicus longus were not evident in the current study for either of the androgen treated groups Trenbolone treated animals increased LABC muscle mass to a simila r degree as testosterone treated animal, without increased serum levels of T, and DHT. Trenbolone is capable of binding to the androgen receptor with three times the affinity of T, specific binding to the androgen receptor may partially explain the hypertr ophic response in the LABC. However, the exercise intervention failed to maintain or hypertrophy the LABC, muscle while the soleus, plantaris, semimembranosus, and flexor hallicus longus muscles remained unchanged throughout the study. These results are similar to other published studies examining the responsiveness of skeletal muscle tissues to anabolic agents. The hypertrophy recorded for the LABC in T and TR treated animals is similar to results found in younger animals in our laboratory ( 11 40 ) The responsiveness of the LABC to androgens can partially be explained by its 3x greater androgen receptor density comp ared to other skeletal muscles in the rat with approximatel y 75% of the levator ani muscle fibers expressing androgen receptors ( 42 ) Since TR treated animals have low circulating levels of T and DHT, hypertrophic responses may be directly related to binding of TR to the androgen receptor since TR binds with a 3x greater affinity than T and with the

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113 same affinity as DHT ( 35 ) mechanisms possi bly induced by trenbolone or its metabolites possibly having additional inhibitory actions on glucocorticoids Interestingly, whole body progressive resistance training in ORX induced males did not maintain LABC muscle mass, nor did it cause hypertrophy in the primary muscle involved in ladder climbing the flexor hallicus longus. Whereas other studies in intact animals found significant hypertrophy to this muscle in intact males over a similar time course ( 62 ) In the hypogonadal state, resistance training may not be able to induce hypertrophic responses without adequate hormonal support. Anabolic Effects on Bone Hypogonadism in elderly men is associated with a g reater risk of fractures ( 15 68 ) loss of functional independence, and nearly double the one year mortality ri sk compared to women ( 18 ) Borst and colleagues identified that the Fisher F344 and Brown Norway rats were model s for displaying high turnover osteopenia following ORX ( 60 ) I ncreases in catabolic bone and muscle effects are evident in bone as early as two weeks following hormone ablation in younger rod ents ( 60 ) In the current study, we had no changes between any groups in gross measures of tibial and femoral bone mass or length The current study also examined changes in cortical and trabecular bone utilizing pQCT In this study animals were aged ten months, prior to study initiation, which generally denotes a mature skeleton and the large changes in length and mass seen in younger animals was not expected. Six weeks following orchiectomy significa nt loss to cortical bone mass occurs However, previous studies with testosterone undecanoate and testosterone enanthate attenuated changes in high turnover osteopenia in younger animals. Testosterone

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114 administration resulted in reductions of both serum osteo calcin and Trap 5b concentrations levels to below that of SHAM and ORX animals Similar results were found by Yarrow and colleagues in three month old animals receiving the same dosage of testosterone during a four week long intervention ( 11 40 ) Testosterone treated animals also had higher trabecular content, trabecular density and cortical density compared to both O RX groups at the 5mm evaluation site following six weeks of the intervention Testosterone treated animals also tended to have stronger femoral neck strength, a common fracture site in hypogondal elderly men Similarly, TR treated animals had significantly elevated trabecular content, trabecular density, and cortical density at the 5mm evaluation site compared to ORX controls Both androgens were able to maintain trabecular content, trabecular density and cortical density at the 5mm site at the level of SHA M animals, preventing the high turnover osteopenia experienced by the ORX animals. Trenbolone treated animals had stronger femoral neck strength values compared to ORX+EX animals and tended to have increased femoral neck strength compared to ORX+V controls Progressive resistance training without optimal hormone support may lead to impairments in bone formation, which warrants further attention. Intraskeletal Testosterone and DHT Concentrations Administration of 7.0 mg week 1 of testosterone enanthate resul ted in a four fold increase in intraskeletal T concentration above ORX conditions, and 3 fold above SHAM conditions. Yarrow and colleagues evaluated three month old Fischer 344 rats after 28 days of treatment and reported similar concentrations for both th e SHAM and ORX values of intraskeletal T to our ten month old rats evaluated after 42 days of treatment In contrast, three month old rats administered 7.0 mg week 1 of testosterone enanthate

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115 had intraskeletal concentrations of 27.4 ng/g ; whereas we obser ved intraskeletal T concentration of 11.4 ng/g. Notably, the administration of trenbolone enanthate did not impact intraskeletal levels of T Future studies should examine the time course of T depletion, the responsiveness to testosterone replacement and i ts effects on bone morphology and strength. Administration of 7.0 mg week 1 resulted in elevated levels of serum DHT of 3 fold above SHAM and ORX conditions Yarrow and colleagues reported elevations in intraskeletal DHT concentrations of 11,554 pg/g follo wing weekly dosage of 7.0 mg of testost erone enanthate for 28 days. In the current study, we observed DHT concentrations of 12,413 pg/g in ten month old rodents follow ing a 42 day treatment period. Trenbolone administration did not alter intraskeletal DHT concentration. In conclusion, older animals appear to have a greater conversion of T to DHT in bone Intraskeletal androgen levels appear to be conserved following 42 days of orchiectomy. Anabolic Effects on the Prostate Benign prostate hyperplasia is a co mmonly reported side effect of administration of testosterone ( 6 9 12 53 136 ) Several large clinical trials have exclusionary criteria for prostate symptomology and track its growth over the course of a study In a n ongoing study, Borst and colleagues have administered a 5 alpha reductase inhibitor in tandem with testosterone replacement therapy in an attempt to minimize prostate growth ( 8 30 ) Similar to results from human testosterone replacement trials and supraphysiological administration of testosterone in younger orchiectomized rodents ( 40 ) prostate ma ss was significantly increase d by superphysiological testosterone in our 10 month old orchidectimized male rats. Testosterone treatment resulted in prostate

