Age-related loss of muscle mass and strength.

Age-related muscle wasting and increased frailty are major socioeconomic as well as medical problems. In the quest to extend quality of life it is important to increase the strength of elderly people sufficiently so they can carry out everyday tasks and to prevent them falling and breaking bones that are brittle due to osteoporosis. Muscles generate the mechanical strain that contributes to the maintenance of other musculoskeletal tissues, and a vicious circle is established as muscle loss results in bone loss and weakening of tendons. Molecular and proteomic approaches now provide strategies for preventing age-related muscle wasting. Here, attention is paid to the role of the GH/IGF-1 axis and the special role of the IGFI-Ec (mechano growth factor/MGF) which is derived from the IGF-I gene by alternative splicing. During aging MGF levels decline but when administered MGF activates the muscle satellite (stem) cells that "kick start" local muscle repair and induces hypertrophy.

Mechano Growth Factor E peptide (MGF-E), derived from an isoform of IGF-1, activates human muscle progenitor cells and induces an increase in their fusion potential at different ages.

Loss of muscle mass and strength is a major problem during aging and the expression of Mechano Growth Factor (MGF), a member of the IGF-1 (insulin-like Growth Factor 1) super family, has been shown to be both exercise and age dependent. MGF, also called IGF-1Ec, has a unique E domain with a 49bp insert in humans (52bp in rodents; IGF-1Eb), which results in a reading frame shift during the IGF-1 gene splicing to produce a distinct mature isoform. We have studied the effects of the MGF-24aa-E peptide on proliferation and differentiation of primary human muscle cell cultures isolated from healthy subjects of different ages. We found that MGF-E peptide significantly increases the proliferative life span and delays senescence of satellite cells isolated from neonatal and young adult but not from old adult muscle, hypertrophy associated with a significant decrease in the percentage of reserve cells was observed in all cultures. It is concluded that the MGF-24aa-E peptide alone has a marked ability to enhance satellite cell activation, proliferation and fusion for muscle repair and maintenance and could provide a new strategy to combat age related sarcopenia without the oncogenic side effects observed for IGF1.

Growth factors, muscle function, and doping.

This article discusses the inevitable use of growth factors for enhancing muscle strength and athletic performance. Much effort has been expended on developing a treatment of muscle wasting associated with a range of diseases and aging. Frailty in the aging population is a major socioeconomic and medical problem. Emerging molecular techniques have made it possible to gain a better understanding of the growth factor genes and how they are activated by physical activity. The ways that misuse of growth factors may be detected and verified in athletes and future challenges for detecting manipulation of signaling pathways are discussed.

Investigation of MGF mRNA expression in patients with amyotrophic lateral sclerosis using parallel in vivo and in vitro approaches.

In an animal model of ALS, intramuscular administration of MGF, the IGF-I Ec gene splice variant, improved muscle strength and increased both motor unit and motor neuron survival. Here we investigated whether there is a deficit in MGF production in the muscles of patients with ALS. We used complementary in vivo and in vitro techniques to study the IGF-I splice variant response of human muscle to exercise or mechanical stretch. We assessed the levels of MGF and IGF-IEa mRNA in muscle biopsy samples from healthy subjects and patients with ALS, before and after exercise. We used primary muscle cells to build three-dimensional collagen constructs and subjected them to a ramp stretch. Patients with ALS had similar baseline levels of MGF and IGF-IEa mRNA to healthy controls. No up-regulation was seen in either group within a short time of a single bout of low intensity exercise. Three-dimensional human muscle constructs also detected no response to a mechanical stretch from either control subjects or ALS. We conclude that the pathology of ALS does not include a deficit in baseline levels of MGF and IGF-IEa mRNA splice variants in muscle.

Serum IGF-I levels and IGF-I gene splicing in muscle of healthy young males receiving rhGH.

