APS 2006 Refresher Course Sex Hormones and Aging: Skeletal Muscle and Bone Marybeth Brown PT, PhD Center for Gender Physiology, Physical Therapy, University of Missouri-Columbia

APS 2006 Refresher Course Overview of talk…. Estrogen effects- Estrogen effects- –Direct (muscle mass, bone mass) –Indirect (inactivity, increase in fat mass) Testosterone effects- Testosterone effects- –Direct (muscle mass, strength) –Indirect (bone mass, fat mass) –Unintended consequences

APS 2006 Refresher Course Why was the WHI study halted?  Higher incidence of stroke  32 in HRT group  24 in placebo group in 10,000 subjects!!!  Lower incidence of hip fracture, colon cancer

APS 2006 Refresher Course Overview of E 2 effects.. Direct Estrogen effects on skeletal muscle: mass, strength, fatigue and injury Direct Estrogen effects on skeletal muscle: mass, strength, fatigue and injury Indirect Estrogen effects on skeletal muscle: inactivity, fat Indirect Estrogen effects on skeletal muscle: inactivity, fat Rehabilitation implications Rehabilitation implications

APS 2006 Refresher Course E 2 and skeletal muscle mass Skeletal muscle is loaded with estrogen receptors Skeletal muscle is loaded with estrogen receptors –On the membrane –In the cytoplasm –On the nuclear membrane Cell membrane has both ERα and ERß but the specific function of each is not known Cell membrane has both ERα and ERß but the specific function of each is not known –Greater distribution on type II fibers

APS 2006 Refresher Course

Estrogen, muscle and aging Literature rather confusing… Literature rather confusing… –Confounds: age, duration of HRT, conjugated vs. unconjugated estrogens, dose of HRT Observation: appears to be a more rapid decline in strength with age during the menopause. Observation: appears to be a more rapid decline in strength with age during the menopause.

Age in Years Leg Press (lb) Strength losses with age in Master Athletes APS 2006 Refresher Course

Evidence for and against estrogen effects on muscle No: 64 wks of 20-mg estradiol significantly increased bone mass, decreased fat mass by 18% and had no effect on muscle mass. Hansen et al, 2003 No: 64 wks of 20-mg estradiol significantly increased bone mass, decreased fat mass by 18% and had no effect on muscle mass. Hansen et al, 2003 Yes: 12 week double-blind, cross-over design with wash-out phase in between E 2 administration and placebo. Significant increase in lean mass during E 2 phase Yes: 12 week double-blind, cross-over design with wash-out phase in between E 2 administration and placebo. Significant increase in lean mass during E 2 phase Sorensen et al, 2001, Obes Res Sorensen et al, 2001, Obes Res

APS 2006 Refresher Course Recent Meta analysis The strength of evidence (27 papers) supports an anabolic effect of estrogen in (younger*) women. The strength of evidence (27 papers) supports an anabolic effect of estrogen in (younger*) women. The mechanism(s) of action is unclear The mechanism(s) of action is unclear *my emphasis Meeuwsen et al, Maturitas, 2000

APS 2006 Refresher Course Does E 2 effectiveness decrease with age? Young women with normal menstrual cycles show a 12% variation in strength. Muscle strength is highest when serum E 2 levels are at their peak. Young women with normal menstrual cycles show a 12% variation in strength. Muscle strength is highest when serum E 2 levels are at their peak.

