The effects of dihydrotestosterone on amino acid transport in ageing mammalian skeletal muscle fibres
Lead Research Organisation:
University of East Anglia
Department Name: Norwich Medical School
Abstract
Ageing leads to a gradual decline in skeletal muscle mass and strength, commonly referred to as sarcopenia. Sarcopenia occurs mostly in people >80yrs (and rodents >2 years old) and its consequences include inactivity, increased susceptibility to falls and eventually to loss of independence. As the number of people over 80yrs in the United Kingdom is expected to double in the next 20 years, from 2.7 million in 2006 to 5.4 million by 2031, the percentage of the population suffering from sarcopenia and the demands this imposes on the NHS and society in general is expected to rise accordingly. Despite its physical and socioeconomic importance, little is known about the causes of sarcopenia. In this study we propose that it occurs because of a decrease in the ability of skeletal muscle cells to transport amino acids (the building blocks of proteins ) into and out of the cells. We also suggest that this decline is due to a reduction in the number of the transporters/pumps that move amino acids into and out of the cells or is due to a decrease in the concentration of hormones such as the male sex hormones that normally stimulate these pumps.
To test these suggestions we plan to investigate; (1) the effects of age on the number of amino acid transporters and their ability to move amino acids into and out of mouse skeletal muscle cells. (2) Whether treating muscle cells with dihydrotestosterone (DHT), the active metabolite of testosterone, can reverse these age-dependent changes. (3) Finally we will test whether treating mice >560 days old with DHT for 4 weeks reverses/slows down sarcopenia. We will also investigate whether DHT treatment increases the transport amino acids and the synthesis of new proteins.
Three types of experiments will be used in the study. The first type will use mice of different ages (range 60-730 days) kept under normal laboratory conditions. Small muscle fibre bundles isolated from the edl and soleus muscles of each age group will then be used to investigate the effects of age on the expression and physiological function of various amino acid transporters and the cellular and molecular mechanisms underlying these changes.
Most previous studies have investigated the effects of testosterone treatment on skeletal muscle mass and strength and no studies have investigated those of DHT treatment. Therefore, the second experiment will investigate the effects of treating young, old and senescent mice with DHT for at least 4 weeks. Small muscle fibre bundles isolated from the edl and soleus of treated and untreated mice will then be used to investigate the effects of DHT treatment on skeletal muscle mass, strength, the expression and function of various amino acid transporters, the uptake of isoleucine (Ile) and MeAIB and the incorporation of Ile into proteins.
In the third experiment, the acute effects of DHT treatment on force production, amino acid transport and incorporation into proteins in muscle fibre bundles isolated from the edl and soleus of senescent mice will be investigated. The cellular and molecular mechanisms underlying these effects will also be investigated.
Most of these experiments will be performed using CD1 mice because they are inexpensive, have similar genetic variations as normal mouse and human populations and suffer from severe sarcopenia from 24 months onwards.
The results from these experiments will provide important information on the role a decline in amino acid transport and reduced bioavailability of anabolic steroids, play in sarcopenia. They will also increase our understanding of the changes that occur in skeletal muscle with ageing and the mechanisms underlying these changes. They will also provide information on whether DHT treatment can delay/slow the development of sarcopenia as well as the mechanisms underlying its effects. These findings will be important in the development of new ways of managing sarcopenia in the future.
To test these suggestions we plan to investigate; (1) the effects of age on the number of amino acid transporters and their ability to move amino acids into and out of mouse skeletal muscle cells. (2) Whether treating muscle cells with dihydrotestosterone (DHT), the active metabolite of testosterone, can reverse these age-dependent changes. (3) Finally we will test whether treating mice >560 days old with DHT for 4 weeks reverses/slows down sarcopenia. We will also investigate whether DHT treatment increases the transport amino acids and the synthesis of new proteins.