OBJECTIVE: Elevated growth hormone (GH) levels lead to increased circulating insulin-like growth factor-I (IGF-I), but the effects on localised muscle IGF-I splice variant expression is not known. The effects of rhGH administration, with or without an acute bout of high resistance exercise, were measured on serum IGF-I and on the mRNA levels of IGF-I splice variants in the vastus lateralis muscle of healthy young men. DESIGN: The study was a randomised double blind trial with a crossover design. Seven subjects were randomly assigned to a group receiving daily injections of rhGH (0.075IU kg(-1)day(-1)) or placebo for a two week period. Following a one month washout, the groups were reversed. RESULTS: Administration of rhGH increased circulating IGF-I from 31.8+/-3.2 to 109+/-5.4 nmol/L (p<0.05). There was no effect of the exercise bout. RNA was extracted from muscle biopsies obtained from exercised and non-exercised legs 2.5h after the cessation of the exercise. Transcript expression was measured using Real-time QPCR. There was no effect of either exercise or rhGH administration on IGF-I 5' (Class 1 or Class 2) or 3' (IGF-IEa, or MGF) transcripts. CONCLUSION: Although rhGH administration has an effect on liver IGF-I expression, as shown by increase in circulating IGF-I, muscle IGF-I expression is unaffected in young healthy subjects with normal GH profile. The findings contrast with those of a previous study in which GH deficient elderly men showed higher muscle IGF-I 3' splice variant levels following rhGH administration with and without resistance training. Unlike in the liver, muscle Class1 and 2 IGF-I expression do not change significantly following administration of rhGH.

Mechano-growth factor, an IGF-I splice variant, rescues motoneurons and improves muscle function in SOD1(G93A) mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motoneuron degeneration. Although viral delivery of IGF-I has shown therapeutic efficacy in the SOD1(G93A) mouse model of ALS, clinical trials of IGF-I in ALS patients have led to conflicting results. Here we examine the effects of an IGF-I splice variant, mechano-growth factor (MGF) which has previously been shown to have greater neuroprotective effects than IGF-I in a number of models of neurodegeneration. A mammalian expression plasmid containing either MGF or, for comparison, the IGF-I cDNA sequence was delivered to the hindlimb muscles of SOD1(G93A) mice at 70 days of age, at symptom onset. Treatment with either IGF-I or MGF resulted in a significant improvement in hindlimb muscle strength, and an increase in motor unit and motoneuron survival. Significantly more motoneurons survived in MGF treated mice.

A proteomic approach combining MS and bioinformatic analysis for the detection and identification of biomarkers of administration of exogenous human growth hormone in humans.

An integrated MS-based proteomic approach is described that combines MALDI-MS and LC-MS with artificial neural networks for the identification of protein and peptide biomarkers associated with recombinant human growth hormone (rhGH) administration. Serum from exercised males administered with rhGH or placebo was analysed using ELISA to determine insulin-like growth factor-I concentrations. Diluted serum from rhGH- and placebo-treated subjects was analysed for protein biomarkers by MALDI-MS, whereas LC-MS was used to analyse tryptically digested ACN-depleted serum extracts for peptide biomarkers. Ion intensities and m/z values were used as inputs to artificial neural networks to classify samples into rhGH- and placebo-treated groups. Six protein ions (MALDI-MS) correctly classified 96% of samples into their respective groups, with a sensitivity of 91% (20 of 22 rhGH treated) and specificity of 100% (24 of 24 controls). Six peptide ions (LC-MS) were also identified and correctly classified 93% of samples with a sensitivity of 90% (19 of 21 rhGH treated) and a specificity of 95% (20 of 21 controls). The peptide biomarker ion with the highest significance was sequenced using LC-MS/MS and database searching and found to be associated with leucine-rich α-2-glycoprotein.

Growth factors, muscle function and doping.

Recently much interest has been shown in developing a treatment of muscle wasting associated with a range of diseases as well as in ageing, which are major medical and socioecomonic problems. Emerging molecular techniques have made it possible to gain a better understanding of the growth factor genes involved and how they are activated by physical activity including the IGF-I gene that can be spliced to give rise to different isoforms, one of which is called MGF that activates muscle progenitor cells that provide the extra nuclei required for muscle hypertrophy, repair and maintenance. This fact that MGF 'kick starts' the hypertrophy process clearly has potential for abuse and has already attracted the attention of body builders.

Mechano-growth factor reduces loss of cardiac function in acute myocardial infarction.

