The ability of insulin like growth factor 1 (IGF-1) to prevent the pathophysiology associated with amyotrophic lateral sclerosis (ALS) is currently being explored with animal models and in clinical trials with patients. Several studies have reported positive effects of IGF-1 in reducing motor neuron death, delaying the onset of motor performance decline, and increasing life span, in SOD-1 mouse models of ALS and in one clinical trial. However, a second clinical trial produced no positive results raising questions about the therapeutic efficacy of IGF-1. To investigate the effect of specific and sustained IGF-1 expression in skeletal muscle or central nervous system on motor performance, life span, and motor neuron survival, human-IGF-1-transgenic mice were crossed with the G93A SOD-1 mutant model of ALS. No significant differences were found in onset of motor performance decline, life span, or motor neuron survival in the spinal cord, between SOD+/IGF-1+ and SOD+/IGF-1- hybrid mice. IGF-1 concentration levels, measured by radioimmunoassay, were found to be highly increased throughout life in the central nervous system (CNS) and skeletal muscle of IGF-1 transgenic hybrid mice. Additionally, increased CNS weight in SOD+ mice crossbred with CNS IGF-1 transgenic mice demonstrates that IGF-1 overexpression is biologically active even after the disease is fully developed. Taken together, these results raise questions concerning the therapeutic value of IGF-1 and indicate that further studies are needed to examine the relationship between methods of IGF-1 administration and its potential therapeutic value.
Amyotrophic Lateral Sclerosis; Insulin-like growth factor-1; motor neuron; cell death; skeletal muscle; transgenic; G93A SOD-1; motor performance
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by a selective degeneration of motor neurons, atrophy, and paralysis of skeletal muscle. Although a significant proportion of familial ALS results from a toxic gain of function associated with dominant SOD1 mutations, the etiology of the disease and its specific cellular origins have remained difficult to define. Here, we show that muscle-restricted expression of a localized insulin-like growth factor (Igf) -1 isoform maintained muscle integrity and enhanced satellite cell activity in SOD1G93A transgenic mice, inducing calcineurin-mediated regenerative pathways. Muscle-specific expression of local Igf-1 (mIgf-1) isoform also stabilized neuromuscular junctions, reduced inflammation in the spinal cord, and enhanced motor neuronal survival in SOD1G93A mice, delaying the onset and progression of the disease. These studies establish skeletal muscle as a primary target for the dominant action of inherited SOD1 mutation and suggest that muscle fibers provide appropriate factors, such as mIgf-1, for neuron survival.
Partial denervation or paralysis of adult skeletal muscle is followed by nerve sprouting, probably due to release of diffusible sprout- inducing activity by inactive muscle. Insulin-like growth factors (IGF1 and IFG2) are candidates for muscle-derived sprouting activity, because (a) they induce neurite growth from peripheral neurons in vitro; and (b) their mRNA levels in adult skeletal muscle increase severalfold after denervation or paralysis. We sought to determine whether the presence of elevated levels of IGFs in innervated adult skeletal muscle was sufficient to produce intramuscular nerve growth. Low concentrations of IGFs induced massive neurite growth from enriched embryonic chick motoneurons in vitro. Half-maximal responses required 0.2 nM IGF2 or IGF1, or 20 nM insulin. Similar hormone binding properties of motoneuron processes in vitro were observed. Exposure of adult rat or mouse gluteus muscle in vivo to low quantities of exogenous IGF2 or IGF1 led to intramuscular nerve sprouting. Numbers of sprouts in IGF-exposed muscles were 10-fold higher than in vehicle- exposed or untreated muscles, and 12.2% of the end plates in IGF- exposed muscle (control: 2.7%) had sprouts growing from them. The nerve growth reaction was accompanied by elevated levels of intramuscular nerve-specific growth-associated protein GAP43. Additional properties of IGF-exposed muscle included modest proliferation of interstitial cells and elevated interstitial J1 immunoreactivity. These results suggest that elevated levels of IGFs in denervated or paralyzed muscle might trigger coordinate regenerative reactions, including nerve sprouting and expression of nerve growth-supporting substrate molecules by activated interstitial cells.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor system. Recent work in rodent models of ALS has shown that insulin-like growth factor-1 (IGF-1) slows disease progression when delivered at disease onset. However, IGF-1’s mechanism of action along the neuromuscular axis remains unclear. In this study, symptomatic ALS mice received IGF-1 through stereotaxic injection of an IGF-1-expressing viral vector to the deep cerebellar nuclei (DCN), a region of the cerebellum with extensive brain stem and spinal cord connections. We found that delivery of IGF-1 to the central nervous system (CNS) reduced ALS neuropathology, improved muscle strength, and significantly extended life span in ALS mice. To explore the mechanism of action of IGF-1, we used a newly developed in vitro model of ALS. We demonstrate that IGF-1 is potently neuroprotective and attenuates glial cell–mediated release of tumor necrosis factor-α (TNF-α) and nitric oxide (NO). Our results show that delivering IGF-1 to the CNS is sufficient to delay disease progression in a mouse model of familial ALS and demonstrate for the first time that IGF-1 attenuates the pathological activity of non-neuronal cells that contribute to disease progression. Our findings highlight an innovative approach for delivering IGF-1 to the CNS.
