Sarcopenia Age-Related Muscle Wasting and Weakness: Mechanisms and Treatments P42 doc

Growth and mainte-nance of skeletal muscle mass requires IGF-1 from both circulating secreted from liver and locally produced sources.. When the IGF-1 receptor is absent in skeletal musc

muscle expresses Class 1 isoforms (Shemer et al 1992) Both sources of IGF-1 contribute to growth (see Section 2.2) A detailed critique of different genetically modified mouse models to investigate IGF-1 function lies beyond the scope of this Chapter but is reviewed elsewhere (Shavlakadze et al 2005b; Le Roith et al

2001b) This intriguing aspect of the potential roles of the different IGF-1 transcript isoforms awaits further clarification

Meanwhile, the main focus of biological function is on the mature IGF-1 pro-tein Extracellular IGF-1 protein is sequestered and stabilised by binding to IGF-1 binding proteins (IGFBPs): the IGFBPs maintain control of IGF-1 binding to its receptor There are six structurally related IGFBPs located in the vascular and inter-stitial spaces, they are modified by proteases and their tissue specific pattern of expression affects the bioavailability of IGF-1 In skeletal muscle, the most abun-dant are IGFBP-3 and -5, although -4 and -6 are also present: their availability is also influenced by gender and age (Oliver et al 2005)

The action of IGF-1 is mediated by IGF-1 binding to specific receptors on the cell surface, especially the type 1 IGF-1 receptor (IGF-1R) Binding of IGF-1 to the receptor alters the configuration of the receptor subunits and brings the two intrac-ellular motifs together to result in auto-phosphorylation (activation) of the receptor This initiates a complexity of signalling pathways with effects on, not only protein

Rodent IGF-1 gene and IGF-1 isoforms

Class 1 IGF-1Ea

Class 1 IGF-1Eb

Class 2 IGF-1Ea

Class 2 IGF-1Eb

E-41

Mature IGF-1 B C A D

S-48 S-32 S-32

E-35

E-41

Fig 2 Rodent IGF-1 gene and IGF-1 isoforms Simplified diagram to indicate how different isoforms (only four are shown) result from alternative use of transcription start sites in Exon 1 (Class 1) or Exon 2 (Class 2) and from alternative splicing Exons 3 and Exon 4 code for the 70 amino acids of the mature IGF-1 peptide Exon 4 also contains code corresponding to the amino-terminal portion of the E-domain and Exon 5 and Exon 6 each encode distinct E extension pep-tides, termination codons and 3 ¢-untranslated regions Alternative splicing of the exons 4, 5 and 6

yields Ea and Eb variants Subsequently these isoforms are cleaved to produce the mature func-tional 70 amino acid IGF-1 peptide The significance of the initiation and signalling peptides of these IGF-1 isoforms remains to be fully defined (Based upon Shavlakadze et al 2005a, b )

synthesis and degradation resulting in atrophy/hypertrophy (Shavlakadze and Grounds 2006), but also on apoptosis, myoblast proliferation and muscle differen-tiation (Fig 4) IGF-1 binds with greatest affinity to IGF-1R it also binds to the insulin receptor with decreased affinity Thus there can be some redundancy and overlapping function between these molecules in certain situations

2.2 Growth Hormone and IGF-1 Activity

Circulating IGF-1 (mainly produced by the liver) affects muscle, in addition to the locally produced IGF-1 that acts in an autocrine/paracrine way (Fig 3) The circulating levels of IGF-1 are influenced by various factors including the IGFBPs (especially IGFBP-1 and -2) and Growth Hormone (GH) produced by the anterior