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116 masses nearly double SHAM controls, and greater than 6 fold increase in mass than ORX treatment conditions Whereas, TR treatme nt non significantly reduced prostate mass by 20% compared to SHAM, and were 64% smaller than T treated animals at sacrifice. Anabolic Effects on Hemoglobin Anemia is a common blood disorder defined as a low number of red blood cells Anemic disorders numb er in the hundreds and can be a result of stomach ulcers, medications, cancers, iron deficiency, vitamin B12 and folate deficiencies The reported prevalence 8.1% of anemia in a sample of 2905 men of anemia in the aging popula tion and therapies able to imp rove hemoglobin concentrations without serious side effects would benefit oxygen carrying capacity and energy levels in the aging population ( 154 ). Administration of testosterone elevates red blood cell concentrations in some individuals to pathological le vels, increasing risks of serious adverse events and requiring stringent screening criteria for study inclusion In the present study, we found elevations in blood hemoglobin concentrations following both T and TR administration in 10 month old orchiectomi zed male rats While both drugs increased hemoglobin concentrations, the process took till the fourth week of the experiment to elevate concentrations above SHAM conditions A similar increase in circulating hemoglobin concentrations has been seen by Yarro w and colleagues (unpublished) in younger orchiectomized rats also occurring at the four week time period Since TR exerted a similar response to T while suppressing T and DHT; TR may induce elevations in hemoglobin concentrations through direct binding to the androgen receptor Some possible mechanisms through which TR may induce hemoglobin alterations are through upregulation of erythropoietin, inhibiting hepcidin production ( 139 ) enhancing red blood cell integrity, direct effects on

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117 bone marrow or through another unknown mechanism Progressive resistance training exerted no effect on hemoglobin concentrations during the experimental period. Effects of Testosterone and Trenbolone Enanthate and Weighted Ladder Climbing on Serum markers of T, DHT and TR Serum Testosterone at Sacrifice At sacrifice, nadir (6 days after the last injection) values of T were evaluated in all groups Administration of 7.0mg week 1 of testo sterone enanthate into the quadriceps muscle group resulted in a significant elevation of serum T in middle aged rodents Testosterone administered animals serum T values were greater than SHAM animals and ORX greater than TR treated animals Similar supe rphysiological levels have been reported by Yarrow and colleagues in a 4 week study using 3 month old rats ( 40 ) Serum DHT at Sacrifice At sacrifice, nadir (6 days after the last injection) values of DH T were evaluated in all groups. Administration of 7.0mg week 1 o f testosterone enanthate into the quadriceps muscle group resulted in a significant elevation of serum DH T in middle aged rodents. Testosterone administered animals serum DHT values wer e greater than SHAM, ORX and greater ORX+ TR treated animals Similar le vels of serum DHT have been reported by Yarrow and colleagues in a 4 week study using 3 month old rats ( 40 ) Serum TR at Sacrifice At sacrifice, nadir (6 days removed from last injection) values of T R were evaluated in all groups. Administration of 1 .0mg week 1 of t renbolone enanthate into the quadriceps muscle group resulted in a significant elevation of serum T R in middle aged rodents T renbolone administered animals serum TR values were greater than SHAM

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118 ORX and TR treated animals Similar levels of TR have been r eported by Yarrow and colleagues in a 4 week study using 3 month old rats ( 40 ) Effects of Testosterone and Trenbolone Enanthate and Weighted Ladder Climbing on Retroperitoneal Fat Pad and Kidney Mass Retroperitoneal Fat Pad Mass Increasing adiposity is associated w ith cardiovascular risk, insulin resistance, diabetes, fatigue and the metabolic syndrome Orchiectomized rodents exhibited an increase in visceral adiposity during the experimental period Notably, castrated male rats engaging in weighted ladder climbing three times a week, also increased visceral adiposity stores during the experimental period In contrast animals treated with supraphysiologic levels of T, experienced significant decreases in visceral adiposity during the study Similar effects have been shown by Yarrow and colleagues in younger animals ( 40 ) Trenbolone administered animals reduced visceral adiposity stores below the levels of SHAM and ORX conditions The lipolytic effect seen from the androgen group could be attributable to binding of the androgen receptor, increased physical or foraging activity Kidney Mass at Sacrifice Borst and colleagues have demonstrated increased kidney mass following androgen administration in younger rodents Both T and TR treated animals conserved kidney mass at SHAM leve ls compared to ORX treated animals ORX treated animals reduction in kidney mass of similar reductions have been shown in younger animals ( 40 ) Disease states also show a reduction in kidney mass and function Although kidney mass was reduced changes in renal cleara nce and physiologic function were not assessed in the present investigation

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119 Effects of Testosterone and Trenbolone Enanthate and Weighted Ladder Climbing on Centralized and Internalized Myonuclei Sinha Hikim and colleagues previously reported an increase in satellite cell significant changes in satellite cell populations following doses replacing testosterone at levels consistent with a eugonadal state ( 64 ) In the present study, muscle fibers were evaluated in a small number of animals treated with a superphysiologic dose of testosterone and trenbolone Although not significant, the presence of centralized and internalized nuclei in these treated animals ma y indicate the possibility of muscle regeneration and renewal In contrast the exercising group without androgen replacement experienced a trend towards slightly declining popula tions of centralized and internalized nuclei T renbolone and testosterone treatment may initiate or commit myonuclear populations for regeneration, or renewal, however due to the small populations sampled these results must be interpreted with caution Ef fects of Testosterone and Trenbolone Enanthate and Weighted Ladder Climbing on Pax7+ Nuclei in the Semimembranosus and Flexor Hallicus Longus Thompson and colleagues produced one of the seminal articles evaluating the induction of cellular modifications to satellite cells following trenbolone administration ( 35 ) Similar ly preliminary findings from the current investigation seem to suggest trenbolone may possibly induce Pax 7 activation of satellite cells in castrated male rats Similarly superphysiologic doses of testosterone tended to have i ncreases in satellite cell Pax 7+ nuclei Interestingly, weighted ladder climbing in castrated male rats seems to result in a reduction in Pax 7+ nuclei number It has been reported that the functional importance of Pax 7+ may diminish after 21 days of age and may be critical for early stage muscle development Resistance training induces post exercise GH, and IGF 1