Current methods of gene transfer for heart disease include injection into heart muscle or intracoronary coronary delivery, approaches that typically provide limited expression and are cumbersome to apply. To circumvent these problems, we selected a transgene, insulin-like growth factor-I (IGF-I), which may, in theory, have favorable effects on heart function when secreted from a remote site. We examined the feasibility and efficacy of skeletal muscle injection of adeno-associated virus 5 encoding IGF-I under Tet regulation (AAV5.IGFI-tet) to treat heart failure. Myocardial infarction (MI) was induced in rats by coronary occlusion; 1 week later, rats with impaired left ventricular (LV) function received 2×1012 genome copies (GC) of AAV5.IGFI-tet in the anterior tibialis muscle, and 4 weeks later, were randomly assigned to receive doxycycline in drinking water to activate IGF-I expression (IGF-On; n=10), or not to receive doxycycline (IGF-Off; n=10). Ten weeks after MI (5 weeks after activation of IGF-I expression), LV size and function were assessed by echocardiography and physiological studies. IGF-On rats showed reduced LV end-systolic dimension (p=0.03) and increased LV ejection fraction (p=0.02). In addition, IGF-On rats showed, before and during dobutamine infusion, increases in cardiac output (p=0.02), stroke work (p=0.0001), LV + dP/dt (p<0.0001), LV relaxation (LV – dP/dt; p=0.03), and systolic arterial blood pressure (p=0.0003). Mean arterial pressure and systemic vascular resistance were unchanged. Activation of IGF-I expression reduced cardiac fibrosis (p=0.048), apoptosis (p<0.0001), and caspase-3/7 activity (p=0.04). Serum IGF-I was increased 5 weeks after transgene activation (p=0.008). These data indicate that skeletal muscle injection of AAV5.IGFI-tet enables tetracycline-activated expression, increases serum IGF-I levels, and improves function of the failing heart.
Lai and colleagues demonstrate that a single intramuscular injection of an AAV5 vector encoding insulin-like growth factor I (IGF-I) under the control of a tetracycline-inducible promoter is able to treat heart failure in a rat model of myocardial infarction. Specifically, the therapy leads to increased circulating levels of IGF-I and reduced cardiac apoptosis and fibrosis.
Insulin-like growth factor (IGF)-I is a critical protein for cell development and growth. Alternative splicing of the igf1 gene gives rise to multiple isoforms. In rodents, proIGF-IA and proIGF-IB have different carboxy-terminal extensions called the E-peptides (EA and EB) and upon further posttranslational processing, produce the identical mature IGF-I protein. Rodent EB has been reported to have mitogenic and motogenic effects independent of IGF-I. However, effects of EA or EB on mature IGF-I, or whether proIGF-IA and proIGF-IB have different properties, have not been addressed. To determine whether the presence of EA or EB affected the distribution and stability of mature IGF-I protein, transient transfections of cDNAs encoding murine IGF-IA, IGF-IB, and mature IGF-I were performed in C2C12 cells, a skeletal muscle cell line. IGF-I secretion was measured by enzyme-linked immunosorbent assay of the media, and did not differ between expression of proIGF-IA, proIGF-IB, or mature IGF-I expression. Next, epitope-tagged constructs were transfected to determine cellular distribution of IGF-I, EA, and EB in the cells throughout the culture. IGF-I was detected in significantly fewer nontransfected cells in cultures transfected with mature IGF-I compared with transfection of proIGF-IA or proIGF-IB. These results demonstrate that EA and EB are not required for IGF-I secretion but that they increase cell entry of IGF-I from the media. This study provides evidence that the EA and EB may modulate IGF-I in addition to having independent activity.
BACKGROUND: Perturbation in a level of any peptide from insulin-like growth factor (IGF) family (ligands, receptors, and binding proteins) seems to be implicated in lung cancer formation; IGF ligands and IGF-I receptor through their mitogenic and anti-apoptotic action, and the mannose 6-phosphate/insulin-like growth factor II receptor (M6-P/IGF-IIR) possibly as a tumor suppressor. MATERIALS AND METHODS: To determine the identity, role, and mutual relationship of IGFs in lung cancer growth and maintenance, we examined IGF's gene (by RT-PCR) and protein (by immunohistochemistry) expression in 69 human lung carcinoma tissues. We also examined IGF-I receptor numbers (Scatchard analysis) and IGF-II production and release (by Western blot) in IGF-II/IGF-IR mRNA positive and negative lung carcinomas. Finally, the potential role of IGF-IR and IGF-II as growth promoting factors in lung cancer was studied using antisense oligodeoxynucleotides that specifically inhibit IGF-IR and IGF-II mRNA. RESULTS: Thirty-two tumors were positive for IGF-I, 39 for IGF-II, 48 for IGF-IR, and 35 for IGFBP-4 mRNA. Seventeen tumors were concomitantly positive for all four IGFs, whereas 34 were positive for IGF-II, IGF-IR, and IGFBP-4 mRNA. An elevated amount of IGF-II peptide was secreted into the growth medium of cell cultures established from five different IGF-II/IGF-IR mRNA positive lung cancer tissues. The cells also expressed elevated numbers of IGF-IR. Nine IGF-II-negative and 19 IGF-II-positive lung cancers of different stages were selected, and M6-P/ IGF-II receptor was determined immunohistochemically. Most of the IGF-II-negative tumors were strongly positive for M6-P/IGF-IIR. IGF-II-positive tumors were mostly negative for M6-P/IGF-II receptors. Antisense oligodeoxynucleotides to IGF-II significantly inhibited, by 25-60%, the in vitro growth of all six lung cancer cell lines. However, the best results (growth inhibition of up to 80%) were achieved with concomitant antisense treatment (to IGF-IR and IGF-II). CONCLUSION: Our data suggest that lung cancer cells produce IGF-IR and IGF-II, which in turn stimulates their proliferation by autocrine mechanism. Cancer cell proliferation can be abrogated or alleviated by blocking the mRNA activity of these genes indicating that an antisense approach may represent an effective and practical cancer gene therapy strategy.