Fig 3 Overview of systemic regulation of IGF-1 by growth hormone (GH) Growth and mainte-nance of skeletal muscle mass requires IGF-1 from both circulating (secreted from liver) and locally produced sources The major regulation is attributed to locally produced IGF-1 acting in

an autocrine/paracrine manner Synthesis and secretion of IGF-1 is regulated by GH, which is secreted from the anterior pituitary gland IGF-1 is able to regulate secretion of GH in a classical feedback mechanism (See text for details)

pituitary gland The effects of GH are mediated in large part by IGF-1 in what has been termed the somatomedin hypothesis, although specific actions of GH to induce fusion of myoblasts independently of IGF-1 have been shown (Sotiropoulos

et al 2006) In the revised version of this hypothesis, GH stimulates synthesis and secretion of IGF-1 from the liver, which circulates in blood to downstream targets

In addition, GH stimulates autocrine and paracrine actions of IGF-1 in peripheral tissues, the most likely of which is skeletal muscle (Isgaard et al 1989; Le Roith

et al 2001a; Kaplan and Cohen 2007) In fact autocrine/paracrine actions of IGF-1 predominate over endocrine originating from liver and this was convincingly dem-onstrated when liver specific deletion of IGF-1 failed to inhibit growth of mice despite a 75% reduction in concentrations of IGF-1 in blood (Sjogren et al 1999; Yakar et al 1999) A more recent study has demonstrated that endocrine derived IGF-1 is important and contributes about 30% to adult body size (Stratikopoulos

et al 2008) It is important to note that in mature organism, there is negative feed-back of GH secretion by IGF-I (Giustina and Veldhuis 1998; McMahon et al 2001) which in turn regulates liver production of IGF-I (Fig 3)

GH activates the transcription factor Stat5b, but redundancy with Stat5a is also noted (Teglund et al 1998; Herrington et al 2000) Global deletion of Stat5b pre-vents sexually dimorphic growth in mice and a naturally occurring mutation retarded growth in a girl (Udy et al 1997; Kofoed et al 2003) The importance of

GH acting via autocrine/paracrine stimulation of IGF-1 in skeletal muscle was demonstrated in two elegant studies Targeted deletion of Stat5a and 5b from skel-etal muscle resulted in reduced expression of IGF-1 in skelskel-etal muscle and stunted growth of mice despite normal expression in, and availability of IGF-1 from liver (Klover and Hennighausen 2007) Furthermore, growth of skeletal muscle requires the presence of local IGF-1 and the IGF-1 receptor When the IGF-1 receptor is absent in skeletal muscle, GH does not stimulate growth of skeletal muscle, despite

an increase in circulating concentrations of IGF-1 (Kim et al 2008)

Overall, it appears that IGF-1 plays a major role in growth of all tissues with both endocrine and paracrine sources of IGF-1 playing vital roles in hypertrophy of skeletal muscle (discussed below) In adult muscles, IGF-1 may play a lesser role

in homeostasis of muscle mass The big questions are ‘to what extent does IGF-1

contribute to sarcopenia ’ and ‘can elevated IGF-1 prevent or reverse sarcopenia’.

3 IGF-1 Signalling in Skeletal Muscle

3.1 Classic IGF-1 Signalling, with a Focus on Protein

Synthesis and Degradation

IGF-1 acts via a transmembrane tyrosine kinase receptor to exert its anabolic effect:

it is thought that IGF-1 stimulates muscle growth by promoting myoblast prolifera-tion and their fusion into the myofibres as well as by increasing differentiaprolifera-tion and

protein accretion in the mature myofibres (Florini et al 1991, 1996; Engert et al

1996) Several intracellular signalling pathways mediate the pleiotropic effects of IGF-1 Studies using cultured muscle cells link the mitogenic effect of IGF-1 to the mitogen-activated protein kinase (MAPK) pathway (Coolican et al 1997) and the anabolic effect of IGF-1 on protein accretion to the PI3K/Akt/mTOR pathway (Rommel et al 2001) The effects mediated by these pathways in vivo are very complex and inter-connected

