Paula H. A. Ronkainen

Postmenopausal Hormone Replacement Therapy Modifies Skeletal Muscle Composition and Function: A Study with Monozygotic Twin Pairs

We investigated whether long-term hormone replacement therapy (HRT) is associated with mobility and lower limb muscle performance and composition in postmenopausal women. Fifteen 54- to 62-yr-old monozygotic female twin pairs discordant for HRT were recruited from the Finnish Twin Cohort. Habitual (HWS) and maximal (MWS) walking speeds over 10 m, thigh muscle composition, lower body muscle power assessed as vertical jumping height, and maximal isometric hand grip and knee extension strengths were measured. Intrapair differences (IPD%) with 95% confidence intervals (CI) were calculated. The mean duration of HRT use was 6.9 +/- 4.1 yr. MWS was on average 7% (0.9 to 13.1%, P = 0.019) and muscle power 16% (-0.8 to 32.8%, P = 0.023) greater in HRT users than in their cotwins. Thigh muscle cross-sectional area tended to be larger (IPD% = 6%, 95% CI: -0.07 to 12.1%, P = 0.065), relative muscle area greater (IPD% = 8%, CI: 0.8 to 15.0%, P = 0.047), and relative fat area smaller (IPD% = -5%, CI: -11.3 to 1.2%, P = 0.047) in HRT users than in their sisters. There were no significant differences in maximal isometric strengths or HWS between users and nonusers. Subgroup analyses revealed that estrogen-containing therapies (11 pairs) significantly decreased total body and thigh fat content, whereas tibolone (4 pairs) tended to increase muscle cross-sectional area. This study showed that long-term HRT was associated with better mobility, greater muscle power, and favorable body and muscle composition among 54- to 62-yr-old women. The results indicate that HRT is a potential agent in preventing muscle weakness and mobility limitation in older women.

Differential influence of peripheral and systemic sex steroids on skeletal muscle quality in pre‐ and postmenopausal women

Aging is associated with gradual decline of skeletal muscle strength and mass often leading to diminished muscle quality. This phenomenon is known as sarcopenia and affects about 30% of the over 60‐year‐old population. Androgens act as anabolic agents regulating muscle mass and improving muscle performance. The role of female sex steroids as well as the ability of skeletal muscle tissue to locally produce sex steroids has been less extensively studied. We show that despite the extensive systemic deficit of sex steroid hormones in postmenopausal compared to premenopausal women, the hormone content of skeletal muscle does not follow the same trend. In contrast to the systemic levels, muscle tissue of post‐ and premenopausal women had similar concentrations of dehydroepiandrosterone and androstenedione, while the concentrations of estradiol and testosterone were significantly higher in muscle of the postmenopausal women. The presence of steroidogenetic enzymes in muscle tissue indicates that the elevated postmenopausal steroid levels in skeletal muscle are because of local steroidogenesis. The circulating sex steroids were associated with better muscle quality while the muscle concentrations reflected the amount of infiltrated fat within muscle tissue. We conclude that systemically delivered and peripherally produced sex steroids have distinct roles in the regulation of neuromuscular characteristics during aging.

Effects of combined hormone replacement therapy or its effective agents on the IGF-1 pathway in skeletal muscle

OBJECTIVES To investigate the effects of combined hormone replacement therapy (HRT) and its effective agents on the IGF-1 signaling pathway. DESIGN AND METHODS To examine the effects of HRT on skeletal muscle in vivo, we utilized pre- and post-intervention samples from a randomized double blinded trial with 50-57-year-old women. The intervention included the year-long use of either HRT preparation (2 mg 17β-estradiol, E₂; 1mg norethisterone acetate, NETA, n=10) or placebo (CO, n=9). Microarray technology and quantitative PCR (qPCR) were used to study the expression of insulin-like growth factor I (IGF-1) and its splice variants as well as IGF-1 receptor, Akt1, mTOR, FOXO1, FOXO3, atrogin, estrogen receptors and androgen receptor in muscle samples. Serum concentrations of IGF-1, E(2) and testosterone were measured. C2C12 myotubes were fed with E₂ or NETA followed by analyzing the expression of essentially the same gene transcripts as in human samples by qPCR and phosphorylation of Akt and mTOR by Western blotting. RESULTS The gene expression of IGF-1 and its splice variant, IGF-1Ec (also known as the mechano growth factor or MGF), mTOR, FOXO3, and AR was up-regulated among the HRT users compared to the CO (P<0.05), while Akt1 was down-regulated (P<0.05). The change in the level of IGF-1Ec transcript correlated positively with muscle size at post-intervention (r=0.5, P<0.05). In C2C12 myotubes, no statistically significant effects of either E₂ or NETA at the level of gene transcripts studied were identified. The amount of phosphorylated Akt appeared to respond to NETA, albeit the response was not statistically significant. Phosphorylation of mTOR did not respond to either of the treatments. CONCLUSION Year-long postmenopausal HRT was found to affect the expression of the genes along the IGF-1 signaling cascade reflecting the higher muscle mass compared to the CO women. By using cell culture model we were, however, unable to confirm the possible differential role of E₂ and NETA. It appears that the synchronous presence of both effective agents of the HRT or the presence of yet unidentified microenvironmental factors providing proper paracrine signals naturally existing in the intact muscle tissue is critical for appropriate signaling via sex steroid-IGF-1 axis to occur.