The insulin-like growth factor-1 (IGF-1) gene encodes two isoforms, IGF-1Ea and IGF-1Eb. Both isoforms can regulate skeletal muscle growth and strength. It has been suggested that IGF-Eb may be more potent in promoting skeletal muscle hypertrophy. Precise contractile force regulation is particularly important in the oculomotor system. However, expression of these isoforms in mammalian extraocular muscles (EOMs) is unknown. Here, we examined their expression in rabbit EOMs and the innervating nerve, two potential sources for myogenic growth factors, and compared isoform expression between EOMs and limb skeletal muscles.
Expression of IGF-1 isoforms was quantified by real-time RT-PCR in adult rabbit EOMs, trochlear and ophthalmic nerves, and compared with expression in rabbit limb skeletal muscles. The presence of mature IGF-1 peptide in the muscles was further examined by Western blot.
Both IGF-1Ea and IGF-1Eb were expressed in the EOM and the trochlear nerve. Both isoforms were expressed at significantly higher levels (9- fold) in EOM than in limb skeletal muscle. Transcripts of IGF-1 isoforms, of IGF-1 receptor and of IGF binding proteins showed a gradient distribution along the EOM from proximal to distal. The mature IGF-1 protein showed the same gradient distribution in the EOM.
Expression of relatively abundant amounts of both IGF-1 splicing isoforms in EOMs, and at a significantly higher level than in limb skeletal muscle, underscores the potential relevance of these myogenic growth factors in EOM plasticity and force regulation.
Extraocular Muscle; Insulin-like Growth Factor-1; Mechano-Growth Factor; Oculomotor System; Real-time PCR; Splicing Isoform; Housekeeping Gene
The protracted absence of muscle activation initiates complex cellular and molecular reactions aimed at restoring functional neuromuscular transmission and preventing degenerative processes. A central aspect of these reactions is the sprouting of intramuscular nerves in the vicinity of inactivated muscle fibers. Sprouts emerging from terminal nerve branches and nodes of Ranvier can reestablish functional contacts with inactive muscle fibers, and this is an essential restorative process in pathological conditions of the neuromuscular system. Due to their rapid upregulation in inactive skeletal muscle fibers and their ability to induce nerve sprouting in adult muscle, insulin-like growth factors (IGFs) are candidate signaling molecules to promote restorative reactions in the neuromuscular system. In this study we have exploited the high affinity and specificity of IGF-binding protein 4 (IGF-BP4) and IGF-BP5 for IGF1 and IGF2 to determine whether these growth factors are involved in the nerve sprouting reaction in paralyzed skeletal muscle. In tissue culture experiments with sensory- and motoneurons we demonstrate that the neurite promoting activity of IGF1 is blocked by IGF-BP4, and that a similar IGF-BP-sensitive activity is detected in muscle extracts from paralyzed, but not from control muscle. In in vivo experiments, we show that local delivery of IGF-BP4 to Botulinum toxin A-paralyzed skeletal muscle effectively prevents nerve sprouting in that muscle. Our findings indicate that muscle IGFs play an essential role in intramuscular nerve sprouting. In addition, these findings suggest that IGFs are major signaling factors from inactivated muscle to promote local restorative reactions, including interstitial cell proliferation and nerve sprouting.
Recent studies in experimental animals show that insulin-like growth factor 1 (IGF1) plays a trophic role during development and tissue injury and that microglia are important sources of IGF1. However, little information is available regarding the expression, regulation, and function of IGF1 and related proteins in human brain cells. In the current study, we examined the expression of IGF1 and IGF2 in human microglia in vivo and in vitro.
Expression of IGF1 and IGF2 was examined by immunohistochemistry in post-mortem human brain sections derived from HIV+ and HIV− brains. In primary cultures of human fetal microglia, IGF1 and IGF2 mRNA and protein expression was examined by Q-PCR, ELISA, and Western blot analysis. Additionally, the role of IGF1 and IGF2 in neuroprotection was examined in primary human neuronal glial cultures.
Immunohistochemistry of human brain tissues showed that nonparenchymal cells (vessels and meninges), as well as parenchymal microglia and macrophages were positive for IGF1, in both HIV encephalitis and control brains, while IGF2 was undetectable. Cultured microglia expressed IGF1 mRNA and produced pg/ml levels of IGF1 protein; this was significantly suppressed by proinflammatory mediators, such as lipopolysaccharide (LPS), poly(I:C), and IFNγ. The Th2 cytokines IL-4 and IL-13 had no significant effect, but the cAMP analog (dibutyryl cAMP) significantly increased IGF1 production. In contrast, microglial IGF2 mRNA and protein (determined by Western blot) were upregulated by LPS. IGF1 receptor (IGF1R) immunoreactivity was predominantly expressed by neurons, and both IGF1 and IGF2 significantly protected neurons from cytokine (IL-1/IFNγ) induced death.