Signalling through the PI3K/Akt pathway plays a fundamental role in control-ling skeletal muscle mass and metabolism (Fig 4) A particular emphasis has been placed on this pathway because it may increase protein synthesis as well as block protein degradation (reviewed in Glass 2005; Shavlakadze and Grounds 2006) Over-expression of constitutively active Akt increases myofibre cross sectional area

IGF-1

mTOR

Amino acids

Protein synthesis

FOXO

Atrophy genes MuRF1, MAFbx

Protein degradation

FOXO

IRS-1 Vit D

VDR

Src Shc

MAPK

Cell Proliferation

AKT

Klotho

Lysosomal autophagy

Lysosomal

autophagy genes

Amino acids Mechanical

loading

Fig 4 Key molecules in IGF-1 signalling pathway in skeletal muscle This highly simplified diagram indicates signalling downstream of the IGF-1 receptor Akt plays a central role as activa-tion (phosphorylaactiva-tion) results in increased protein synthesis and inhibiactiva-tion of protein degradaactiva-tion; this net signalling leads to muscle growth (hypertrophy) Exercise (loading and stretch) and amino acids (from ingested protein) also increase protein synthesis by direct activation of mTOR signal-ling Muscle wasting (atrophy) is not a simple reversal of the Akt/mTOR signalling pathway Instead, atrophy results from other pathways e.g TNF-mediated (not shown) that directly activate the atrophy related genes in the nucleus (by mechanisms independent of FOXO) and also inhibit Akt phosphorylation, hence FOXO is not phosphorylated and remains in the nucleus to activate the atrophy related genes (MuRF1 and MAFbx) The insert tentatively indicates interactions of Klotho and vitamin D with IGF-1 signalling (Based in part on Shavlakadze and Grounds ( 2006 ) and Arthur et al ( 2008 ))

caused by activation of the protein synthesis pathway (Bodine et al 2001; Lai et al

2004) In addition, Akt activation appears to antagonize signalling that leads to muscle atrophy: for example, over-expression of the constitutively active (genetic activation) Akt was sufficient to block muscle wasting following short term (7 days) denervation (Bodine et al 2001) Not much is known about the regulation of protein synthesis and degradation pathways in old muscle Some results suggest diminished responsiveness of old muscle to signalling stimuli controlling protein translation, which may determine a limited ability of old muscle to hypertrophy (Thomson and Gordon 2006; Hwee and Bodine 2009) In response to functional over-loading (caused by synergetic muscle ablation), old rat muscles upregulate Akt, however signal transmission to downstream targets involved in protein synthe-sis machinery is impaired (Hwee and Bodine 2009) Studies in humans demonstrate that protein synthesis rates decrease with age but can be dramatically stimulated by resistance exercise (Yarasheski 2003) The benefits of exercise for the elderly are widely recognised and such mechanical stress (Hornberger et al 2004) can act downstream of IGF-1 via mTOR to increase protein synthesis in a similar manner

to that of amino acids (Fig 4) The extent to which IGF can boost this mTOR-mediated signalling stimulation of protein synthesis (initiated by mechanical load-ing or amino acids), especially in the elderly, remains to be determined

Activation of the PI3K/Akt pathway not only increases protein synthesis, but can also counteract the protein degradation in catabolic states and reduce loss of muscle protein (myofibre atrophy) A common molecular mechanism that increases protein breakdown is revealed by microarray analysis of skeletal muscle undergoing atro-phy induced by different factors (e.g fasting, cancer, acute diabetes, renal failure) and involves induction of the muscle-specific ubiquitin E3-ligases Atrogin-1 and MuRF1, also referred to as atrophy related genes Expression of MAFbx and MuRF1 is suppressed by activation of the PI3K/Akt signalling (Fig 4) and it has been extensively shown that this pathway can counteract the protein degradation in catabolic states and reduce loss of muscle protein (myofibre atrophy) (Bodine et al