Three types of experiments will be used in the study. The first type will use mice of different ages (range 60-730 days) kept under normal laboratory conditions. Small muscle fibre bundles isolated from the edl and soleus muscles of each age group will then be used to investigate the effects of age on the expression and physiological function of various amino acid transporters and the cellular and molecular mechanisms underlying these changes.
Most previous studies have investigated the effects of testosterone treatment on skeletal muscle mass and strength and no studies have investigated those of DHT treatment. Therefore, the second experiment will investigate the effects of treating young, old and senescent mice with DHT for at least 4 weeks. Small muscle fibre bundles isolated from the edl and soleus of treated and untreated mice will then be used to investigate the effects of DHT treatment on skeletal muscle mass, strength, the expression and function of various amino acid transporters, the uptake of isoleucine (Ile) and MeAIB and the incorporation of Ile into proteins.
In the third experiment, the acute effects of DHT treatment on force production, amino acid transport and incorporation into proteins in muscle fibre bundles isolated from the edl and soleus of senescent mice will be investigated. The cellular and molecular mechanisms underlying these effects will also be investigated.
Most of these experiments will be performed using CD1 mice because they are inexpensive, have similar genetic variations as normal mouse and human populations and suffer from severe sarcopenia from 24 months onwards.
The results from these experiments will provide important information on the role a decline in amino acid transport and reduced bioavailability of anabolic steroids, play in sarcopenia. They will also increase our understanding of the changes that occur in skeletal muscle with ageing and the mechanisms underlying these changes. They will also provide information on whether DHT treatment can delay/slow the development of sarcopenia as well as the mechanisms underlying its effects. These findings will be important in the development of new ways of managing sarcopenia in the future.
Technical Summary
The hypotheses to be tested in this study are that; (1) sarcorpenia (the gradual decline in skeletal muscle mass and strength with age) is caused by the progressive failure in amino acid transport into and out of skeletal muscle fibres especially type 2 fibres. (2) This decline in amino acid transport is due to an age-dependent reduction in the number of amino acid transporters or their reduced/altered sensitivity to stimulation by anabolic factors such as the male sex steroids.
To test these hypotheses, we will perform the following investigates; (1) the effects of age on the expression and physiological functions of SNAT2, LAT1 and LAT2 in fast- (type 2) and slow- (type 1) twitch fibres isolated from mice aged between 60 and 730 days. (2) The cellular and molecular mechanisms underlying these effects. (3) The effects of treating type 1 and 2 fibres isolated from mice ~730 days old with DHT on force production, amino acid transporter expression and function as well as the cellular and molecular mechanism underlying these effects. (4) The effects of treating mice >560 days old with DHT for 4 weeks on skeletal muscle mass, strength, the expression and function of SNAT2, LAT1 and LAT2 and protein synthesis. (5) The cellular and molecular mechanisms underlying these effects.
The results from these experiments will provide important information on the role a decline in amino acid transport and reduced bioavailability of anabolic steroids, play in sarcopenia. They will also increase our understanding of the changes that occur in skeletal muscle with ageing and the mechanisms underlying these changes. They will also provide information on whether DHT treatment can delay/slow the development of sarcopenia as well as the mechanisms underlying its effects. The findings from this study will have important implications on in the management of sarcopenia in the future.
To test these hypotheses, we will perform the following investigates; (1) the effects of age on the expression and physiological functions of SNAT2, LAT1 and LAT2 in fast- (type 2) and slow- (type 1) twitch fibres isolated from mice aged between 60 and 730 days. (2) The cellular and molecular mechanisms underlying these effects. (3) The effects of treating type 1 and 2 fibres isolated from mice ~730 days old with DHT on force production, amino acid transporter expression and function as well as the cellular and molecular mechanism underlying these effects. (4) The effects of treating mice >560 days old with DHT for 4 weeks on skeletal muscle mass, strength, the expression and function of SNAT2, LAT1 and LAT2 and protein synthesis. (5) The cellular and molecular mechanisms underlying these effects.