Effects of 32-Year Leisure Time Physical Activity Discordance in Twin Pairs on Health (TWINACTIVE Study): Aims, Design and Results for Physical Fitness

The physically active lifestyle is associated with low future morbidity and mortality, but the causality between physical activity and health is not always clear. As some inherited biological characteristics and childhood experiences may cause selection bias in observational studies, we sought to take them into account by identifying 16 twin pairs (7 MZ, 9 DZ, mean age 60 years) discordant for leisure time physical activity habits for thirty years. We conducted detailed health-related examinations among these twin pairs. Our main aims were to study the effects of physical activity and genes on fitness and body composition, with special reference to body fat compartments, metabolic syndrome components and related diseases and risk factor levels, status of arteries, structure and function of the heart, bone properties, and muscle and fat tissue-related mechanisms linked to physical activity and chronic disease development. Our physical activity assessments showed that inactive co-twins were on average 8.8 MET hours/day less active than their active co-twins through out their midlife (2.2+/-2.3 vs. 11.0+/-4.1 MET h/day, p< .001). Follow-up fitness tests showed that physically inactive co-twins were less fit than their active co-twins (estimated VO(2peak) 26.4+/-4.9 vs. 32.5+/-5.5 ml/kg/min, p< .001). Similar differences were found in both MZ and DZ pairs. On the basis of earlier epidemiological observations on nonrelated individuals, these physical activity and fitness differences are large enough to cause differences in many mechanisms and risk factors related to the development of chronic diseases and to permit future analyses.

Combination of hormone replacement therapy and high physical activity is associated with differences in Achilles tendon size in monozygotic female twin pairs

Estrogen concentration has been suggested to play a role in tendon abnormalities and injury. In physically active postmenopausal women, hormone replacement therapy (HRT) has been suggested to decrease tendon diameter. We hypothesized that HRT use and physical activity are associated with Achilles tendon size and tissue structure. The study applied cotwin analysis of fourteen 54- to 62-yr-old identical female twin pairs with current discordance for HRT use for an average of 7 yr. Achilles tendon thickness and cross-sectional areas were determined by ultrasonography, and tendon structural organization was analyzed from the images using linear discriminant analysis (LDA). Maximal voluntary and twitch torques from plantar flexor muscles were measured. Serum levels of estradiol, estrone, testosterone, and sex hormone binding globulin were analyzed. Total daily metabolic equivalent score (MET-h/day) was calculated from physical activity questionnaires. Results showed that, in five physically active (MET > 4) pairs, the cotwins receiving HRT had greater estradiol level (P = 0.043) and smaller tendon cross-sectional area than their sisters (63 vs. 71 mm(2), P = 0.043). Among all pairs, Achilles tendon thickness and cross-sectional area did not significantly differ between HRT using and nonusing twin sisters. Intrapair correlation for Achilles tendon thickness was high, despite HRT use discordance (r = 0.84, P < 0.001). LDA distinguished different tendon structure only from two of six examined twin pairs who had a similar level of physical activity. In conclusion, the effect of HRT on Achilles tendon characteristics independent of genetic confounding may be present only in the presence of sufficient physical activity. In physically active twin pairs, the higher level of estrogen seems to be associated with smaller tendon size.

Baculovirus-mediated immediate-early gene expression and nuclear reorganization in human cells

Baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), has the ability to transduce mammalian cell lines without replication. The general objective of this study was to detect the transcription and expression of viral immediate‐early genes in human cells and to examine the interactions between viral components and subnuclear structures. Viral capsids were seen in large, discrete foci in nuclei of both dividing and non‐dividing human cells. Concurrently, the transcription of viral immediate‐early transregulator genes (ie‐1, ie‐2) and translation of IE‐2 protein were detected. Quantitative microscopy imaging and analysis showed that virus transduction altered the size of promyelocytic leukaemia nuclear bodies, which are suggested to be involved in replication and transcription of various viruses. Furthermore, altered distribution of the chromatin marker Draq5™ and histone core protein (H2B) in transduced cells indicated that the virus was able to induce remodelling of the host cell chromatin. To conclude, this study shows that the non‐replicative insect virus, baculovirus and its proteins can induce multiple changes in the cellular machinery of human cells.