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120 secretion that may partially induce satellite cell activation, under normal hormonal control. The exact impact of reduced satellite cell numbe r on the organism, and potential for recovery, renewal, and regeneration throughout out the lifespan or following injury during the hypogonadal state warrants further investigation. Effects of ORX on Weighted Ladder Climbing Hornberger and colleagues demon strated an increase in weighted ladder climbing weight, muscular hypertrophy, and specific tension over a period of 8 weeks in intact animals without changes to myosin heavy chain concentrations or short term energy substrates of ATP, ADP and creatinine ph osphate ( 62 ) In the present study, hypogonadal animals participating in weighted ladder climbing improved weight carried throughout the study, however weight carried was significantly lower than our previous experiments with intact animals (unpublished observations) ORX animals increased the weight carried without the concomitant muscular hypertrophy reported by Hornberger and colleagues Although not statisti cally significant the primary muscle of movement (i.e., flexor hallicus longus) during the climbing activity experienced a reduction in satellite cell number, central and internalized nuclei, with a minor loss in hindlimb muscle wet weight These prelimin ary findings may assist in the development of optimized resistance training programs for hypogonadal individuals. Effects of Weighted Ladder Climbing, Testosterone Enanthate, and Trenbolone Enanthate on Muscle Fiber Cross sectional Area of the Flexor Halli cus Longus Weighted ladder climbing may promote increases in Type IIa cross sectional area in the flexor hallicus longus, whereas androgen treatments do not appear to preserve Type IIa cross sectional area compared to ORX Similar results have been observe d with a 23% increase in muscle mass following eight weeks of weighted ladder climbing

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121 in intact rodents (61, 62). In Type IIbx, testosterone appears to partially attenuate the loss in cross sectional area following ORX, while weighted ladder climbing appe ars to reduce cross sectional area in these fibers Type I muscle fibers represented less than 0.5% of muscle cross sectional area and were excluded from analysis. In conclusion, weighted ladder climbing and testosterone administration may exert differing effects on the preservation of Type II muscle fibers of the FHL following ORX, however these results must be interpreted cautiously due to the small number of animals sampled. Effects of Weighted Ladder Climbing, Testosterone Enanthate, and Trenbolone Enan thate on Muscle Fiber Cross sectional Area of the Flexor Hallicus Longus Weighted ladder climbing appeared to reduce muscle cross sectional area of the se mimembranosus compared to ORX. The Type IIa fibers of the semimembranosus appear to be partially re sis tant to the effects of ORX. Type IIbx fibers appear to be decreased following ORX, and this decrement is partially prevented by trenbolone administration Type I muscl e fibers represented less than 0.5 % of muscle cross sectional area and were excluded from analysis. Future research should examine the individual and combined effects of androgen administration and progressive resistance training on slow and fast twitch muscle fibers. Conclusion In conclusion, we have examined the impact of two anabolic and an drogenic agents on skeletal muscle, bone, intraskeletal horomone concentrations, and adipose tissue in middle aged ORX male rats The present investigation determined that intramuscular injections of testosterone, and trenbolone enanthate result in elevati ons in plasma concentrations of both substances In contrast to testosterone, trenbolone administration decreased DHT and testosterone Interestingly, both substances caused

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122 significant hypertrophy the LABC complex, however trenbolone administration result ed in prostate reduction compared to a doubling of prostate mass in testosterone treated animals Notably, progressive resistance exercise failed to prevent the typical pattern of osteopenia and sarcopenia seen in the castrated male rat model Similarly, p reliminary evidence suggests that progressive resistance training in the absence of normal levels of testosterone impair skeletal muscle satellite cell disposition, mechanisms involved in skeletal muscle hypertrophy, and markers of bone health Administrat ion of testosterone enanthate elevates intraskeletal concentration of testosterone and dihydrotestosterone although the clinical implications of intraskeletal androgen levels are unknown Future work is planned to evaluate the intraskeletal presence of tren bolone and estrogen concentration and their association with pQCT and biomechanical strength measurements. Future investigations should be directed at elucidating the exercise pe rformance, hemoglobin, muscular hypertrophy, and bone health.

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123 REFERENCES 1. Tenover, J.S., Androgen replacement therapy to reverse and/or prevent age associated sarcopenia in men. Baillieres Clin Endocrinol Metab, 1998. 12 (3): p. 419 25. 2. Emmelot Vonk, M.H., et al., Effect of testosterone supplementation on functional mobility, cognition, and other parameters in older men: a randomized controlled trial. Jama, 2008. 299 (1): p. 39 52. 3. Borst, S.E., Interventions for sarcopenia and mu scle weakness in older people. Age Ageing, 2004. 33 (6): p. 548 55. 4. Pande, I. and R.M. Francis, Osteoporosis in men. Best Pract Res Clin Rheumatol, 2001. 15 (3): p. 415 27. 5. Bhasin, S., et al., Testosterone therapy in men with androgen deficiency syndro mes: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 95 (6): p. 2536 59. 6. Bhasin, S., et al., Older men are as responsive as young men to the anabolic effects of graded doses of testosterone on the skeletal muscle. J Clin Endocr inol Metab, 2005. 90 (2): p. 678 88. 7. Amory, J.K., et al., Exogenous testosterone or testosterone with finasteride increases bone mineral density in older men with low serum testosterone. J Clin Endocrinol Metab, 2004. 89 (2): p. 503 10. 8. Borst, S.E., J. H. Lee, and C.F. Conover, Inhibition of 5alpha reductase blocks prostate effects of testosterone without blocking anabolic effects. Am J Physiol Endocrinol Metab, 2005. 288 (1): p. E222 7. 9. Borst, S.E. and T. Mulligan, Testosterone replacement therapy for older men. Clin Interv Aging, 2007. 2 (4): p. 561 6. 10. Turner, R.T., et al., Failure of isolated rat tibial periosteal cells to 5 alpha reduce testosterone to 5 alpha dihydrotestosterone. J Bone Miner Res, 1990. 5 (7): p. 775 9. 11. Yarrow, J.F., et al., Supraphysiological testosterone enanthate administration prevents bone loss and augments bone strength in gonadectomized male and female rats. Am J Physiol Endocrinol Metab, 2008. 295 (5): p. E1213 22. 12. Calof, O.M., et al., Adverse events associated with testosterone replacement in middle aged and older men: a meta analysis of randomized, placebo controlled trials. J Gerontol A Biol Sci Med Sci, 2005. 60 (11): p. 1451 7.