2001) The extent to which age-related muscle mass loss is dependent on Atrogin-1 and MuRF1 gene expression is not known While some studies report elevation of atrophy related genes in old muscle (Raue et al 2007) others show suppression of their expression (Edstrom et al 2006)

3.2 Inflammation, TNF and ROS

Various factors are known to inhibit IGF-1 signalling and these include factors associated with inflammation (e.g TNF) and obesity (e.g diglycerides) Many cytokines are altered during inflammation and the pro-inflammatory cytokines tumour necrosis factor (TNF) and interleukin-1 (IL-1) are strongly associated with catabolism and muscle atrophy Such cytokines are responsible for muscle protein degradation in more severe cases of inflammation, such as cancer cachexia, sepsis and AIDS (Reviewed in (Tisdale 2005, 2009)) One of the main mechanisms by

which TNF and IL-1 cause myofibre atrophy is by elevating protein degradation, due to increased atrogene expression mediated by nuclear factor–kappa beta (NFkB) (Tisdale 2005; Messina et al 2006) Muscle wasting produced by TNF is associated with induction of oxidative stress (Tisdale 2005) that can modulate a complexity of interacting signalling pathways to result in muscle atrophy (Reviewed

in (Arthur et al 2008)) TNF can also directly interfere with IGF-1 signalling: TNF may inhibit IGF-1 dependent events by down-regulation of IGF-1 synthesis (Frost

et al 2003) and inhibition of signalling pathways downstream of the IGF-1 receptor leads to decreased protein synthesis and further up-regulation of atrophy related genes (Broussard et al 2003, 2004; Strle et al 2004) Activation of C-Jun N-Terminal Kinase (JNK) appears to play role in both of these processes (Frost

et al 2003; Grounds et al 2008) Because of such cross-talk between TNF and IGF-1 signalling, changes in relative amounts of these cytokines during ageing are important to consider and inverse changes of TNF and IGF-1 are well documented with age Ageing results in chronic low-grade increases in circulating inflammatory cytokines and high plasma levels of TNF and IL-6 are strongly associated with morbidity and mortality in elderly humans (Bruunsgaard and Pedersen 2003; Sandmand et al 2003) However, in some situations IL-6 is clearly anti-inflamma-tory and can decrease systemic TNF levels and it is well documented that exercise increases muscle production of IL-6 and elevates systemic IL-6 (Pedersen 2006,

2007) The fine balance between these cytokines and others appears critical for modulating the precise inflammatory response Human studies show that in the elderly, systemic low-grade inflammation with increased TNF and IL-6 can con-tribute to loss of muscle mass and strength (Visser et al 2002; Schaap et al 2006)

In contrast, serum levels of GH and IGF-1 decrease in old humans and rats (Ullman

et al 1990; Grounds 2002) (discussed in more detail below) Thus, attempts to minimize muscle wasting in various clinical conditions have focused on both anti-inflammatory drugs to block TNF action and development of strategies to deliver IGF-1 to skeletal myofibres

3.3 Lipids

Impaired IGF-1 signalling also results from high levels of lipids within muscles; this contributes to insulin resistance and type 2 diabetes that is of increasing preve-lance in association with obesity and the ageing population (Reviewed (Shavlakadze and Grounds 2006)) It is suggested that a high fat diet activates S6K1 to inhibit signalling downstream of IRS1 (by phosphorylating IRS1 at Ser307 and Ser636/639) and thus suppresses insulin signalling and leads to insulin resistance (Um et al

2004) In addition, increased lipid content within human myofibres correlates with skeletal muscle insulin resistance, and is independent of total body adiposity (Goodpaster and Brown 2005) This correlation is pronounced in patients with type

