Hormone Replacement Therapy Associated White Blood Cell DNA Methylation and Gene Expression are Associated With Within-Pair Differences of Body Adiposity and Bone Mass.

The loss of estrogen during menopause causes changes in the female body, with wide-ranging effects on health. Estrogen-containing hormone replacement therapy (HRT) leads to a relief of typical menopausal symptoms, benefits bone and muscle health, and is associated with tissue-specific gene expression profiles. As gene expression is controlled by epigenetic factors (including DNA methylation), many of which are environmentally sensitive, it is plausible that at least part of the HRT-associated gene expression is due to changes in DNA methylation profile. We investigated genome-wide DNA methylation and gene expression patterns of white blood cells (WBCs) and their associations with body composition, including muscle and bone measures of monozygotic (MZ) female twin pairs discordant for HRT. We identified 7,855 nominally significant differentially methylated regions (DMRs) associated with 4,044 genes. Of the genes with DMRs, five (ACBA1, CCL5, FASLG, PPP2R2B, and UHRF1) were also differentially expressed. All have been previously associated with HRT or estrogenic regulation, but not with HRT-associated DNA methylation. All five genes were associated with bone mineral content (BMC), and ABCA1, FASLG, and UHRF1 were also associated with body adiposity. Our study is the first to show that HRT associates with genome-wide DNA methylation alterations in WBCs. Moreover, we show that five differentially expressed genes with DMRs associate with clinical measures, including body fat percentage, lean body mass, bone mass, and blood lipids. Our results indicate that at least part of the known beneficial HRT effects on body composition and bone mass may be regulated by DNA methylation associated alterations in gene expression in circulating WBCs.

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

Ageing is associated with a decline in muscle mass and strength leading to increased physical dependency in old age. Postmenopausal women experience a greater decline than men of similar age in parallel with the decrease in female sex steroid hormone production. We recruited six monozygous female twin pairs (55-59 years old) where only one twin pair was on hormone replacement therapy (HRT use = 7.8 ± 4.3 years) to investigate the association of HRT with the cytoplasmic volume supported by individual myonuclei (myonuclear domain (MND) size,) together with specific force at the single fibre level. HRT use was associated with a significantly smaller (∼27%; P < 0.05) mean MND size in muscle fibres expressing the type I but not the IIa myosin heavy chain (MyHC) isoform. In comparison to non-users, higher specific force was recorded in HRT users both in muscle fibres expressing type I (∼27%; P < 0.05) and type IIa (∼23%; P < 0.05) MyHC isoforms. These differences were fibre-type dependent, i.e. the higher specific force in fast-twitch muscle fibres was primarily caused by higher force per cross-bridge while slow-twitch fibres relied on both a higher number and force per cross-bridge. HRT use had no effect on fibre cross-sectional area (CSA), velocity of unloaded shortening (V0) and relative proportion of MyHC isoforms. In conclusion, HRT appears to have significant positive effects on both regulation of muscle contraction and myonuclei organization in postmenopausal women.

Are skeletal muscle FNDC5 gene expression and irisin release regulated by exercise and related to health?

Recently, contradictory findings have been reported concerning the function of irisin and its precursor gene, skeletal muscle FNDC5, in energy homeostasis, and the associated regulatory role of exercise and PGC-1α. We therefore evaluated whether muscle FNDC5 mRNA and serum irisin are exercise responsive and whether PGC-1α expression is associated with FNDC5 expression. The male subjects in the study performed single exercises: (1) 1 h low-intensity aerobic exercise (AE) (middle-aged, n = 17), (2) a heavy-intensity resistance exercise (RE) bout (young n = 10, older n = 11) (27 vs. 62 years), (3) long-term 21 weeks endurance exercise (EE) training alone (twice a week, middle-aged, n = 9), or (4) combined EE and RE training (both twice a week, middle-aged, n = 9). Skeletal muscle mRNA expression was analysed by quantitative PCR and serum irisin by ELISA. No significant changes were observed in skeletal muscle PGC-1α, FNDC5 and serum irisin after AE, EE training or combined EE + RE training. However, a single RE bout increased PGC-1α by 4-fold in young and by 2-fold in older men, while FNDC5 mRNA only increased in young men post-RE, by 1.4-fold. Changes in PGC-1α or serum irisin were not consistently accompanied by changes in FNDC5. In conclusion, for the most part, neither longer-term nor single exercise markedly increases skeletal muscle FNDC5 expression or serum irisin. Therefore their changes in response to exercise are probably random and not consistent excluding the confirmation of any definitive link between exercise and FNDC5 expression and irisin release in humans. Moreover, irisin and FNDC5 were not associated with glucose tolerance and being overweight, or with metabolic disturbances, respectively. Finally, factor(s) other than PGC-1α and transcription may regulate FNDC5 expression.