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124 13. Lin, J.T. and J.M. Lane, Osteoporosis: a review. Clin Orthop Relat Res, 2004(425): p. 126 34. 14. Ray, W.A., et al., A randomized trial of a consultation service to reduce falls in nursing homes. Jama, 1997. 278 (7): p. 557 62. 15. Burge, R., et al., Incidence and economic burden of osteoporosis related fractures in the United States, 20 05 2025. J Bone Miner Res, 2007. 22 (3): p. 465 75. 16. Cummings, S.R., D.M. Black, and S.M. Rubin, Lifetime risks of hip, Colles', or vertebral fracture and coronary heart disease among white postmenopausal women. Arch Intern Med, 1989. 149 (11): p. 2445 8. 17. Ismail, A.A., et al., Risk factors for vertebral deformities in men: relationship to number of vertebral deformities. European Vertebral Osteoporosis Study Group. J Bone Miner Res, 2000. 15 (2): p. 278 83. 18. Bass, E., et al., Risk adjusted mortality rates of elderly veterans with hip fractures. Ann Epidemiol, 2007. 17 (7): p. 514 9. 19. Gardner, M.J., et al., Osteoporosis and skeletal fractures. Hss J, 2006. 2 (1): p. 62 9. 20. Szulc, P., et al., Low skeletal muscle mass is associated with poor structur al parameters of bone and impaired balance in elderly men -the MINOS study. J Bone Miner Res, 2005. 20 (5): p. 721 9. 21. Tinetti, M.E., et al., A multifactorial intervention to reduce the risk of falling among elderly people living in the community. N Engl J Med, 1994. 331 (13): p. 821 7. 22. Baumgartner, R.N., et al., Epidemiology of sarcopenia among the elderly in New Mexico. Am J Epidemiol, 1998. 147 (8): p. 755 63. 23. Melton, L.J., 3rd, Excess mortality following vertebral fracture. J Am Geriatr Soc, 200 0. 48 (3): p. 338 9. 24. Cruz Jentoft, A.J., et al., Understanding sarcopenia as a geriatric syndrome. Curr Opin Clin Nutr Metab Care. 13 (1): p. 1 7. 25. Rolland, Y., et al., Cachexia versus sarcopenia. Curr Opin Clin Nutr Metab Care. 14 (1): p. 15 21. 26. J anssen, I., et al., The healthcare costs of sarcopenia in the United States. J Am Geriatr Soc, 2004. 52 (1): p. 80 5. 27. Cruz Jentoft, A.J., et al., Sarcopenia: European consensus on definition and diagnosis: Report of the European Working Group on Sarcope nia in Older People. Age Ageing. 39 (4): p. 412 23.

PAGE 125

125 28. Kalyani, R.R., S. Gavini, and A.S. Dobs, Male hypogonadism in systemic disease. Endocrinol Metab Clin North Am, 2007. 36 (2): p. 333 48. 29. Yarrow, J.F., et al., Early phase neuroendocrine responses an d strength adaptations following eccentric enhanced resistance training. J Strength Cond Res, 2008. 22 (4): p. 1205 14. 30. Borst, S.E., et al., Anabolic effects of testosterone are preserved during inhibition of 5alpha reductase. Am J Physiol Endocrinol Me tab, 2007. 293 (2): p. E507 14. 31. Allan, G., et al., A selective androgen receptor modulator with minimal prostate hypertrophic activity restores lean body mass in aged orchidectomized male rats. J Steroid Biochem Mol Biol, 2008. 110 (3 5): p. 207 13. 32. Bhasin, S., L. Woodhouse, and T.W. Storer, Proof of the effect of testosterone on skeletal muscle. J Endocrinol, 2001. 170 (1): p. 27 38. 33. Bauer, E.R., et al., Characterisation of the affinity of different anabolics and synthetic hormones to the human an drogen receptor, human sex hormone binding globulin and to the bovine progestin receptor. Apmis, 2000. 108 (12): p. 838 46. 34. Wilson, V.S., et al., In vitro and in vivo effects of 17beta trenbolone: a feedlot effluent contaminant. Toxicol Sci, 2002. 70 (2) : p. 202 11. 35. Thompson, S.H., et al., Trenbolone alters the responsiveness of skeletal muscle satellite cells to fibroblast growth factor and insulin like growth factor I. Endocrinology, 1989. 124 (5): p. 2110 7. 36. Santidrian, S., J.R. Thompson, and V. R. Young, Effect of trienbolone acetate on the rate of myofibrillar protein breakdown in young adrenalectomized male rate treated with corticosterone. Arch Farmacol Toxicol, 1981. 7 (3): p. 333 40. 37. Scheffler, J.M., et al., Effect of repeated administrat ion of combination trenbolone acetate and estradiol implants on growth, carcass traits, and beef quality of long fed Holstein steers. J Anim Sci, 2003. 81 (10): p. 2395 400. 38. Johnson, B.J., et al., Effect of a combined trenbolone acetate and estradiol im plant on feedlot performance, carcass characteristics, and carcass composition of feedlot steers. J Anim Sci, 1996. 74 (2): p. 363 71. 39. Johnson, B.J., et al., Stimulation of circulating insulin like growth factor I (IGF I) and insulin like growth factor binding proteins (IGFBP) due to administration of a combined trenbolone acetate and estradiol implant in feedlot cattle. J Anim Sci, 1996. 74 (2): p. 372 9. 40. Yarrow, J.F., et al., 17{beta} hydroxyestra 4,9,11 trien 3 one (Trenbolone) Exhibits Tissue Sele ctive Anabolic Activity: Effects on Muscle, Bone, Adiposity, Hemoglobin, and Prostate. Am J Physiol Endocrinol Metab.