Our study in human brain tissues and cells indicates that microglia are important sources of neurotrophic growth factors IGF1 and IGF2, and that microglial activation phenotypes can influence the growth factor expression. Importantly, our results suggest that chronic neuroinflammation and upregulation of proinflammatory cytokines could lead to neurodegeneration by suppressing the production of microglia-derived neuronal growth factors, such as IGF1.
Brain; Cytokines; Growth factors; HIV; Human; IGF1; IGF2; Inflammation; LPS; Microglia
Brain ischemia leads to muscle inactivity-induced atrophy and may exacerbate motor function deficits. Intramuscular insulin-like growth factor I (IGF-I) injection has been shown to alleviate the brain ischemia-induced muscle atrophy and thus improve the motor function. Motor function is normally gauged by the integrity and coordination of the central nervous system and peripheral muscles. Whether brain ischemic regions are adaptively changed by the intramuscular IGF-I injection is not well understood. In this study, the effect of intramuscular IGF-I injection was examined on the central nervous system of brain ischemic rats. Rats were divided into 4 groups: sham control, brain ischemia control, brain ischemia with IGF-I treatment, and brain ischemia with IGF-I plus IGF-I receptor inhibitor treatment. Brain ischemia was induced by right middle cerebral artery occlusion. IGF-I and an IGF-1 receptor inhibitor were injected into the affected calf and anterior tibialis muscles of the treated rats for 4 times. There was an interval of 2 days between each injection. Motor function was examined and measured at the 24 hours and 7 days following a brain ischemia. The affected hind-limb muscles, sciatic nerve, lumbar spinal cord, and motor cortex were collected for examination after euthanizing the rats. IGF-I expression in the central nervous system and affected muscles were significantly decreased after brain ischemia. Intramuscular IGF-I injection increased the IGF-I expression in the affected muscles, sciatic nerve, lumbar spinal cord, and motor cortex. It also increased the p-Akt expression in the affected motor cortex. Furthermore, intramuscular IGF-I injection decreased the neuronal apoptosis and improved the motor function. However, co-administration of the IGF-I receptor inhibitor eliminated these effects. Intramuscular IGF-I injection after brain ischemia attenuated or reversed the decrease of IGF-I in both central and peripheral tissues, and these effects could contribute to neuroprotection and improve motor function.
The actions of insulin-like growth factor 1 (IGF-1) on protein turnover and of the IGF-1 receptor (IGF-1R) were examined in skeletal muscle of rats with chronic renal failure (CRF) and sham operated (SO), pair-fed controls. Acidemia was prevented in CRF rats with NaHCO3. Serum IGF-1 and skeletal muscle IGF-1 and IGF-1 mRNA were reduced in CRF rats. Dose-response studies revealed impaired stimulation of protein synthesis and suppressed inhibition of protein degradation by IGF-1 in epitrochlearis muscle of CRF rats. Neither IGF-1 analogues with low affinity to IGF binding proteins nor proteinase inhibitors obliterated the IGF-1 resistance. In CRF rats, skeletal muscle IGF-1R mRNA was increased; displacement ligand binding studies and affinity labeling of the IGF-1R alpha subunit indicated increased total skeletal muscle IGF-1R number with normal affinity. However, both autophosphorylation of the IGF-1R beta subunit (i.e., IGF-1R tyrosine kinase) and the IGF-1R tyrosine kinase activity towards exogenous insulin receptor substrate-1, a natural substrate for IGF-1R tyrosine kinase, were reduced in CRF fats. These data indicate that in skeletal muscle of CRF rats there is resistance to the IGF-1 effects on protein synthesis and degradation and decreased IGF-1 and IGF-1 mRNA levels; IGF-1R mRNA and number are increased; but activity of IGF-1R tyrosine kinase is impaired. This postreceptor defect may be a cause of the skeletal muscle resistance to IGF-1 in CRF.
The insulin-like growth factor signalling pathway is an important regulator of skeletal muscle growth. We examined the mRNA expression of components of the insulin-like growth factor (IGF) signalling pathway as well as Fibroblast Growth Factor 2 (FGF2) during maturation of myotubes in primary cell cultures isolated from fast myotomal muscle of Atlantic salmon (Salmo salar). The transcriptional regulation of IGFs and IGFBP expression by amino acids and insulin-like growth factors was also investigated. Proliferation of cells was 15% d−1 at days 2 and 3 of the culture, increasing to 66% d−1 at day 6. Three clusters of elevated gene expression were observed during the maturation of the culture associated with mono-nucleic cells (IGFBP5.1 and 5.2, IGFBP-6, IGFBP-rP1, IGFBP-2.2 and IGF-II), the initial proliferation phase (IGF-I, IGFBP-4, FGF2 and IGF-IRb) and terminal differentiation and myotube production (IGF2R, IGF-IRa). In cells starved of amino acids and serum for 72 h, IGF-I mRNA decreased 10-fold which was reversed by amino acid replacement. Addition of IGF-I and amino acids to starved cells resulted in an 18-fold increase in IGF-I mRNA indicating synergistic effects and the activation of additional pathway(s) leading to IGF-I production via a positive feedback mechanism. IGF-II, IGFBP-5.1 and IGFBP-5.2 expression was unchanged in starved cells, but increased with amino acid replacement. Synergistic increases in expression of IGFBP5.2 and IGFBP-4, but not IGFBP5.1 were observed with addition of IGF-I, IGF-II or insulin and amino acids to the medium. IGF-I and IGF-II directly stimulated IGFBP-6 expression, but not when amino acids were present. These findings indicate that amino acids alone are sufficient to stimulate myogenesis in myoblasts and that IGF-I production is controlled by both endocrine and paracrine pathways. A model depicting the transcriptional regulation of the IGF pathway in Atlantic salmon muscle following feeding is proposed.
Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by the mutation of the survival motor neuron 1 (SMN1) gene and deficiency of the SMN protein. Severe SMA mice have abnormal motor function and small, immature myofibers early in development suggesting that SMN protein deficiency results in retarded muscle growth. Insulin-like growth factor 1 (IGF-1) stimulates myoblast proliferation, induces myogenic differentiation and generates myocyte hypertrophy in vitro and in vivo. We hypothesized that increased expression of IGF-1 specifically in skeletal muscle would attenuate disease features of SMAΔ7 mice. SMAΔ7 mice overexpressing a local isoform of IGF-1 (mIGF-1) in muscle showed enlarged myofibers and a 40% increase in median survival compared with mIGF-1-negative SMA littermates (median survival = 14 versus 10 days, respectively, log-rank P = 0.025). Surprisingly, this was not associated with a significant improvement in motor behavior. Treatment of both mIGF-1NEG and mIGF-1POS SMA mice with the histone deacetylase inhibitor, trichostatin A (TSA), resulted in a further extension of survival and improved motor behavior, but the combination of mIGF-1 and TSA treatment was not synergistic. These results show that increased mIGF-1 expression restricted to muscle can modulate the phenotype of SMA mice indicating that therapeutics targeted to muscle alone should not be discounted as potential disease-modifying therapies in SMA. IGF-1 may warrant further investigation in mild SMA animal models and perhaps SMA patients.
Insulin-like growth factor-1 (IGF-1) is essential to hippocampal neurogenesis and the neuronal response to hypoxia/ischemia injury. IGF (IGF-1 and -2) signaling is mediated primarily by the type 1 IGF receptor (IGF-1R) and modulated by six high-affinity binding proteins (IGFBP) and the type 2 IGF receptor (IGF-2R), collectively termed IGF system proteins. Defining the precise cells that express each is essential to understanding their roles. With the exception of IGFBP-1, we found that mouse hippocampus expresses mRNA for each of these proteins during the first 2 weeks of postnatal life. Compared to postnatal day 14 (P14), mRNA abundance at P5 was higher for IGF-1, IGFBP-2, -3, and -5 (by 71%, 108%, 100%, and 98%, respectively), lower for IGF-2, IGF-2R, and IGFBP-6 (by 65%, 78%, and 44%, respectively), and unchanged for IGF-1R and IGFBP-4. Using laser capture microdissection (LCM), we found that granule neurons and pyramidal neurons exhibited identical patterns of expression of IGF-1, IGF-1R, IGF-2R, IGFBP-2, and -4, but did not express other IGF system genes. We then compared IGF system expression in mature granule neurons and their progenitors. Progenitors exhibited higher mRNA levels of IGF-1 and IGF-1R (by 130% and 86%, respectively), lower levels of IGF-2R (by 72%), and similar levels of IGFBP-4. Our data support a role for IGF in hippocampal neurogenesis and provide evidence that IGF actions are regulated within a defined in vivo milieu.
insulin-like growth factor (IGF); hippocampus; dentate gyrus; laser capture microdissection (LCM)
To assess the role of insulin-like growth factors (IGFs) in growth and transformation of normal (myometrium) and tumorous smooth muscle cell (SMC) tissues, in situ hybridization (ISH) analysis for insulin-like growth factor I and II (IGF-I and IGF-II) mRNAs was combined with detection of IGF peptides, their receptors and IGF binding protein-3 (IGFBP-3). mRNAs for both IGFs were detected in smooth muscle cells in normal, benign and malignant SMC tissues, together with the IGF peptides, both IGF receptors and IGFBP-3. This suggests an autocrine role for both IGFs. Leiomyomas had higher IGF-I peptide levels and higher levels of type I IGF receptors than myometrium, supporting the idea that IGFs play a role in the growth and transformation of these tumours. Low-grade leiomyosarcomas contained more IGF-II mRNAs than myometrium and leiomyoma, fewer type II IGF/mannose 6-phosphate receptors and less IGFBP-3 than myometrium and, in addition, fewer IGF-I mRNAs and type I IGF receptors than leiomyoma. Intermediate- and high-grade leiomyosarcomas had intermediate levels of IGF-II mRNAs and peptide, ranging between those in myometrium and low-grade leiomyosarcomas. Thus, growth and transformation of leiomyosarcomas may be regulated by IGF-II, although more markedly in low-grade than in high-grade leiomyosarcomas. In conclusion, the various categories of SMC tissues are associated with a distinct expression pattern of the IGF system. This suggests that each category of SMC tumours arises as a distinct entity and that there is no progression of transformation in these tissues.