APS 2006 Refresher Course Older women- less effect? Kenny et al : Incidence of sarcopenia (age- related muscle mass loss) was the same in 189 women years old who had been using ERT for at least 2 years as those who were not ERT users. Kenny et al : Incidence of sarcopenia (age- related muscle mass loss) was the same in 189 women years old who had been using ERT for at least 2 years as those who were not ERT users. Brown et al: HRT did not augment gains in muscle strength or FFM in response to weight-bearing exercise. 16 vs. 17% strength increase Brown et al: HRT did not augment gains in muscle strength or FFM in response to weight-bearing exercise. 16 vs. 17% strength increase

APS 2006 Refresher Course Our recent work with OVX To amplify the potential anabolic effect of female sex hormones, we used hind limb unweighting (HLU) in rats To amplify the potential anabolic effect of female sex hormones, we used hind limb unweighting (HLU) in rats –Model of weightlessness, bed rest Ho: if ovarian hormones, notably E 2, influence muscle mass and function, OVX + HLU rats should experience > decline than intact rats. Ho: if ovarian hormones, notably E 2, influence muscle mass and function, OVX + HLU rats should experience > decline than intact rats.

APS 2006 Refresher Course OVX cont’d Additionally, we hypothesized that OVX rats would recover from HLU more slowly or to a lesser extent than intact rats and in OVX rats given E 2 supplementation Additionally, we hypothesized that OVX rats would recover from HLU more slowly or to a lesser extent than intact rats and in OVX rats given E 2 supplementation

APS 2006 Refresher Course Hind limb unweigting

Results APS 2006 Refresher Course

Total Akt Akt –p(Ser437) Sham OVX Cont HLU Rec APS 2006 Refresher Course

What to conclude? Data suggest that failure to recover muscle mass in OVX rats was ovarian hormone mediated, particularly E 2 Data suggest that failure to recover muscle mass in OVX rats was ovarian hormone mediated, particularly E 2 Data also suggest that conditions which result in low E 2 (hysterectomy, trauma, bed rest) may impact recovery from the loss of muscle mass and strength that occurs with inactivity Data also suggest that conditions which result in low E 2 (hysterectomy, trauma, bed rest) may impact recovery from the loss of muscle mass and strength that occurs with inactivity

APS 2006 Refresher Course Bouts of inactivity result in incremental losses in muscle

APS 2006 Refresher Course Indirect effects of estrogen Estrogen has marked behavioral influence on spontaneous activity Estrogen has marked behavioral influence on spontaneous activity –Level of the hyothalamus Estrogen also affects basal metabolic rate such that OVX or hysterectomy results in weight gain without a significant increase in food intake Estrogen also affects basal metabolic rate such that OVX or hysterectomy results in weight gain without a significant increase in food intake

APS 2006 Refresher Course ¶ * + * + ¶ # + # ¶ # * # OVX HRT Spontaneous daily running distance Intact HRT

APS 2006 Refresher Course Running activity and phase of the estrus cycle Figure from Eckel et al.

APS 2006 Refresher Course ¶ * + ¶ # + ¶ # * gonadectomyHRT Intact OVX HRT Body weight changes with hormonal status

APS 2006 Refresher Course ¶ + + ~ ¶ # * ~ # # * + gonadectomyHRT Food intake varies with hormonal status

APS 2006 Refresher Course Estrogen and fat Estrogen regulates the amount and distribution of adipose tissues Estrogen regulates the amount and distribution of adipose tissues –Central adiposity with menopause or hysterectomy Increase in body fat mass with low hormone values Increase in body fat mass with low hormone values –Which contributes to further inactivity! Note: inactivity predisposes to low estrogen values Note: inactivity predisposes to low estrogen values

APS 2006 Refresher Course Sex hormone suppression Subjects: 14 healthy, young females with mean age of 29±5 years Subjects: 14 healthy, young females with mean age of 29±5 years Six days of GnRHant Six days of GnRHant Resting energy expenditure was significantly reduced from 1405± 42 to 1334± 36 kcal Resting energy expenditure was significantly reduced from 1405± 42 to 1334± 36 kcal –A 5% decline in 6 days! Day et al, 2005 J Clin Endocrinol Metab

APS 2006 Refresher Course More body mass= less activity=more body mass Which subsequently contributes to more inactivity  less estrogen Which subsequently contributes to more inactivity  less estrogen –Muscle atrophy –Less muscle strength –Higher predisposition to muscle injury –Functional decline 450g250g Distance (km)