PAGE 126

126 41. Yarrow, J.F., S.C. McCoy, and S.E. Borst, Tissue selectivity and potential clinical applications of trenbolone (17beta hydroxyestra 4, 9,11 trien 3 one): A potent anabolic steroid with reduced androgenic and estrogenic activity. Steroids. 75 (6): p. 377 89. 42. Freyberger, A., H. Ellinger Ziegelbauer, and F. Krotlinger, Evaluation of the rodent Hershberger bioassay: testing of coded chemic als and supplementary molecular biological and biochemical investigations. Toxicology, 2007. 239 (1 2): p. 77 88. 43. Gracia, T., et al., Modulation of steroidogenic gene expression and hormone production of H295R cells by pharmaceuticals and other environm entally active compounds. Toxicol Appl Pharmacol, 2007. 225 (2): p. 142 53. 44. Compston, J., Secondary causes of osteoporosis in men. Calcif Tissue Int, 2001. 69 (4): p. 193 5. 45. Compston, J.E., Sex steroids and bone. Physiol Rev, 2001. 81 (1): p. 419 447. 46. Vanderschueren, D., et al., The aged male rat as a model for human osteoporosis: evaluation by nondestructive measurements and biomechanical testing. Calcif Tissue Int, 1993. 53 (5): p. 342 7. 47. Vanderschueren, D. and L. Vandenput, Androgens and oste oporosis. Andrologia, 2000. 32 (3): p. 125 30. 48. Liegibel, U.M., et al., Concerted action of androgens and mechanical strain shifts bone metabolism from high turnover into an osteoanabolic mode. J Exp Med, 2002. 196 (10): p. 1387 92. 49. Cadore, E.L., et a l., Hormonal responses to resistance exercise in long term trained and untrained middle aged men. J Strength Cond Res, 2008. 22 (5): p. 1617 24. 50. Ryan, A.S., et al., Regional bone mineral density after resistive training in young and older men and women. Scand J Med Sci Sports, 2004. 14 (1): p. 16 23. 51. Pfeifer, M., et al., Effects of a long term vitamin D and calcium supplementation on falls and parameters of muscle function in community dwelling older individuals. Osteoporos Int, 2009. 20 (2): p. 315 22 52. Araujo, A.B., et al., Association between testosterone and estradiol and age related decline in physical function in a diverse sample of men. J Am Geriatr Soc, 2008. 56 (11): p. 2000 8. 53. Bhasin, S., et al., Testosterone therapy in adult men with an drogen deficiency syndromes: an endocrine society clinical practice guideline. J Clin Endocrinol Metab, 2006. 91 (6): p. 1995 2010.

PAGE 127

127 54. Lewis, M.I., et al., Skeletal muscle adaptations to testosterone and resistance training in men with COPD. J Appl Physiol 2007. 103 (4): p. 1299 310. 55. Storer, T.W., et al., Changes in muscle mass, muscle strength, and power but not physical function are related to testosterone dose in healthy older men. J Am Geriatr Soc, 2008. 56 (11): p. 1991 9. 56. Sullivan, D.H., et al. Effects of muscle strength training and testosterone in frail elderly males. Med Sci Sports Exerc, 2005. 37 (10): p. 1664 72. 57. Turner, R.T., D.S. Colvard, and T.C. Spelsberg, Estrogen inhibition of periosteal bone formation in rat long bones: down regu lation of gene expression for bone matrix proteins. Endocrinology, 1990. 127 (3): p. 1346 51. 58. Turner, R.T., et al., Dehydroepiandrosterone reduces cancellous bone osteopenia in ovariectomized rats. Am J Physiol, 1990. 258 (4 Pt 1): p. E673 7. 59. Sehmisc h, S., et al., Vitex agnus castus as prophylaxis for osteopenia after orchidectomy in rats compared with estradiol and testosterone supplementation. Phytother Res, 2008. 60. Borst, S.E. and C.F. Conover, Orchiectomized Fischer 344 male rat models body comp osition in hypogonadal state. Life Sci, 2006. 79 (4): p. 411 5. 61. Lee, S., et al., Viral expression of insulin like growth factor I enhances muscle hypertrophy in resistance trained rats. J Appl Physiol, 2004. 96 (3): p. 1097 104. 62. Hornberger, T.A., Jr. and R.P. Farrar, Physiological hypertrophy of the FHL muscle following 8 weeks of progressive resistance exercise in the rat. Can J Appl Physiol, 2004. 29 (1): p. 16 31. 63. Menkes, A., et al., Strength training increases regional bone mineral density and bone remodeling in middle aged and older men. J Appl Physiol, 1993. 74 (5): p. 2478 84. 64. Sinha Hikim, I., et al., Effects of testosterone supplementation on skeletal muscle fiber hypertrophy and satellite cells in community dwelling older men. J Clin End ocrinol Metab, 2006. 91 (8): p. 3024 33. 65. Vincent, K.R., et al., Resistance exercise and physical performance in adults aged 60 to 83. J Am Geriatr Soc, 2002. 50 (6): p. 1100 7. 66. Seftel, A., Male hypogonadism. Part II: etiology, pathophysiology, and di agnosis. Int J Impot Res, 2006. 18 (3): p. 223 8. 67. Seftel, A.D., Male hypogonadism. Part I: Epidemiology of hypogonadism. Int J Impot Res, 2006. 18 (2): p. 115 20.