BACKGROUND: Mechano-growth factor (MGF) is a splice-variant of IGF-I sharing an identical mature region, but with a different E domain. Our objective was to determine if MGF would reduce the area of 'at-risk' myocardium and improve cardiac function in the post-infarct heart. METHODS: Infarcts were induced by injection of microspheres. In experiment 1, sheep were treated with vehicle, 200 nM each of mature IGF-I, MGF E domain, or full-length MGF. In experiment 2, sheep were treated with vehicle or 200 nM of MGF E domain alone. Cardiac function was assessed using echocardiography and sheep were killed eight days post-MI. Evans Blue dye was injected before death to stain the compromised myocardium. Immunohistochemistry was used to assess the abundance of pAkt(T308) and cleaved caspase 3. RESULTS: In experiment 1, cardiac function improved in sheep treated with the MGF E domain, while in experiment 2, MGF E domain preserved cardiac function and there was 35% less compromised cardiac muscle than controls. Furthermore, immunostaining of cleaved caspase 3 was absent in MGF E domain-treated hearts, suggesting that MGF E domain reduced infarct expansion. CONCLUSIONS: We conclude that the E domain of MGF protects the myocardium against ischaemia, thus improving cardiac function post-MI.

Role of insulin-like growth factor binding protein-4 in prevention of colon cancer.

BACKGROUND: Insulin-like growth factors (IGFs) are important for the proliferation of cancer cells. One of their binding proteins, known as insulin-like growth factor binding protein -4 (IGFBP-4) is well known for its inhibitory action on IGFs in vitro. We assessed the effect of IGFBP-4 in prevention of development of colon cancer in vivo. METHODS: Nude mice were subcutaneously inoculated with HT-29 colon cancer cells and they were also simultaneously injected either gene construct containing mammalian expression vector pcDNA3 with or without IGFBP-4 gene or phosphate buffered saline. The effect was assessed 4 weeks later by evaluating the tumours for mitosis, necrosis, apoptosis, and expressions of IGFBP-4, Bcl-2 and Bax proteins. RESULTS: The results showed that the IGFBP-4 gene therapy did not prevent the tumour establishment but it increased the tumour apoptosis which was associated with an increase in Bcl-2 and Bax expressions. The IGFBP-4 protein was low in tumours which received IGFBP-4 gene construct which may be due to a feed back mechanism of IGFBP-4 upon its own cells. CONCLUSION: IGFBP-4 gene therapy in the form localised gene transfer did not prevent colon cancer initiation and establishment but it resulted in increased apoptosis and Bax protein expression and a decrease in tumour cellular mitosis.

Loss of muscle strength during aging studied at the gene level.

Age-related muscle wasting and increased frailty is a major socioeconomic as well as a major medical problem. In our quest to extend the quality of life it is important to increase the strength of elderly people sufficiently so they can carry out everyday tasks and prevent them falling and breaking bones that are brittle because of osteoporosis. Muscles generate the mechanical strain that contributes to the maintenance of other musculoskeletal tissues and a vicious cycle is established when the muscles start to produce less force resulting in more bone loss and weakening of tendons. Another aspect that is less well appreciated is that muscle acts as a dynamic, metabolic store. In a traumatic situation, muscle provides amino acids to aid tissue repair processes and maintaining acid-base balance. At the present time there are strategies in addition to exercise for preventing age-related muscle wasting and these are briefly reviewed. Here, more attention is paid to the role of the growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis and the discovery of mechano-growth factor (MGF). This is derived from the IGF-1 gene by alternative splicing and in the young is responsible for increasing contractile strength in response to exercise by activating the muscle satellite (stem) cells that kick-start local muscle repair and induce hypertrophy. Recent studies including gene transfer of this part of the IGF-1 gene and unique MGF peptides offer the prospect of treating muscle wasting during the aging process as well as muscle cachexia associated with many diseases.

The IGF-I splice variant MGF increases progenitor cells in ALS, dystrophic, and normal muscle.