Hormone replacement therapy improves contractile function and myonuclear organization of single muscle fibres from postmenopausal monozygotic female twin pairs

•  The ageing‐related impairment of muscle function and consequent falls and fall‐related injuries have severe negative effects on morbidity and mortality in old age, with women being more negatively affected than men. •  The effects of hormone replacement therapy (HRT) on regulation of muscle contraction and myonuclear organization were investigated in monozygous postmenopausal twin pairs where only one twin was an HRT‐user. •  HRT treatment improved single fibre force‐generating capacity (specific force), without affecting fibre size and speed of contraction, due to fibre type‐specific effects on force and number of force‐generating cross‐bridges. •  HRT had a significant effect on the myonuclear organization in slow‐twitch muscle fibres, improving the synthetic capacity of the myonuclei and optimizing transport of proteins. •  Significant positive effects on regulation of muscle contraction and myonuclear organization were observed at the cellular level in response to HRT with consequences for quality of life in postmenopausal women.

Muscle function in monozygotic female twin pairs discordant for hormone replacement therapy.

Introduction: Postmenopausal monozygotic twin pairs discordant for hormone replacement therapy (HRT) provide an advantageous study design controlling for genetic background for elucidating the relationships between aging, sex hormone levels, muscle strength, contractile capacity, and fatigability. Methods: Thirteen postmenopausal monozygotic twin pairs discordant for HRT were measured for maximal voluntary torque (MVC) and twitch characteristics using electrical stimulation before and after intermittent dynamic plantarflexor exercise until exhaustion. Results: Peak twitch torque was 32% higher in HRT users than in their non‐HRT, genetically identical sisters (P = 0.002), but MVC did not differ. There were no differences in the activation level or twitch time characteristics between the co‐twins. Fatigue caused decreases in MVC (P = 0.001), twitch torque (P = 0.001), time to peak (P = 0.013), and half‐relaxation time (P = 0.001) similarly in HRT users and non–HRT users. Conclusion: In early postmenopausal women, involuntary but not voluntary force‐generating mechanisms of the plantarflexors are augmented by the use of HRT. Muscle Nerve, 2011

Global gene expression profiles in skeletal muscle of monozygotic female twins discordant for hormone replacement therapy

Aging is accompanied by inexorable loss of muscle tissue. One of the underlying causes for this is the massive change in the hormonal milieu of the body. The role of a female sex steroid – estrogen – in these processes is frequently neglected, although the rapid decline in its production coincides with a steep deterioration in muscle performance. We recruited 54‐ to 62‐year‐old monozygotic female twin pairs discordant for postmenopausal hormone replacement therapy (HRT, n = 11 pairs; HRT use 7.3 ± 3.7 years) from the Finnish Twin Cohort to investigate the association of long‐term, estrogen‐based HRT with skeletal muscle transcriptome. Pathway analysis of muscle transcript profiles revealed significant HRT‐induced up‐regulation of a biological process related to regulation of cell structure and down‐regulation of processes concerning, for example, cell–matrix interactions, energy metabolism and utilization of nutrients (false discovery rate < 0.15). Lending clinical relevance to the findings, these processes explained a significant fraction of the differences observed in relative proportion of muscle within thigh and in muscle performance (R2 = 0.180–0.257, P = 0.001–0.023). Although energy metabolism was affected through down‐regulation of the transcripts related to succinate dehydrogenase complex in mitochondria, no differences were observed in mtDNA copy number or oxidative capacity per muscle cross section. In conclusion, long‐term use of HRT was associated with subtle, but significant, differences in muscle transcript profiles. The better muscle composition and performance among the HRT users appeared to be orchestrated by improved regulatory actions on cytoskeleton, preservation of muscle quality via regulation of intramuscular extracellular matrix and a switch from glucose‐oriented metabolism to utilization of fatty acids.

Hormone therapy is associated with better body composition and adipokine/glucose profiles: a study with monozygotic co-twin control design.

Objective The aim of this study was to evaluate the possibility of preventing the metabolic health consequences of postmenopausal hypogonadism with the use of long-term hormone therapy (HT). Methods We used a monozygotic co-twin control design including 10 twin pairs (aged 56-62 y) discordant for HT (duration of HT, 2-10 y). In addition, 14 premenopausal women (aged 29-35 y) who did not use HT were studied to evaluate the differences in metabolic health between the premenopausal and postmenopausal states. Body composition was determined, and waist-to-hip ratio was used as an estimate for fat distribution. Serum sex steroids, sex hormone-binding globulin, and serum lipid and glucose profiles were analyzed. The serum levels of adiponectin, monocyte chemotactic protein-1, and leptin, as well as their local transcript levels in adipose tissue, skeletal muscle, and leukocytes, were measured. Results Long-term HT was associated with a healthier amount and distribution of body fat. No difference was seen in serum lipid concentrations between HT users and their nonusing identical twin sisters, but fasting serum glucose and glycated hemoglobin levels were 5% and 3% lower in HT users than in nonusers, respectively. Among the adipokines analyzed, the most notable finding was a 15% lower level of monocyte chemotactic protein-1 in HT users, particularly with respect to its suggested mediator role between obesity and insulin resistance. Conclusions Long-term HT is associated with healthier amount and distribution of body fat and better adipocytokine profile, with concomitant signs of improved insulin sensitivity.