2 diabetes where myofibres display insulin resistance and significantly increased lipid content (Goodpaster et al 2001) It is suggested that increased lipid deposition

in myofibres per se does not affect insulin sensitivity, but rather represents a marker for the increase of other lipid molecules (such as ceramide, diglyceride, or long-chain acyl-CoA) that may induce defects in the insulin-signalling pathway and muscle insulin resistance [Reviewed (Goodpaster et al 2001; Goodpaster and Brown 2005)] Insulin sensitivity may also be influenced by the oxidative capacity

of skeletal muscle (Reviewed (Goodpaster et al 2001; Goodpaster and Brown

2005)) Ageing is associated with increased fat within myofibres, with healthy non-diabetic subjects showing increasing intramyocellular triacylgycerols with age and this correlates with insulin resistance (Cree et al 2004)

4 Loss of IGF-1 in Ageing Animals

4.1 GH/IGF-1 Axis in Ageing

Concentrations of GH and IGF-1 in blood and GH receptor and IGF-1 mRNA in skeletal muscle decline steadily with age in humans, sheep and rodents (Oldham

et al 1996; Martin et al 1997; Corpas et al 1993; Dardevet et al 1994; Florini et al

1985; Maggio et al 2006; O’Connor et al 1998) In particular, secretion of GH becomes more irregular with age with a decrease in total secretion over a 24 h period and concentrations of IGF-1 decline at a rate of 2 ng per ml per year from

20 to 100 years (O’Connor et al 1998; Maggio et al 2006; Ho et al 1987; Veldhuis

et al 1995) The decline in GH/IGF-1 axis is also correlated with a decline in cog-nitive function, suggesting a causal relationship and a role in neuroprotection (Ceda

et al 2005)

The impact of IGF-1 on the nervous (and other) systems must be considered with respect to maintenance of skeletal muscle mass and function, although detailed examination of this topic lies beyond the scope of this review Loss of motorneu-rone function in the central nervous system will result in loss of axons and neuro-muscular synapses, with subsequent denervation of myofibres (MacIntosh et al

2006) To some extent this problem may be initially countered by sprouting of surviving motorneurones to form new neuromuscular synapses, but even this leads

to some diminution of contractile capacity Progressive motorneurone loss over time will result in permanent denervation with severe myofibre atrophy and loss of function (MacIntosh et al 2006; Edstrom et al 2007) This aspect of sarcopenia provides quite different potential targets for therapeutic interventions but will not

be considered further in this Chapter

While concentrations of IGF-1 decrease in blood, changes in expression of IGF-1 and IGF-1 receptor mRNA is less clear IGF-1Ea and MGF mRNA were not changed in biopsy samples taken from vastus lateralis muscles of young (<30 years) and old (>60 years) humans before and shortly after leg extension exercises

at 80% of one maximum repetition (RM) or before and after eccentric cycling exercise Expression of MGF, but not IGF-1Ea mRNA increased in these muscles

of young men after concentric, and in both young and old men after eccentric

exercise (Hameed et al 2003, 2008) In support, others confirm no change in expression of either IGF-1 transcripts with ageing, but show that expression of both

is increased 24 h after concentric exercise at 1-RM and further increased after 16 weeks of exercise (three times per week) (Petrella et al 2006) MGF is more sensi-tive to exercise and/or injury, which explains the observed increase measured within hours after an acute bout, while there is a delay in IGF-1Ea and the increase

in both transcripts after 16 weeks is consistent with an adaptive response to resis-tance training (Goldspink 2005) In contrast, others do not distinguish between splice-variants of IGF-1 and have observed a decrease in IGF-1 mRNA in muscles

of elderly compared with young men (Marcell et al 2001; Welle et al 2002; Dennis

et al 2008; Leger et al 2008)

IGF-1 receptor numbers decrease in skeletal muscle over 12 months in rats In addition, IGF-1 mRNA also decreased with age and the binding capacity of recep-tors was reduced, which is consistent with a decrease in the function of IGF-1 in muscle (Dardevet et al 1994) Despite the progressive loss of skeletal muscle mass, ageing rat muscles retain the ability to recruit satellite cells and there is an increased density of cell nuclei with centrally located myonuclei in nascent myofibres There was no downregulation of IGF-1 or of IGF-1 receptor mRNA and there was increased mRNA expression of myogenic regulatory factors (Edstrom and Ulfhake