PAGE 128

128 68. Anderson, F.H., et al., Sex hormones and osteoporosis in men. Calcif Tissue Int, 1998. 62 (3): p. 185 8. 69. Koopman, R. and L.J. van Loon, Aging, exercise and muscle protein metabolism. J Appl Physiol, 2009. 70. Moore, L.M., et al., Real time polymerase chain reaction to follow the response of muscle to training. Artif Organs, 2008. 32 (8): p 630 3. 71. Gao, W., Androgen receptor as a therapeutic target. Adv Drug Deliv Rev. 62 (13): p. 1277 84. 72. Hellstrom, W.J., D. Paduch, and C.F. Donatucci, Importance of hypogonadism and testosterone replacement therapy in current urologic practice: a rev iew. Int Urol Nephrol. 73. Harman, S.M., et al., Longitudinal effects of aging on serum total and free testosterone levels in healthy men. Baltimore Longitudinal Study of Aging. J Clin Endocrinol Metab, 2001. 86 (2): p. 724 31. 74. Bhasin, S., et al., Testo sterone replacement increases fat free mass and muscle size in hypogonadal men. J Clin Endocrinol Metab, 1997. 82 (2): p. 407 13. 75. Wang, C., et al., Sublingual testosterone replacement improves muscle mass and strength, decreases bone resorption, and inc reases bone formation markers in hypogonadal men -a clinical research center study. J Clin Endocrinol Metab, 1996. 81 (10): p. 3654 62. 76. Wang, C., et al., Pharmacokinetics of transdermal testosterone gel in hypogonadal men: application of gel at one site versus four sites: a General Clinical Research Center Study. J Clin Endocrinol Metab, 2000. 85 (3): p. 964 9. 77. Page, S.T., et al., Exogenous testosterone (T) alone or with finasteride increases physical performance, grip strength, and lean body mass in older men with low serum T. J Clin Endocrinol Metab, 2005. 90 (3): p. 1502 10. 78. Page, S.T., et al., Testosterone administration suppresses adiponectin levels in men. J Androl, 2005. 26 (1): p. 85 92. 79. Snyder, P.J., et al., Effect of testosterone treatm ent on bone mineral density in men over 65 years of age. J Clin Endocrinol Metab, 1999. 84 (6): p. 1966 72. 80. Snyder, P.J., et al., Effect of testosterone treatment on body composition and muscle strength in men over 65 years of age. J Clin Endocrinol Met ab, 1999. 84 (8): p. 2647 53.

PAGE 129

129 81. Bryant, T.C., et al., Effects of ractopamine and trenbolone acetate implants with or without estradiol on growth performance, carcass characteristics, adipogenic enzyme activity, and blood metabolites in feedlot steers and heifers. J Anim Sci. 88 (12): p. 4102 19. 82. Walker, D.K., et al., Effects of ractopamine and sex on serum metabolites and skeletal muscle gene expression in finishing steers and heifers. J Anim Sci. 88 (4): p. 1349 57. 83. Baxa, T.J., et al., Additive effe cts of a steroidal implant and zilpaterol hydrochloride on feedlot performance, carcass characteristics, and skeletal muscle messenger ribonucleic acid abundance in finishing steers. J Anim Sci. 88 (1): p. 330 7. 84. Gonzalez, J.M., et al., Effect of ractop amine hydrochloride and trenbolone acetate on longissimus muscle fiber area, diameter, and satellite cell numbers in cull beef cows. J Anim Sci, 2007. 85 (8): p. 1893 901. 85. Pottier, J., et al., Differences in the biotransformation of a 17 beta hydroxylat ed steroid, trenbolone acetate, in rat and cow. Xenobiotica, 1981. 11 (7): p. 489 500. 86. Danhaive, P.A. and G.G. Rousseau, Evidence for sex dependent anabolic response to androgenic steroids mediated by muscle glucocorticoid receptors in the rat. J Steroi d Biochem, 1988. 29 (6): p. 575 81. 87. Meyer, H.H. and M. Rapp, Estrogen receptor in bovine skeletal muscle. J Anim Sci, 1985. 60 (1): p. 294 300. 88. Vernon, B.G. and P.J. Buttery, Protein turnover in rats treated with trienbolone acetate. Br J Nutr, 1976. 36 (3): p. 575 9. 89. Vernon, B.G. and P.J. Buttery, The effect of trenbolone acetate with time on the various responses of protein synthesis of the rat. Br J Nutr, 1978. 40 (3): p. 563 72. 90. Vernon, B.G. and P.J. Buttery, The time course of the response of rat protein metabolism to trenbolone acetate. Proc Nutr Soc, 1978. 37 (2): p. 56A. 91. Wolden Hanson, T., B.T. Marck, and A.M. Matsumoto, Troglitazone treatment of aging Brown Norway rats improves food intake and weight gain after fasting without increas ing hypothalamic NPY gene expression. Exp Gerontol, 2002. 37 (5): p. 679 91. 92. Gray, L.E., Jr., J. Furr, and J.S. Ostby, Hershberger assay to investigate the effects of endocrine disrupting compounds with androgenic or antiandrogenic activity in castrate immature male rats. Curr Protoc Toxicol, 2005. Chapter 16 : p. Unit16 9.