Sex hormones and skeletal muscle weakness.

Human ageing is accompanied with deterioration in endocrine functions the most notable and well characterized of which being the decrease in the production of sex hormones. Current research literature suggests that low sex hormone concentration may be among the key mechanism for sarcopenia and muscle weakness. Within the European large scale MYOAGE project, the role of sex hormones, estrogens and testosterone, in causing the aging-related loss of muscle mass and function was further investigated. Hormone replacement therapy (HRT) in women is shown to diminish age-associated muscle loss, loss in fast muscle function (power), and accumulation of fat in skeletal muscle. Further HRT raises the protein synthesis rate in skeletal muscle after resistance training, and has an anabolic effect upon connective tissue in both skeletal muscle and tendon, which influences matrix structure and mechanical properties. HRT influences gene expression in e.g. cytoskeletal and cell-matrix proteins, has a stimulating effect upon IGF-I, and a role in IL-6 and adipokine regulation. Despite low circulating steroid-hormone level, postmenopausal women have a high local concentration of steroidogenic enzymes in skeletal muscle.

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.

Functional characterization of B class MADS-box transcription factors in Gerbera hybrida.

According to the classical ABC model, B-function genes are involved in determining petal and stamen development. Most core eudicot species have B class genes belonging to three different lineages: the PI, euAP3, and TM6 lineages, although both Arabidopsis and Antirrhinum appear to have lost their TM6-like gene. Functional studies were performed for three gerbera (Gerbera hybrida) B class MADS-box genes--PI/GLO-like GGLO1, euAP3 class GDEF2, and TM6-like GDEF1--and data are shown for a second euAP3-like gene, GDEF3. In phylogenetic analysis, GDEF3 is a closely related paralogue of GDEF2, and apparently stems from a duplication common to all Asteraceae. Expression analysis and transgenic phenotypes confirm that GGLO1 and GDEF2 mediate the classical B-function since they determine petal and stamen identities. However, based on assays in yeast, three B class heterodimer combinations are possible in gerbera. In addition to the interaction of GGLO1 and GDEF2 proteins, GGLO1 also pairs with GDEF1 and GDEF3. This analysis of GDEF1 represents the first functional characterization of a TM6-like gene in a core eudicot species outside Solanaceae. Similarly to its relatives in petunia and tomato, the expression pattern and transgenic phenotypes indicate that GDEF1 is not involved in determination of petal identity, but has a redundant role in regulating stamen development.

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.

Muscular transcriptome in postmenopausal women with or without hormone replacement.

The loss of muscle mass and strength with aging is well characterized, but our knowledge of the molecular mechanisms underlying the development of sarcopenia remains incomplete. Although menopause is often accompanied with first signs of age-associated changes in muscle structure and function, the effects of hormone replacement therapy (HRT) or menopause-related decline in estrogen production in the muscles of postmenopausal women is not well understood. Furthermore the knowledge of the global transcriptional changes that take place in skeletal muscle in relation to estrogen status has thus far been completely lacking. We used a randomized double-blinded study design together with an explorative microarray experiment to characterize possible effects of continuous, combined HRT and estrogen deprivation on the skeletal muscle of fifteen women. Here, we report the differential response of both Gene Ontology-annotated biological processes and some individual genes responding differentially to the use or non-use of HRT. Our results revealed transcription level changes in, for example, muscle protein and energy metabolism. In particular, the ubiquitine-proteosome system was found to be effected at several levels. HRT seemed to partially counteract the postmenopause-related transcriptional changes. Our results suggest that during the early postmenopausal years, when there is no counteracting medication available, muscle transcriptome changes notably, whereas HRT appears to slow down this phenomenon and could therefore aid in maintaining proper muscle mass and function after menopause.