2005): since neither the abundance of IGF-1 protein nor the receptor binding capac-ity were assessed in this study, it is possible that the bioavailabilcapac-ity of IGF-1 was reduced in these aged rat muscles A further factor affecting the ability to regener-ate skeletal muscle during ageing is the decline in the number of motorneurons Ageing motor neurons and the failure to innervate newly formed myofibres is con-sistent with the preferential loss of type II myofibres (Edstrom et al 2007) IGF-1 also promotes angiogenesis and there is a 25% decline in the number of capillaries

in elderly subjects While it is unclear if the decline in capillary density with age is linked to IGF-1, the decline in IGF-1 bioavailability is associated with a reduction

in multiple facets of skeletal muscle integrity which could, collectively, contribute

to sarcopenia (Rogers and Evans 1993; Rabinovsky and Draghia-Akli 2004)

4.2 Nutrition

IGF-1 is secreted as it is synthesised and is directly regulated by nutrition and GH (Schwander et al 1983) Fasting decreases the rate of transcription and the abun-dance of protein and IGF-1 mRNA (Isley et al 1983; Hayden et al 1994) In addi-tion, undernutrition of sheep and cattle (30% of maintenance) is associated with reduced concentrations of IGF-1 in blood and reduced IGF-1 mRNA in skeletal muscle (Breier et al 1986; Jeanplong et al 2003) Paradoxically, there is increased secretion of GH in ruminants and humans during short-term fasting and undernutri-tion, yet secretion of IGF-1 is decreased and secretion in response to exogenous GH

is blunted, which is consistent with refractoriness to GH (Breier et al 1986, 1988; Thissen et al 1994)

The influence of nutrition on the synthesis and secretion of IGF-1 may be a pivotal determinant of the loss of muscle mass during ageing Appetite is progres-sively reduced at a linear rate of 0.5–1% per year from the age of 20–80 and, in conjunction, secretion of IGF-1 is reduced (Wurtman et al 1988; Hallfrisch et al

1990; Briefel et al 1995; Morley 1997; Chapman et al 2002; Chapman 2006,

2007) Despite the progressive decrease in quantity consumed, the proportions of fat, carbohydrate and protein in the diet remain similar (Wurtman et al 1988) Both the energy and protein composition of a diet independently influence secretion of IGF-1 When diets are deficient in either component, secretion of IGF-1 is sup-pressed and secretion is further supsup-pressed when both are inadequate (Isley et al

1983) However, when the protein composition of the diet is restored to greater than 0.9 g per kg BWT per day concentrations of IGF-1 are increased in elderly (>50 years) subjects (Khalil et al 2002; Dawson-Hughes et al 2004) Increasing the protein intake beyond approximately 0.9 g per kg per day does not have any further effect on secretion of IGF-1, which is consistent with the RDA of 0.8 g per kg BWT per day (Roughead et al 2003) However, it has been suggested that this figure is too low and should be increased to 1.6 g per kg BWT per day particularly for people who are active, athletes and the elderly (Evans 2004; Phillips 2006; Campbell and Leidy 2007; Wolfe et al 2008; Paddon-Jones and Rasmussen 2009)

In support, the incidence of protein-energy malnutrition in the elderly has been reported to be 15% in community-dwelling persons, up to 12% of homebound patients, up to 65% of hospitalised patients and up to 85% of institutionalised per-sons (Morley 1997) Therefore, it has been suggested that when overall food intake

is marginal the elderly may benefit from acquiring a greater proportion of energy from the protein portion of the diet with each meal (Evans 2004; Wolfe et al 2008; Paddon-Jones and Rasmussen 2009)