APS 2006 Refresher Course OSTEOBLAST OSTEOCLAST Prostaglandins PTH Vitamin D TNF TGB-ß IL-1 ESTROGEN IL-4 IL-13 Il-4 Il-8 Calcitonin ESTROGEN Glucocorticoids Prostaglandins Calcium Phosphates BONE MATRIX Estrogen and Bone Metabolism Stimulation

APS 2006 Refresher Course Estrogen, bone mineral and the menopause Unequivocal effects Unequivocal effects –Menopause: ~10% decline in bone mass in 5 years –HRT during menopause prevents decline in bone

APS 2006 Refresher Course HRT effects on bone with age Age in Years BMD g/cm 2

APS 2006 Refresher Course Exercise also a modifier of bone loss Some evidence that HRT and exercise effects on bone are additive in post- menopausal women Some evidence that HRT and exercise effects on bone are additive in post- menopausal women

APS 2006 Refresher Course HRT and exercise are additive Villareal et al,, 2003 N=28 women 75 yrs and older N=28 women 75 yrs and older 9 mos of exercise (home or center) 9 mos of exercise (home or center) –Flexibility vs. resistance and endurance Lumbar spine increases in BMD were 1.5% in controls and 3.5% in exercise group Lumbar spine increases in BMD were 1.5% in controls and 3.5% in exercise group Total body BMD increases were 1.5% in exercise group, 0.2% in controls Total body BMD increases were 1.5% in exercise group, 0.2% in controls

APS 2006 Refresher Course Males and Testosterone Continuous decline in testosterone throughout the lifetime. Age in Years T2 values (ng/dL) Hypogonadal

APS 2006 Refresher Course Other factors that affect T 2 levels Obesity Obesity Inactivity (space travel, bed rest) Inactivity (space travel, bed rest) Trauma (SCI, head trauma, fracture) Trauma (SCI, head trauma, fracture) Diet?? Diet?? Disease Disease Drugs Drugs

APS 2006 Refresher Course T 2 and skeletal muscle Skeletal muscle has many androgen receptors (AR) Skeletal muscle has many androgen receptors (AR) AR receptive to IGF-1 and GH in addition to T 2 AR receptive to IGF-1 and GH in addition to T 2 AR also receptive to estrogen? AR also receptive to estrogen?

APS 2006 Refresher Course Direct effects of T 2 Hypgonadal men given T 2 for 8 weeks Hypgonadal men given T 2 for 8 weeks Muscle mass increased 7% (DEXA) Muscle mass increased 7% (DEXA) Muscle strength also increased 30% Muscle strength also increased 30% Results indicate marked T 2 influence on muscle mass and strength Results indicate marked T 2 influence on muscle mass and strength

APS 2006 Refresher Course Normal men made hypo- gonadal Hayes et al, eugonadal men given GnRH which caused T 2 levels to plummet to almost undetectable levels. 8 eugonadal men given GnRH which caused T 2 levels to plummet to almost undetectable levels. T 2 given back in varying doses: 25, 50, 125, 300, 600 mg T 2 given back in varying doses: 25, 50, 125, 300, 600 mg Muscle mass and strength primary outcome measures Muscle mass and strength primary outcome measures

APS 2006 Refresher Course T 2 effects on strength Testosterone dose mg % increase in strength

APS 2006 Refresher Course Conclusions Seems to be a minimum T 2 value for an effect on skeletal muscle Seems to be a minimum T 2 value for an effect on skeletal muscle Unintended consequences- 55 adverse events Unintended consequences- 55 adverse events –PSA above 4 µg/ml –Hematocrit >54% –edema

APS 2006 Refresher Course T 2 effects in older men Bhasin et al, 2004 Same design as for young men Same design as for young men GnRH agonist to suppress endog. T 2 GnRH agonist to suppress endog. T 2 Subjects: men yrs, n=60 Subjects: men yrs, n=60 Doses: 25, 50, 125, 300, 600 mg Doses: 25, 50, 125, 300, 600 mg Study duration: 20 weeks Study duration: 20 weeks Primary outcomes: muscle mass, strength Primary outcomes: muscle mass, strength