PAGE 130

130 93. Erben, R.G., et al., Androgen deficiency induces high turnover osteopenia in aged male rats: a sequential histomorphometric study. J Bone Miner Res, 2000. 15 (6): p. 1085 98. 94. Gu nness, M. and E. Orwoll, Early induction of alterations in cancellous and cortical bone histology after orchiectomy in mature rats. J Bone Miner Res, 1995. 10 (11): p. 1735 44. 95. Reim, N.S., et al., Cortical bone loss in androgen deficient aged male rats is mainly caused by increased endocortical bone remodeling. J Bone Miner Res, 2008. 23 (5): p. 694 704. 96. Falahati Nini, A., et al., Relative contributions of testosterone and estrogen in regulating bone resorption and formation in normal elderly men. J C lin Invest, 2000. 106 (12): p. 1553 60. 97. Ramamani, A., M.M. Aruldhas, and P. Govindarajulu, Differential response of rat skeletal muscle glycogen metabolism to testosterone and estradiol. Can J Physiol Pharmacol, 1999. 77 (4): p. 300 4. 98. Hourde, C., et al., Androgen replacement therapy improves function in male rat muscles independently of hypertrophy and activation of the Akt/mTOR pathway. Acta Physiol (Oxf), 2009. 195 (4): p. 471 82. 99. Fiatarone, M.A., et al., Exercise training and nutritional supple mentation for physical frailty in very elderly people. N Engl J Med, 1994. 330 (25): p. 1769 75. 100. Layne, J.E. and M.E. Nelson, The effects of progressive resistance training on bone density: a review. Med Sci Sports Exerc, 1999. 31 (1): p. 25 30. 101. Ne lson, M.E., et al., Effects of high intensity strength training on multiple risk factors for osteoporotic fractures. A randomized controlled trial. Jama, 1994. 272 (24): p. 1909 14. 102. Hunter, G.R., J.P. McCarthy, and M.M. Bamman, Effects of resistance tr aining on older adults. Sports Med, 2004. 34 (5): p. 329 48. 103. Izquierdo, M., et al., Differential effects of strength training leading to failure versus not to failure on hormonal responses, strength, and muscle power gains. J Appl Physiol, 2006. 100 (5) : p. 1647 56. 104. Ratamess, N.A., et al., Androgen receptor content following heavy resistance exercise in men. J Steroid Biochem Mol Biol, 2005. 93 (1): p. 35 42. 105. Kraemer, W.J., et al., Acute hormonal responses to heavy resistance exercise in younger and older men. Eur J Appl Physiol Occup Physiol, 1998. 77 (3): p. 206 11.

PAGE 131

131 106. Kraemer, W.J., et al., Effects of heavy resistance training on hormonal response patterns in younger vs. older men. J Appl Physiol, 1999. 87 (3): p. 982 92. 107. Ahtiainen, J.P., et al., Muscle hypertrophy, hormonal adaptations and strength development during strength training in strength trained and untrained men. Eur J Appl Physiol, 2003. 89 (6): p. 555 63. 108. Gotshalk, L.A., et al., Hormonal responses of multiset versus single set heavy resistance exercise protocols. Can J Appl Physiol, 1997. 22 (3): p. 244 55. 109. Vingren, J.L., et al., Effect of resistance exercise on muscle steroidogenesis. J Appl Physiol, 2008. 105 (6): p. 1754 60. 110. Tchaikovsky, V.S., J.V. Astratenkova, and O.B. Basharina, The effect of exercises on the content and reception of the steroid hormones in rat skeletal muscles. J Steroid Biochem, 1986. 24 (1): p. 251 3. 111. Aizawa, K., et al., Sex differences in steroidogenesis in skeletal muscle following a s ingle bout of exercise in rats. J Appl Physiol, 2008. 104 (1): p. 67 74. 112. Saitoglu, M., et al., Osteoporosis risk factors and association with somatotypes in males. Arch Med Res, 2007. 38 (7): p. 746 51. 113. Scane, A.C., A.M. Sutcliffe, and R.M. Francis Osteoporosis in men. Baillieres Clin Rheumatol, 1993. 7 (3): p. 589 601. 114. Reznik, M., Thymidine 3H uptake by satellite cells of regenerating skeletal muscle. J Cell Biol, 1969. 40 (2): p. 568 71. 115. Moss, F.P. and C.P. Leblond, Satellite cells as the source of nuclei in muscles of growing rats. Anat Rec, 1971. 170 (4): p. 421 35. 116. Yablonka Reuveni, Z., et al., Defining the transcriptional signature of skeletal muscle stem cells. J Anim Sci, 2008. 86 (14 Suppl): p. E207 16. 117. Olguin, H.C. and B.B. Olwin, Pax 7 up regulation inhibits myogenesis and cell cycle progression in satellite cells: a potential mechanism for self renewal. Dev Biol, 2004. 275 (2): p. 375 88. 118. Seale, P. and M.A. Rudnicki, A new look at the origin, function, and "stem cell" status of muscle satellite cells. Dev Biol, 2000. 218 (2): p. 115 24. 119. Seale, P., et al., Pax7 is required for the specification of myogenic satellite cells. Cell, 2000. 102 (6): p. 777 86. 120. Morrison, J.I., et al., Salamander limb regeneration involv es the activation of a multipotent skeletal muscle satellite cell population. J Cell Biol, 2006. 172 (3): p. 433 40.

PAGE 132

132 121. Kassar Duchossoy, L., et al., Pax3/Pax7 mark a novel population of primitive myogenic cells during development. Genes Dev, 2005. 19 (12) : p. 1426 31. 122. Relaix, F., et al., Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells. J Cell Biol, 2006. 172 (1): p. 91 102. 123. Relaix, F., et al., A Pax3/Pax7 dependent population of skeletal muscle progenitor cel ls. Nature, 2005. 435 (7044): p. 948 53. 124. Oustanina, S., G. Hause, and T. Braun, Pax7 directs postnatal renewal and propagation of myogenic satellite cells but not their specification. Embo J, 2004. 23 (16): p. 3430 9. 125. Sinha Hikim, I., et al., Testo sterone induced muscle hypertrophy is associated with an increase in satellite cell number in healthy, young men. Am J Physiol Endocrinol Metab, 2003. 285 (1): p. E197 205. 126. Joubert, Y. and C. Tobin, Testosterone treatment results in quiescent satellite cells being activated and recruited into cell cycle in rat levator ani muscle. Dev Biol, 1995. 169 (1): p. 286 94. 127. Doumit, M.E., D.R. Cook, and R.A. Merkel, Testosterone up regulates androgen receptors and decreases differentiation of porcine myogenic satellite cells in vitro. Endocrinology, 1996. 137 (4): p. 1385 94. 128. Kamanga Sollo, E., et al., Potential role of G protein coupled receptor 30 (GPR30) in estradiol 17beta stimulated IGF I mRNA expression in bovine satellite cell cultures. Domest Anim Endocrinol, 2008. 35 (3): p. 254 62. 129. Kamanga Sollo, E., et al., Roles of IGF I and the estrogen, androgen and IGF I receptors in estradiol 17beta and trenbolone acetate stimulated proliferation of cultured bovine satellite cells. Domest Anim Endocrino l, 2008. 35 (1): p. 88 97. 130. Arola, L., et al., Effect of stress and sampling site on metabolite concentration in rat plasma. Arch Int Physiol Biochim, 1980. 88 (2): p. 99 105. 131. Schnell, M.A., et al., Effect of blood collection technique in mice on cl inical pathology parameters. Hum Gene Ther, 2002. 13 (1): p. 155 61. 132. Maurissen, J.P., et al., Factors affecting grip strength testing. Neurotoxicol Teratol, 2003. 25 (5): p. 543 53. 133. Pelker, R.R., et al., Effects of freezing and freeze drying on the biomechanical properties of rat bone. J Orthop Res, 1984. 1 (4): p. 405 11. 134. Leppanen, O., et al., Three point bending of rat femur in the mediolateral direction: introduction and validation of a novel biomechanical testing protocol. J Bone Miner Res, 2006. 21 (8): p. 1231 7.