4.3 Calorie Restriction

Calorie restriction (CR) without malnutrition has been shown to prolong lifespan While introduction of CR before skeletal maturity reduces body mass, there is a reduction in the rate of sarcopenia in rhesus monkeys (Colman et al 2008) Moreover, CR extends lifespan and reduces (50% lower) the number of neoplasms and the incidence of cardiovascular disease in rhesus monkeys (Colman et al

2009) CR of 8% prevented the reduction in CSA of the plantaris muscle in rats and the addition of exercise further protected the demise of this muscle and partially prevented the decline in secretion of IGF-1 (Kim et al 2008) Typically, CR reduces concentrations of IGF-1 in rodents In ad libitum fed mice, concentrations of IGF-1

in blood are increased at 24 weeks of age, while in CR mice, concentrations of IGF-1 in blood are unchanged from young controls (3 weeks) Lean mass was pre-served and the mass of adipose tissue was reduced (Huffman et al 2008) In con-trast, CR (28%) for up to 6 years did not alter concentrations of IGF-1 in blood of healthy subjects (mean age 51 years) Only a decrease in protein content in the diet

reduced concentrations of IGF-1 in blood (Fontana et al 2008) A reduced intake

of protein (0.45 g per kg BWT per day) was also shown to decrease concentrations

of IGF-1 and reduce the CSA of type I myofibres over a 10 week period in elderly women (66–79 years) (Castaneda et al 2000) This reinforces the view that the protein portion of the diet should be increased in the elderly given their reduced appetite (see Section 4.2)

Consistently, two interventions, repression of the GH/IGF-1/insulin axis and caloric restriction, have been shown to increase lifespan in both invertebrates and vertebrate animal model systems Longevity is also associated with reduced levels

of the active metabolite of thyroid hormone (T3), which affects metabolism and body temperature and this benefit is attributed to reduced oxidative stress (Buffenstein and Pinto 2009) Furthermore, since T3 influences myosin isoform composition and skeletal muscle function (the impact differs between various muscles and with gender), decreased concentrations of T3 probably contribute to age-related changes in myofibre types and loss of muscle function (Yu et al 1999) The complex interactions between insulin/IGF-1, GH, T3, vitamin D and Klotho is the subject of an excellent review (Buffenstein and Pinto 2009)

4.4 Klotho

The ageing-suppressor gene Klotho was identified in 1997 and found to extend life-span by 20–30% when overexpressed, and to accelerate ageing when disrupted

in mice (Kuro-o et al 1997; Kurosu et al 2005) The 1,014 amino acid protein is present in three isoforms Firstly, as a single-pass transmembrane protein that serves as a receptor for multiple fibroblasts growth factors and a co-receptor for fibroblast growth factor-23 (FGF23), a bone-derived hormone that suppresses vita-min D synthesis Secondly, the extracellular domain comprising the KL1 and KL2 domains can be enzymatically cleaved by the membrane-anchored proteases ADAM10 and ADAM17, which enables the KL1 and KL2 domains to be secreted

to act in an endocrine manner to regulate insulin and IGF-1 signalling pathways along with Wnt (Chen et al 2007) Thirdly, a splice-variant is translated that includes the KL1 domain, but lacks the KL2 domain, and is directly secreted into blood (Chen et al 2007; Kuro-o 2009)

Klotho null mice grow normally to 3 weeks of age, then stop growing, age pre-maturely and die around 8–9 weeks of age This pathology of accelerated ageing is attributed to elevated concentrations of vitamin D, which in turn, promote the absorption of phosphorus and calcium from food in the intestines Consequently, concentrations of phosphate and calcium are also elevated in blood, which contrib-ute to numerous histological changes Notably, there is ectopic calcification in numerous soft tissues, pulmonary emphysema and decreased bone mineral density The ageing process can be rescued by dietary restriction in vitamin D and phos-phate, which implicates hypervitaminosis D and hyperphosphatemia as key regula-tors of ageing regulated by FGF23-Klotho signalling (Kuro-o et al 1997): discussed