APS 2006 Refresher Course Change in fat-free mass Testosterone dose mg % increase in FFM

APS 2006 Refresher Course Maximum voluntary strength- leg press Testosterone dose mg Change in strength in kg

APS 2006 Refresher Course Unintended consequences 147 adverse events 147 adverse events 12 serious adverse events in 9 men 12 serious adverse events in 9 men Rx discontinued in 4 men taking 600 mg, 3 men taking 300 mg and in 1 man taking 125 mg Rx discontinued in 4 men taking 600 mg, 3 men taking 300 mg and in 1 man taking 125 mg

APS 2006 Refresher Course Serious Adverse events Hematocrit >54% (n=6) Hematocrit >54% (n=6) Leg edema with sob (n=1) Leg edema with sob (n=1) Urinary retention (n=1) Urinary retention (n=1) Prostate cancer (n=2) Prostate cancer (n=2) Hematuria with elevated PSA (n=1) Hematuria with elevated PSA (n=1)

APS 2006 Refresher Course Other findings… On the positive side.. On the positive side.. –Fat mass decreased in a dose-dependent manner –No change in aggression On the not so positive side.. On the not so positive side.. –HDLs plummeted –PSAs increased

APS 2006 Refresher Course What to conclude? An androgen receptor modulator with anabolic properties that are free of dose-limiting adverse effects of testosterone is needed An androgen receptor modulator with anabolic properties that are free of dose-limiting adverse effects of testosterone is needed

APS 2006 Refresher Course E 2 and T 2 in men van den Beld et al, men yrs of age 403 men yrs of age Ho: decreases in bone mass, FFM and muscle strength related to fall in endogenous T 2 and E 2 Ho: decreases in bone mass, FFM and muscle strength related to fall in endogenous T 2 and E 2 Measures: serum T 2, SHBG-T, E 2 Measures: serum T 2, SHBG-T, E 2

APS 2006 Refresher Course Results Total and free T 2 positively related with muscle strength and bone mass Total and free T 2 positively related with muscle strength and bone mass Inverse relationship between fat mass and T 2 Inverse relationship between fat mass and T 2 Non-SHBG-T and free T more strongly related to muscle strength, bone mass and fat mass that total T Non-SHBG-T and free T more strongly related to muscle strength, bone mass and fat mass that total T

APS 2006 Refresher Course Current belief It may be the aromatization of testosterone to estrogen that is important for bone mass in men It may be the aromatization of testosterone to estrogen that is important for bone mass in men Estrogen and testosterone use different cellular pathways to inhibit osteoclastic activity and bone resorption Estrogen and testosterone use different cellular pathways to inhibit osteoclastic activity and bone resorption

APS 2006 Refresher Course Other findings…. E 1 and E 2 also strongly related to BMD E 1 and E 2 also strongly related to BMD The positive relationship between T 2 and BMD was independent of relationship of E 2 and bone mass suggesting a role for both hormones for the maintenance of bone with aging The positive relationship between T 2 and BMD was independent of relationship of E 2 and bone mass suggesting a role for both hormones for the maintenance of bone with aging

APS 2006 Refresher Course Summary Falling T 2 with age is associated with loss in lean muscle and bone mass Falling T 2 with age is associated with loss in lean muscle and bone mass T 2 supplementation probably not warranted T 2 supplementation probably not warranted Inactivity likely a major factor contributing to lower T 2 values at all ages Inactivity likely a major factor contributing to lower T 2 values at all ages Exercise increases T 2 levels in young men- not clear if the same occurs in older men Exercise increases T 2 levels in young men- not clear if the same occurs in older men Appalling how little is known about functions that are so fundamental Appalling how little is known about functions that are so fundamental