Skeletal muscle dysfunction in patients with heart failure (HF) has been linked to impaired growth hormone (GH)/insulin-like growth factor (IGF)-1 signaling. We hypothesized that ventricular assist device (VAD) implantation reverses GH/IGF-1 axis dysfunction and improves muscle metabolism in HF.
Blood and rectus abdominis muscle samples were collected during VAD implantation and explantation from patients with HF and controls. Clinical data were obtained from medical records, biomarkers measured by enzyme-linked immunosorbent assay (ELISA), and gene expression analyzed by reverse transcription and real-time polymerase chain reaction (RT-PCR). Grip strength was assessed by dynamometry. Oxidative capacity was measured using oleate oxidation rates. Muscle fiber type and size were assessed by histology.
Elevated GH (0.27 ± 0.27 versus 3.6 ± 7.7 ng/ml in HF; p = 0.0002) and lower IGF-1 and insulin-like growth factor binding protein (IGFBP)-3 were found in HF (IGF-1, 144 ± 41 versus 74 ± 45 ng/ml in HF, p < 0.05; and IGFBP-3, 3,880 ± 934 versus 1,935 ± 862 ng/ml in HF, p = 0.05). The GH/IGF-1 ratio, a marker of GH resistance, was elevated in HF (0.002 ± 0.002 versus 0.048 ± 0.1 pre-VAD; p < 0.0039). After VAD support, skeletal muscle expression of IGF-1 and IGFBP-3 increased (10-fold and 5-fold, respectively; p < 0.05) accompanied by enhanced oxidative gene expression (CD36, CPT1, and PGC1α) and increased oxidation rates (+1.37-fold; p < 0.05). Further, VAD implantation increased the oxidative muscle fiber proportion (38 versus 54 %, p = 0.031), fiber cross-sectional area (CSA) (1,005 ± 668 versus 1,240 ± 670 μm2, p < 0.001), and Akt phosphorylation state in skeletal muscle. Finally, hand grip strength increased 26.5 ± 27.5 % at 180 days on-VAD (p < 0.05 versus baseline).
Our data demonstrate that VAD implantation corrects GH/IGF-1 signaling, improves muscle structure and function, and enhances oxidative muscle metabolism in patients with advanced HF.
Skeletal muscle; Heart failure; Cardiovascular surgery; Metabolism; Growth factors
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects both upper and lower motor neurons (MN) resulting in weakness, paralysis and subsequent death. Insulin-like growth factor-I (IGF-I) is a potent neurotrophic factor that has neuroprotective properties in the central and peripheral nervous systems. Due to the efficacy of IGF-I in the treatment of other diseases and its ability to promote neuronal survival, IGF-I is being extensively studied in ALS therapeutic trials. This review covers in vitro and in vivo studies examining the efficacy of IGF-I in ALS model systems and also addresses the mechanisms by which IGF-I asserts its effects in these models, the status of the IGF-I system in ALS patients, results of clinical trials, and the need for the development of better delivery mechanisms to maximize IGF-I efficacy. The knowledge obtained from these studies suggests that IGF-I has the potential to be a safe and efficacious therapy for ALS.
Amyotrophic lateral sclerosis; insulin-like growth factor-I; treatment
Muscle is an important target tissue for insulin-like growth factor (IGF) action. The presence of specific, high affinity IGF receptors, as well as the expression of IGF peptides and binding proteins by muscle suggest that a significant component of IGF action in this tissue is mediated through autocrine and/or paracrine mechanisms. To explore autocrine/paracrine action of IGFs in muscle, we studied the regulation of the IGF-I receptor and the expression of IGF peptides during differentiation of the mouse BC3H-1 muscle cell line. Differentiation from myoblasts to myocytes was associated with a 60% decrease in IGF-I receptor sites determined by Scatchard analysis. Analysis of mRNA abundance and protein labeling studies indicated that the decrease in IGF-I receptor sites was associated with similar reductions in IGF-I receptor gene expression and receptor biosynthesis. IGF-II peptide gene expression was detected in myoblasts and increased 15-fold with differentiation; the increase in IGF-II gene expression preceded the decrease in IGF-I receptor gene expression. In contrast, IGF-I peptide gene expression was low in myoblasts and decreased slightly with differentiation. To explore the potential role of endogenous IGF-II in the differentiation-associated decrease in IGF-I receptor expression, we investigated the effects of IGF-II treatment in myoblasts. The addition of IGF-II to undifferentiated myoblasts resulted in downregulation of the IGF-I receptor which was associated with decreased IGF-I receptor biosynthesis and decreased IGF-I receptor mRNA abundance. These studies suggest, therefore, that IGF-I receptor expression during muscle cell differentiation may be regulated, at least in part, through autocrine production of IGF-II.
Human insulin-like growth factor-I (hIGF-I) is a growth factor which is highly resemble to insulin. It is essential for cell proliferation and has been proposed for treatment of various endocrine-associated diseases including growth hormone insensitivity syndrome and diabetes mellitus. In the present study, an efficient plant expression system was developed to produce biologically active recombinant hIGF-I (rhIGF-I) in transgenic rice grains.