PAGE 133

133 135. Bhasin, S., et al., Effects of testosterone supplementation on whole body and regional fat mass and distribution in human immunodeficiency virus infected men with abdominal obesity. J Clin Endocrinol Metab, 2007. 92 (3): p. 1049 57. 136. Bhasin, S. and J.S. Tenover, Age associated sarcopenia -issues in the use of testosterone as an anabolic agent in older men. J Clin Endocrinol Metab, 1997. 82 (6): p. 1659 60. 137. Bhasin, S., et al., Testosterone dose response relationships in he althy young men. Am J Physiol Endocrinol Metab, 2001. 281 (6): p. E1172 81. 138. Bhasin, S., et al., Issues in testosterone replacement in older men. J Clin Endocrinol Metab, 1998. 83 (10): p. 3435 48. 139. Bachman, E., et al., Testosterone suppresses hepcid in in men: a potential mechanism for testosterone induced erythrocytosis. J Clin Endocrinol Metab. 95 (10): p. 4743 7. 140. Dhawan, J., and T.A. Rando, Stem cells in postnatal myogenesis: molecular mechanisms of satellite cell quiescence, activation, and repl enishment Trends Cell Biol, 2005 15 (12): p. 666 73. 141. Tsivitse, S., Notch and Wnt signaling, physiological stimuli and postnatal myogenesis. Int J Biol Sci, 2010. 6 (3): p. 268 81. 142. Hawke TJ and DJ Garry Myogenci satellite cells: physiology to molecular biology. J Appl Physiol, 2001. 91 (2): p. 534 51. 143. Collins CA and TA Partridge. Self renewal of the adult skeletal muscle satellite cell. Cell Cycle, 2005. 4 (10): p. 1338 41. 144. Abou Khalil, R and AS Brack, Musc le stem cells and reversible quiescence: The role of sprouty. Cell Cycle, 2010. 9 (13): E pub 145. Shea KL., et al., Sprouty1 regualtes reversible quiescence of a self renewing adult muscle stem cell pool during regeneration. Cell Stem Cell, 2010. 9 (13): p. 117 2 9. 146. Abou Khalil, R., et al., Autocrine and paracrine angiopoietin 1/Tie 2 signaling promotes satellite cell self renewal. Cell Stem Cell, 2009. 5 (3): 298 309. 147. Snow MH The effects of aging on satellite cells in skeletal muscles of mice and rats. Cell Tissue Res, 1977. 185 (3): p. 399 408. 148. Conboy I., et al., Notch mediated restoration of regenerative potential to aged muscle. Science, 2003. 302 (5850): p. 1575 7.

PAGE 134

134 149. Creuzet, S., et al., MyoD, myogenin, and desmin nls lacZ transgene emphasize the distinct patterns of satellite cell activation in growth and regeneration Exp Cell Res, 1998. 243 (2): p. 241 53. 150. Sakuma K., et al., The adaptive response of transforming growth factor beta 2 and beta RII in the overloaded, regenerating and denervated muscles of rats. Acta Neuropathol, 2000. 99 (2): p. 177 85. 15 1 Florini, JR., et al., Growth hormone and the insulin like growth factor system in myogenesis. Endocr Rev, 1996. 17 (5): p. 481 517. 152. Lawlor MA., et al., Dual control of muscle cell survival by distinct growth factor regu lated signaling pathways. Mol Cell Biol, 2000. 20 (9): p. 3256 65. 153. Sinha Hikim, I., et al., Effects of testosterone supplementation on skeletal muscle fiber hypertrophy and satellite cells in community dwelling older men. J Clin Endocrinol Metab, 2006. 91 (8 ): p. E pub. 154. Endres HG., et al., Prevalence of anemia in elderly patients in primary care: impact on 5 year mortality risk and differences between men and women. Curr Med Res Opin, 2009. 25 (5): p. 1143 58.

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135 BIOGRAPHICAL SKETCH Sean C McCoy was bo rn in Trenton, New Jersey and moved to Collegeville, Pennsylvania where he worked for A reufit Inc, a health and wellness promotions company, while he completed a Bachelor of Science degree in Exercise and Sports Science in 1999 He then went on to coach i Swimming and Diving at Frostburg State University and graduated with a Master of Science degree in Health and Human Performance in 2001 muscle lipid metabolism led him to begin his doctoral work under Dr. Lesley White researching intramyocellular lipid on the 3T magnetic resonance imaging machine In 2004, Dr. White changed her research focus to the effects of resistance training on multiple sclerosis and Sean began his second chapter of academic endeavors During this period of inadequate funding and academic support, Sean worked for the Florida Department of Corrections at Florida State Prison as a correctional officer. Following Dr. White not being offered tenure at the University of Florida, S ean followed her to the University of Georgia to complete his dissertation After one year of no progress at the University of Georgia, Sean returned to the University of Florida under Dr. Stephen Borst and focused on the anabolic properties of testosteron e and trenbolone to attenuate sarcopenia and osteopenia in men with hypogonadism Upon completion of his Ph.D. Sean will be working on anabolic agents in patients with multiple sclerosis.