The results from these experiments will provide important information on the role a decline in amino acid transport and reduced bioavailability of anabolic steroids, play in sarcopenia. They will also increase our understanding of the changes that occur in skeletal muscle with ageing and the mechanisms underlying these changes. They will also provide information on whether DHT treatment can delay/slow the development of sarcopenia as well as the mechanisms underlying its effects. The findings from this study will have important implications on in the management of sarcopenia in the future.
Planned Impact
In this study we plan to investigate whether a failure in amino acid transport into mammalian skeletal muscles is the cause of the age-dependent decline in skeletal muscle mass or whether the age-dependent decline in the plasma concentration of anabolic androgenic steroids is the cause. Both of these questions are important biological challenges with huge implications on the lives of many people from all walks of life including;
1) The elderly and their carers
2) Sports and exercise physiologists
3) Patients suffering from chronic illness
4) Athletes
5) Gerontologists
5) Nutritionists and dieticians
6) Veterinary and animal welfare scientists
7) Pharmacologists
8) The pharmaceutical and food industries
9) The NHS/Government
Muscle mass is a key determinant of health, mobility, quality of life and survival in both humans and animals. Therefore, the loss in skeletal muscle mass due to old age (sarcopenia) is often accompanied by frailty, limited mobility and increased susceptibility to falls. Falls cause pain are a strain on the health economy, and can lead to death in the elderly; whereas frailty is the commonest cause of loss of independent living in those over 80 years old. Furthermore, this is a global problem. Therefore, finding ways of delaying the age at which sarcopenia starts will benefit a large proportion of the worlds' population.
For example, in the UK the number of people in England aged over 65 yrs is expected to increase by 60% from 7.8million in 1996 to 12.4 million by 2031; the number of those over 85yrs is expected to increase by 88% from 0.9million to 1.7 million over the same period. Therefore, the number of frail people dependent on carers in the UK and the demands the elderly impose on the NHS and society in general is expected to rise tremendously. It is this group of people plus all their carers (30% of the UK population) who are potential beneficiaries from any breakthroughs we make in our study. Also, increasing the quality of life through healthy ageing has the potential of saving the NHS and the government millions of pounds.
Delaying ageing will also benefit veterinarians interested in the care of companion animals and the owners of such animals.
Testosterone and its synthetic derivatives (commonly known as anabolic-androgenic steroids, AASs) are widely prescribed for the treatment of sarcopenia (muscle wasting due to old age) and cachexia (muscle wasting due to chronic illness). For example, in 2001 it was estimated that there were over 3 million AAS users in the USA alone. Although the number of AAS users in the UK is relatively small (~100,000), it is expected to grow rapidly as the percentage of the population over 65 years old increases. Thus, if we can show that dihydrotestosterone (DHT) is a better anabolic steroid that testosterone, this will open new opportunities for creating new drugs for combating sarcopenia and cachexia. These findings will be of great interest to the food and pharmaceutical industries.
Identifying a compound that can reduce/limit the effects of age on skeletal muscle mass has huge economic potential. For example, in 2001 the trade in anabolic-androgenic steroids, in the USA alone, was worth over $100million and is currently estimated to be over $1billion.
1) The elderly and their carers
2) Sports and exercise physiologists
3) Patients suffering from chronic illness
4) Athletes
5) Gerontologists
5) Nutritionists and dieticians
6) Veterinary and animal welfare scientists
7) Pharmacologists
8) The pharmaceutical and food industries
9) The NHS/Government
Muscle mass is a key determinant of health, mobility, quality of life and survival in both humans and animals. Therefore, the loss in skeletal muscle mass due to old age (sarcopenia) is often accompanied by frailty, limited mobility and increased susceptibility to falls. Falls cause pain are a strain on the health economy, and can lead to death in the elderly; whereas frailty is the commonest cause of loss of independent living in those over 80 years old. Furthermore, this is a global problem. Therefore, finding ways of delaying the age at which sarcopenia starts will benefit a large proportion of the worlds' population.