Postexercise myostatin and activin IIb mRNA levels: effects of strength training.

PURPOSE: Muscle hypertrophy is likely to result from the cumulative effects of repeated bouts of resistance exercise (RE) on postexercise molecular responses. Therefore, we determined muscle growth- and regeneration-related mRNA expression in response to a single RE bout both before and after a strength-training (ST) period. By means of this novel longitudinal setting, we examined whether postexercise gene expression at the transcriptional level is different in the trained and untrained state. METHODS: Eleven untrained healthy older men and 11 controls (age 62.3 +/- 6.3 yr) volunteered as subjects. Muscle biopsies from the vastus lateralis muscle were taken at rest and 1 and 48 h after five sets of 10-repetition leg press RE both before and after 21 wk of supervised ST. RESULTS: Myostatin and myogenin mRNA expression, determined by real-time RT-PCR, increased (P < 0.05) after ST. Conversely, the single RE bout decreased myostatin mRNA after ST, with the decrease showing a negative correlation (r = -0.65, P < 0.05) with the long-term increase in myostatin during ST. Furthermore, RE before ST increased myogenin mRNA (P < 0.05) and tended to increase after ST (P = 0.08). Myostatin receptor activin IIb mRNA levels were decreased at 1 h after RE in the pre-ST condition (P = 0.05) and also tended to decrease in the post-ST condition (P = 0.07). RE-induced downregulation in myostatin mRNA correlated with the ST-induced increase in total body muscle mass (r = -0.82, P = 0.002). CONCLUSIONS: A single bout of RE in older men can downregulate the expression of myostatin receptor activin IIb mRNA. ST influences the response of myostatin to RE, as short-term RE-induced downregulation of myostatin was observed only after ST. The results also indicate that RE-induced alterations in myostatin mRNA expression may have a role in ST-induced muscle hypertrophy.

Toll-like receptor 5 in obesity: the role of gut microbiota and adipose tissue inflammation.

OBJECTIVE: This study aimed at establishing bacterial flagellin-recognizing toll-like receptor 5 (TLR5) as a novel link between gut microbiota composition, adipose tissue inflammation, and obesity. METHODS: An adipose tissue microarray database was used to compare women having the highest (n = 4, H-TLR) and lowest (n = 4, L-TLR) expression levels of TLR5-signaling pathway genes. Gut microbiota composition was profiled using flow cytometry and FISH. Standard laboratory techniques were used to determine anthropometric and clinical variables. In vivo results were verified using cultured human adipocytes. RESULTS: The H-TLR group had higher flagellated Clostridium cluster XIV abundance and Firmicutes-to-Bacteroides ratio. H-TLR subjects had obese phenotype characterized by greater waist circumference, fat %, and blood pressure (P < 0.05 for all). They also had higher leptin and lower adiponectin levels (P < 0.05 for both). Six hundred and sixty-eight metabolism- and inflammation-related adipose tissue genes were differentially expressed between the groups. In vitro studies confirmed that flagellin activated TLR5 inflammatory pathways, decreased insulin signaling, and increased glycerol secretion. CONCLUSIONS: The in vivo findings suggest that flagellated Clostridium cluster XIV bacteria contribute to the development of obesity through distorted adipose tissue metabolism and inflammation. The in vitro studies in adipocytes show that the underlying mechanisms of the human findings may be due to flagellin-activated TLR5 signaling.