The effects of muscle splice variants of insulin-like growth factor I (IGF-I) on proliferation and differentiation were studied in human primary muscle cell cultures from healthy subjects as well as from muscular dystrophy and ALS patients. Although the initial numbers of mononucleated progenitor cells expressing desmin were lower in diseased muscle, the E domain peptide of IGF-IEc (MGF) significantly increased the numbers of progenitor cells in healthy and diseased muscle. IGF-I significantly enhances myogenic differentiation whereas MGF E peptide blocks this pathway, resulting in an increased progenitor (stem) cell pool and thus potentially facilitating repair and maintenance of this postmitotic tissue.

Insulin-like growth factor binding protein-4 gene therapy increases apoptosis by altering Bcl-2 and Bax proteins and decreases angiogenesis in colorectal cancer.

Insulin-like growth factors are known to inhibit apoptosis and promote tumour angiogenesis. Previously we have shown that insulin-like growth factor binding protein-4 (IGFBP-4) gene therapy increased apoptosis and decreased mitosis in colon cancer. In this experiment we used HT-29 colon cancer cells to induce subcutaneous cancers in nude mice and administered either the mammalian expression vector with IGFBP-4 insert or vector only around the tumour site. Three weeks after gene transfer, tumours were harvested and expressions of Bax, Bcl-2 and IGF-I receptor in tumours were determined by Western blotting and immunofluorescence. Micro-vessel counting was performed by immunostaining with CD34 and von Willebrand antibodies. Results showed that tumours treated with IGFBP-4 gene had higher expression of Bax, lower expression of Bcl-2 and IGF-I receptor. Bcl-2 was localised to tumour cell cytoplasm while Bax was expressed both in the interstitial area and cytoplasm. IGFBP-4 treatment also decreased micro-vessel count in tumour tissues. Micro-vessels were mainly located in the periphery and interstitial area. This experiment shows that IGFBP-4 gene therapy increases tumour apoptosis via altering the expressions of Bcl-2 and Bax and decreasing the angiogenesis in colorectal cancer.

Skeletal muscle IGF-I isoform expression in healthy women after isometric exercise.

CONTEXT: Little is known about the expression of skeletal muscle-specific isoforms of the insulin-like growth factor-I (IGF-I) gene in response to exercise in humans. Data for women are sparse and the influences of age and physical training status are unknown. Here we aimed to describe the expression of mRNA for the IGF-I isoforms in the quadriceps muscles of women at rest and after isometric exercise. OBJECTIVE: To categorize mRNA expression of the IGF-I splice variants IGF-IEa and IGF-IEc (MGF) in healthy women, determine whether isometric exercise stimulates expression, and to determine the duration of the response. DESIGN: Observational study. SETTING: Clinical research facility. PARTICIPANTS: Six healthy women (median age 38 years, range 25-43 years). INTERVENTION: Single bout of maximal isometric knee extension undertaken as 20 sets of 6 contractions. Muscle samples from the lateral mass of the quadriceps were obtained by needle biopsy at baseline, 2.5h and 50h after exercise. MAIN OUTCOME MEASURES: Quantification of mRNAs for IGF-IEa and MGF. RESULTS: mRNA for the IGF-IEa transcript was significantly elevated by 50h post-exercise compared to baseline (p=0.005) and mRNA for MGF was significantly elevated by 2.5h (p=0.026). There were no statistically significant differences between measurements at 2.5h and 50h for either transcript. CONCLUSIONS: We have shown that the upregulation of the muscle specific IGF-IEa and MGF isoforms occurs within 2.5h of a single bout of isometric exercise in women. The upregulation persists for at least 2 days after exercise. We have also demonstrated a potentially safe and effective way of studying the responsiveness of these isoforms to resistance exercise in future studies of older and/or frail individuals.

Biology of insulin-like growth factor binding protein-4 and its role in cancer (review).

Insulin-like growth factor binding protein-4 (IGFBP-4) is an important member of the insulin-like growth factor (IGF) system. The IGFBP-4 has three domains of which the N-terminal sequence is important for the binding of IGF. It acts as a transport protein for IGF-I and IGF-II and modulates their biological effects. There is increasing evidence that IGFBP-4 inhibits IGF-induced cellular growth both in vitro and in vivo. IGFBP-4 can also mediate its actions through a mechanism independent of IGFs. IGFBP-4 level and expression in various tissues are influenced by IGFBP protease, nutrition, several growth factors and hormones. Overexpression of IGFBP-4 in transgenic animal models causes reduced growth of organs containing smooth muscle. Most cancers express IGFBP-4 at levels which correlate with their state of differentiation. However, the effects of IGFBP-4 on tumor growth are uncertain. In vitro studies have shown that overexpression of IGFBP-4 inhibit the growth of some colon cancer cells. Overexpression of IGFBP-4 in vivo has been reported to decrease the growth of prostate cancer. The effect of altered expression of IGFBP-4 in vivo in colon and other cancers needs to be explored as locally available IGFs appear to stimulate mitogenesis.