For example, in the UK the number of people in England aged over 65 yrs is expected to increase by 60% from 7.8million in 1996 to 12.4 million by 2031; the number of those over 85yrs is expected to increase by 88% from 0.9million to 1.7 million over the same period. Therefore, the number of frail people dependent on carers in the UK and the demands the elderly impose on the NHS and society in general is expected to rise tremendously. It is this group of people plus all their carers (30% of the UK population) who are potential beneficiaries from any breakthroughs we make in our study. Also, increasing the quality of life through healthy ageing has the potential of saving the NHS and the government millions of pounds.
Delaying ageing will also benefit veterinarians interested in the care of companion animals and the owners of such animals.
Testosterone and its synthetic derivatives (commonly known as anabolic-androgenic steroids, AASs) are widely prescribed for the treatment of sarcopenia (muscle wasting due to old age) and cachexia (muscle wasting due to chronic illness). For example, in 2001 it was estimated that there were over 3 million AAS users in the USA alone. Although the number of AAS users in the UK is relatively small (~100,000), it is expected to grow rapidly as the percentage of the population over 65 years old increases. Thus, if we can show that dihydrotestosterone (DHT) is a better anabolic steroid that testosterone, this will open new opportunities for creating new drugs for combating sarcopenia and cachexia. These findings will be of great interest to the food and pharmaceutical industries.
Identifying a compound that can reduce/limit the effects of age on skeletal muscle mass has huge economic potential. For example, in 2001 the trade in anabolic-androgenic steroids, in the USA alone, was worth over $100million and is currently estimated to be over $1billion.
Organisations
Publications
Beales ILP
(2014)
Adiponectin inhibits leptin-induced oncogenic signalling in oesophageal cancer cells by activation of PTP1B.
in Molecular and cellular endocrinology
Boncompagni S
(2015)
Membrane glucocorticoid receptors are localised in the extracellular matrix and signal through the MAPK pathway in mammalian skeletal muscle fibres.
in The Journal of physiology
Jefferson M
(2019)
The Autophagy Protein ATG16L1 Is Required for Sindbis Virus-Induced eIF2a Phosphorylation and Stress Granule Formation.
in Viruses
Jones EJ
(2019)
Integrative analysis of Paneth cell proteomic and transcriptomic data from intestinal organoids reveals functional processes dependent on autophagy.
in Disease models & mechanisms
Pearson T
(2021)
Enhanced small neutral but not branched chain amino acid transport after epigenetic sodium coupled neutral amino acid transporter-2 (SNAT2) cDNA expression in myoblasts.
in Journal of cachexia, sarcopenia and muscle
Pérez MH
(2013)
A membrane glucocorticoid receptor mediates the rapid/non-genomic actions of glucocorticoids in mammalian skeletal muscle fibres.
in The Journal of physiology
Treveil A
(2020)
Regulatory network analysis of Paneth cell and goblet cell enriched gut organoids using transcriptomics approaches.