The plant-codon-optimized hIGF-I was introduced into rice via Agrobacterium-mediated transformation. To enhance the stability and yield of rhIGF-I, the endoplasmic reticulum-retention signal and glutelin signal peptide were used to deliver rhIGF-I to endoplasmic reticulum for stable accumulation. We found that only glutelin signal peptide could lead to successful expression of hIGF-I and one gram of hIGF-I rice grain possessed the maximum activity level equivalent to 3.2 micro molar of commercial rhIGF-I. In vitro functional analysis showed that the rice-derived rhIGF-I was effective in inducing membrane ruffling and glucose uptake on rat skeletal muscle cells. Oral meal test with rice-containing rhIGF-I acutely reduced blood glucose levels in streptozotocin-induced and Zucker diabetic rats, whereas it had no effect in normal rats.
Our findings provided an alternative expression system to produce large quantities of biologically active rhIGF-I. The provision of large quantity of recombinant proteins will promote further research on the therapeutic potential of rhIGF-I.
Oryza sativa L., plant bioreactor; transgenic plant; recombinant protein; protein targeting; KDEL; IGF-I
Duchenne muscular dystrophy is an X-linked degenerative disorder of muscle caused by the absence of the protein dystrophin. A major consequence of muscular dystrophy is that the normal regenerative capacity of skeletal muscle cannot compensate for increased susceptibility to damage, leading to repetitive cycles of degeneration–regeneration and ultimately resulting in the replacement of muscle fibers with fibrotic tissue. Because insulin-like growth factor I (IGF-I) has been shown to enhance muscle regeneration and protein synthetic pathways, we asked whether high levels of muscle-specific expression of IGF-I in mdx muscle could preserve muscle function in the diseased state. In transgenic mdx mice expressing mIgf-I (mdx:mIgf+/+), we showed that muscle mass increased by at least 40% leading to similar increases in force generation in extensor digitorum longus muscles compared with those from mdx mice. Diaphragms of transgenic mdx:mIgf+/+ exhibited significant hypertrophy and hyperplasia at all ages observed. Furthermore, the IGF-I expression significantly reduced the amount of fibrosis normally observed in diaphragms from aged mdx mice. Decreased myonecrosis was also observed in diaphragms and quadriceps from mdx:mIgf+/+ mice when compared with age-matched mdx animals. Finally, signaling pathways associated with muscle regeneration and protection against apoptosis were significantly elevated. These results suggest that a combination of promoting muscle regenerative capacity and preventing muscle necrosis could be an effective treatment for the secondary symptoms caused by the primary loss of dystrophin.
IGF-I; muscular dystrophy; satellite cells; regeneration; protein kinase B
Preclinical studies have shown synergistic antitumour activity by inhibition of insulin-like growth factor-1 receptor (IGF-1R) and mTOR. The expression of IGF-1R seems to be crucial for this effect. We investigated the safety and efficacy of the combination of the IGF-1R antibody cixutumumab and the mTOR inhibitor temsirolimus in patients with chemotherapy-refractory bone and soft-tissue sarcomas according to IGF-1R expression by immunohistochemistry.
We undertook a multicentre, open-label, phase 2 study in 19 cancer centres in the USA. Patients aged at least 16 years with a histologically confirmed diagnosis of bone or soft-tissue sarcoma were allocated on the basis of IGF-1R expression by immunohistochemistry to one of three treatment groups: IGF-1R-positive soft-tissue sarcoma (group A), IGF-1R-positive bone sarcomas (group B), or IGF-1R-negative bone and soft-tissue sarcoma (group C). Patients received weekly treatment with cixutumumab (6 mg/kg, intravenous) and temsirolimus (25 mg, intravenous flat dose) in 6-week cycles. A Simon optimal two-stage design was used for every arm. The primary endpoint was progression-free survival (PFS) at 12 weeks by intention-to-treat analysis in the first 54 patients assigned to every treatment arm. Although patients still remain on treatment, this trial has completed enrolment and this represents the final analysis. This study is registered with ClinicalTrials.gov, number NCT01016015.
Between Nov 18, 2009, and April 11, 2012, 388 patients were screened for IGF-1R expression and 54 were assigned to each arm. 17 of 54 patients in the IGF-1R-positive soft-tissue sarcoma group (31%; one-sided 95% CI lower bound 21%; two-sided 90% CI 21–43), 19 of 54 in IGF-1R-positive bone sarcoma group (35%; one-sided 95% CI lower bound 24%; two-sided 90% CI 24–47), and 21 of 54 in the IGF-1R-negative group (39%, one-sided 95% CI lower bound 28%; two-sided 90% CI 28–51) were progression free at 12 weeks. On April 6, 2011, the protocol was amended to include three additional patients in the IGF-1R-positive soft-tissue sarcoma group (total of 57 patients) and nine more in the IGF-1R-negative group (total of 63 patients). There were 2546 adverse events reported during the study, 214 (8%) of which were grade 3–4. The most common grade 3–4 toxicities in the 174 treated patients were anaemia in 16 (9%) patients, hyperglycaemia in 18 (10%), hypophosphataemia in 16 (9%), lymphopenia in 25 (14%), oral mucositis in 19 (11%), and thrombocytopenia in 19 (11%).
The combination of cixutumumab and temsirolimus shows clinical activity in patients with sarcoma and forms a basis for future trials. However, IGF-1R expression by immunohistochemistry is not predictive of clinical outcome after treatment with this combination.