Impairment of IGF-I gene splicing and MGF expression associated with muscle wasting.

The characterisation of a local tissue repair factor (mechano growth factor, MGF) that is produced by exercised and/or damaged muscle by differential splicing of the IGF-I gene provides understanding of how muscle is maintained in the young normal individual. Mechano growth factor, or MGF, is different to the systemic IGF-I as it has an insert of 49 base pairs in exon 5 that introduces a reading frame shift resulting in a C terminal peptide with unique properties. Muscle is a post-mitotic tissue and as cell replacement is not a means of tissue repair there has to be an efficient local repair mechanism otherwise the damaged cells undergo cell death. The extra nuclei for muscle repair and hypertrophy are provided by the muscle satellite (stem) cells. The pool of these stem cells is apparently replenished by the action of MGF, which is produced as a pulse following a mechanical challenge. Unfortunately, the production of MGF is deficient in certain diseases such as in the muscular dystrophies in which the mechanotransduction mechanism, which may involve the dystrophin complex, is defective. In elderly muscles, decreased levels of growth hormone apparently mean that there is less primary RNA transcript of the IGF-I gene to be spliced towards MGF. Consequently, there is an increasing inability to maintain muscle mass during ageing. Delivery of MGF and cDNA or peptide produces marked increases in the strength of normal as well as diseased muscle and, therefore, MGF has considerable potential as a generic means of treating muscle cachexia.

A strong neuroprotective effect of the autonomous C-terminal peptide of IGF-1 Ec (MGF) in brain ischemia.

The ischemic stroke is the third leading cause of death in developed countries. The C-terminal peptide of mechano-growth factor (MGF), an alternatively spliced variant of insulin-like growth factor 1 (IGF-1), was found to function independently from the rest of the molecule and showed a neuroprotective effect in vivo and in vitro. In vivo, in a gerbil model of transient brain ischemia, treatment with the synthetic MGF C-terminal peptide provided very significant protection to the vulnerable neurons. In the same model, ischemia evoked increased expression of endogenous MGF in the ischemia-resistant hippocampal neurons, suggesting that the endogenous MGF might have an important neuroprotective function. In an in vitro organotypic hippocampal culture model of neurodegeneration, the synthetic peptide was as potent as the full-length IGF-1 while its effect lasted significantly longer than that of recombinant IGF-1. While two peptides showed an additive effect, the neuroprotective action of the C-terminal MGF was independent from the IGF-1 receptor, indicating a new mode of action for this molecule. Although MGF is known for its regenerative capability in skeletal muscle, our findings demonstrate for the first time a neuroprotective role against ischemia for this specific IGF-1 isoform. Therefore, the C-terminal MGF peptide has a potential to be developed into a therapeutic modality for the prevention of neuronal damage.

Impairment of IGF-I gene splicing and MGF expression associated with muscle wasting.

An aminopeptidase was purified from bovine skeletal muscle by ammonium sulfate fractionation and by successive chromatographies of DEAE-cellulose, Sehacryl S-200, phenyl-sepharose CL-4B, hydroxyapatite and Hi-Trap chelating HP columns. The aminopeptidase was purified about 14-fold over the crude extract with a yield of 1.0% activity. The molecular mass of the enzyme was found to be 58 kDa on SDS-PAGE. The enzyme activity was enhanced by the addition of some anions, such as Cl(-), NO(3)(-) and SCN(-), which is the most unique property of this enzyme. While, the activity was strongly inhibited by bestatin, PMSF and puromycin, suggesting that it was a serine protease. In addition, this enzyme was identical with leukotriene (LT) A4 hydrolase, converting LTA4 to LTB4.