in Molecular omics
Wendowski EO
(2014)
Dihydrotestosterone rescues the age-dependent decline in protein synthesis in mouse fast-twitch skeletal muscle fibres
in Proceedings of the Physiological Society
| Description | This Project investigates the mechanism underlying age-dependent changes in transporter expression and function and whether DHT treatment can reverse the effects of age on the expression and physiological function of amino acid transporters in skeletal muscle. Dr Mutungi has made significant progress to understand the effect of DHT on expression and function of amino acid transporters in young and old muscle. The change in PI in the final year of this project to Dr Penny Powell will direct the remaining aims relating to gene expression, gene transfer into muscle fibres using viruses and intracellular signalling pathways. Preliminary work so far has shown that the amino acid transporters SNAT 2 and LAT2 decrease in expression as muscles age, and the decrease was greater in fast-twitch than in slow-twitch fibres. The first aim is whether treating muscle cells with dihydrotestosterone (DHT), the active metabolite of testosterone, can reverse these age-dependent changes. In experiments so far, the effects of testosterone and DHT on expression transporters SNAT2, LAT2, androgen receptor, 5 alpha reductase 1 and 2, 17 beta dehydoreductase and aromatase were investigated in C2C12 myoblasts and myotubes. SNAT2, LAT2 and MyoD were expressed in myoblast but not myotubes, even after drug treatment, whereas COX IV, a marker of mitochondria, and aromatase were higher in myotubes, but not increased by DHT or testosterone. In vivo experiments in progress in young and old mice use I.P pellets containing a physiological relevant concentration of DHT to look at the effect of steroids on the specified target proteins listed above. In vitro, isolated primary FDB fibres from young mice treated with testosterone and DHT showed a small enhancement of 5 alpha reductase expression, but LAT2 was not detectable by Western blot, and SNAT2 levels were low. Signalling through STAT3, p38 and ERK was not altered. Current studies compare expression in fast (EDL) verses slow (soleus) muscle from young and old mice, and shown increases in 5 alpha reductase 1 and 2 in ageing EDL muscle, and a decrease in soleus muscle |
| Exploitation Route | We will ask whether gene transfer of SNAT2 into aging muscles will restore function. Furthermore, we will test the aims of understanding the signalling mechanisms underlying the effects of DHT on amino acid uptake and protein synthesis in small skeletal muscle fibre by analysing RNA levels by qPCR and microarrays. Since target protein levels are low in muscle, the next series of experiments are now underway to investigate if there is a selective loss of gene expression during muscle ageing. Primers have been designed for RT-PCR to detect mRNA encoding target muscle proteins in C2C12 cells. Quantitative RT-PCR will analyse relative levels of mRNAs in normal and DHT-treated myoblasts and myotubes. Subsequently, fibres isolated from fast twitch and slow twitch muscle from young and old mice mRNA levels will be measued by qRT-PCR. We have supplies of old mice to isolate RNA for qPCR of the selected genes. Functional activity of the transporters in old mice will be investigated using C14 isoleucine uptake in isolated fibres and compared with young mice. We will also look at the respiratory function of the mitochondria in control and treated muscle of young and old mice using the Oxytherm system. Individual fibers are also being stained on coverslips. It appears that SNAT2 disappears from fast-twich fibres, resulting in fibre senescence. To prove that replacement of SNAT2 in ageing muscle will restore function, we will clone SNAT2 cDNA from muscle RNA and construct lentivirus gene transfer vectors to express this protein in post-mitotoic myotubes and muscle fibres in vitro. Dr Powell has shown that lentiviruses can be used to transduce muscle fibres using beta-galactosidase expression. The functional activity of the ectopically expressed SNAT2 cDNA and the effect of DHT, will be measured by amino acid transport and protein synthesis as above. In addition, muscle RNA isolated from treated young and old mice will be used to obtain profiles of cytokine and mTOR pathways by RNA microarray analysis, to demonstrate the effects of DHT on activation of the MAPK, AKT and other pathways in senescent skeletal muscle fibres |
| Sectors | Healthcare,Manufacturing, including Industrial Biotechology |
| Description | Studentship |
| Amount | £78,000 (GBP) |
| Organisation | Seeb |
| Sector | Hospitals |
| Country | Oman |
| Start | 10/2013 |
| End | 08/2016 |
| Description | Training Day: Medicine of The Elderly Doctors |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Professional Practitioners |
| Results and Impact | Approximately 40 Medicine for the Elderly doctors from Norfolk region attended a training day at the Norwich and Norfolk University Hospital which ended with a questions and discussions on how to meet the challenges of an ageing society especially in the East of England. Several innovative and novel ways of meeting the health needs of the elderly in the region were discussed. |
| Year(s) Of Engagement Activity | 2013 |