National Cancer Institute and Cycle for Survival Fund, Memorial Sloan-Kettering Cancer Center.
Insulin-like growth factor 2 (IGF2) was recently found to play a critical role in memory consolidation in rats and mice, and hippocampal or systemic administration of recombinant IGF2 enhances memory. Here, using a gene therapy-based approach with adeno-associated virus (AAV), we show that IGF2 overexpression in the hippocampus of aged wild-type mice enhances memory and promotes dendritic spine formation. Furthermore, we report that IGF2 expression decreases in the hippocampus of patients with Alzheimer's disease, and this leads us to hypothesize that increased IGF2 levels may be beneficial for treating the disease. Thus, we used the AAV system to deliver IGF2 or IGF1 into the hippocampus of the APP mouse model Tg2576 and demonstrate that IGF2 and insulin-like growth factor 1 (IGF1) rescue behavioural deficits, promote dendritic spine formation and restore normal hippocampal excitatory synaptic transmission. The brains of Tg2576 mice that overexpress IGF2 but not IGF1 also show a significant reduction in amyloid levels. This reduction probably occurs through an interaction with the IGF2 receptor (IGF2R). Hence, IGF2 and, to a lesser extent, IGF1 may be effective treatments for Alzheimer's disease.
Alzheimer's disease; IGF1; IGF2; IGF2R; synaptic plasticity
Heart failure (HF), a debilitating disease in a growing number of adults, exerts structural and neurohormonal changes in both cardiac and skeletal muscles. However, these alterations and their affected molecular pathways remain uncharacterized. Disease progression is known to transform skeletal muscle fiber composition by unknown mechanisms. In addition, perturbation of specific hormonal pathways, including those involving skeletal muscle insulin-like growth factor-1 (IGF-1) and insulin-like growth factor-binding protein-5 (IGFB-5) appears to occur, likely affecting muscle metabolism and regeneration. We hypothesized that changes in IGF-1 and IGFB-5 mRNA levels correlate with the transformation of single–skeletal muscle fiber myosin heavy chain isoforms early in disease progression, making these molecules valuable markers of skeletal muscle changes in heart failure.
Materials and methods
To investigate these molecules during “early” events in HF patients, we obtained skeletal muscle biopsies from New York Heart Association (NYHA) Class II HF patients and controls for molecular analyses of single fibers, and we also quantified isometric strength and muscle size.
There were more (P < 0.05) single muscle fibers coexpressing two or more myosin heavy chains in the HF patients (30% ± 7%) compared to the control subjects (13% ± 2%). IGF-1 and IGFBP-5 expression was fivefold and 15-fold lower in patients with in HF compared to control subjects (P < 0.05), respectively. Strikingly, there was a correlation in IGF-1 expression and muscle cross-sectional area (P < 0.05) resulting in a decrease in whole-muscle quality (P < 0.05) in the HF patients, despite no significant decrease in isometric strength or whole-muscle size.
These data indicate that molecular alterations in myosin heavy chain isoforms, IGF-1, and IGFB-5 levels precede the gross morphological and functional deficits that have previously been associated with HF, and may be used as a predictor of functional outcome in patients.
muscle quality; hybrid fibers; IGF1 and IGFBP-5
The insulin-like growth factor-1 receptor (IGF-1R) pathway is known to play a role in the acquisition of resistance to epidermal growth factor receptor (EGFR)-specific tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC). However, its exact role in TKI resistance has so far remained unclear. Here, we interrogated the hypothesis that the IGF-1R may serve as a biomarker for, and may play a role in, intrinsic resistance to the EGFR-specific TKl gefitinib in NSCLC.
Total-IGF-1R and phosphorylated (p)-IGF-1R expression levels were related to gefitinib sensitivity in 23 NSCLC cell lines. This sensitivity was re-evaluated after knocking down IGF-1R expression and after IGF-1R up-regulation through exogenous IGF-1 expression. The utility of IGF-1R expression as a predictive biomarker was also evaluated by immunohistochemistry (IHC) in 98 primary NSCLC samples from patients treated with gefitinib.
Seventeen of the cell lines tested were resistant to gefitinib, whereas 3 cell lines were sensitive. The three remaining cell lines showed intermediate values. Thirteen resistant cell lines were found to be positive for total-IGF-lR expression, while all the sensitive cell lines were negative, resulting in a positive predictive value (PPV) of 81 % for total-IGF-lR to predict resistance. Seven resistant cell lines exhibited high p-IGF-1R levels, whereas all 3 sensitive cell lines were negative for p-IGF-1R, resulting in a PPV of 100 % for p-IGF-1R to predict resistance. Neither a knock-down of IGF-lR expression nor an activation of the IGF1-R pathway through exogenous IGF-1 expression affected gefitinib sensitivity. In primary NSCLC tissues, IGF-1R expression was found to be significantly higher in patients with progressive disease, i.e., showing gefitinib resistance, as compared to those with a complete or partial response.
IGF-1R acts as a predictor for resistance to gefitinib in NSCLC cell lines and NSCLC patients, but does not seem to play a role in the intrinsic resistance to this drug. High total-IGF-1R and p-IGR-1R levels may predict such a resistance. Since the underlying mechanism does not appear to be related to proliferation induction, alternative pathways should be explored.
NSCLC; EGFR; IGF-1R; Therapy resistance; Biomarkers