Myofibroblasts protect myoblasts from intrinsic apoptosis associated with differentiation via β1 integrin‐PI3K/Akt pathway

Skeletal myoblasts withdrawing from cell cycle is a prerequisite for myodifferentiation, while upon proliferation/differentiation transformation, a large portion of myoblasts will undergo apoptosis. Skeletal fibroblasts, residing in muscle tissue both during and post myogenesis, have been proofed to play pivotal roles in muscle development, while their effect on myoblast apoptosis being coincident with differentiation has not been reported. Using a membrane insert co‐culture system, we studied it and found that the mitochondrial pathway played a crucial role in myoblast apoptosis during differentiation, and fibroblasts promoted not only cell cycle withdrawal but also myoblast survival in a paracrine fashion, which was coupled with upregulations of β1 integrin, phosphorylated Akt and anti‐apoptotic protein Bcl2. To determine the effect of β1 integrin in the process, we transfected myoblasts with siRNA specific for β1 integrin before co‐culture and found that β1 integrin knockdown abolished anti‐apoptotic ability of myoblasts and inhibited Akt activation and Bcl2 expression. Blockage of PI3K/Akt pathway with wortmannin also seriously impaired the protective effect of fibroblasts on myoblasts and fibroblast‐induced Bcl2 expression. The data demonstrated that fibroblasts protected myoblasts from intrinsic apoptosis associated with differentiation, and β1 integrin‐PI3K/Akt pathway activation was required for the process.     

Influences of the environment on the endocrine and paracrine fish growth hormone–insulin‐like growth factor‐I system

Insulin‐like growth factor‐I (IGF‐I) is a key component of the complex system that regulates differentiation, development, growth and reproduction of fishes. The IGF‐I gene is mainly expressed in the liver that represents the principal source of endocrine IGF‐I but also in numerous other organs where the hormone most probably acts in an autocrine–paracrine manner. The primary stimulus for synthesis and release of IGF‐I is growth hormone (GH) from the anterior pituitary. Thus, in analogy to mammals, it is usual to speak of a fish ‘GH–IGF‐I axis'. The GH–IGF‐I system is affected by changes in the environment and probably represents a target of endocrine disrupting compounds (EDC) that impair many physiological processes in fishes. Thus, the review deals with the influences of changes in different environmental factors, such as food availability, temperature, photoperiod, season, salinity and EDCs, on GH gene expression in pituitary, IGF‐I gene expression in liver and extrahepatic sites and the physiological effects resulting from the evoked alterations in endocrine and local IGF‐I. Environmental influences certainly interact with each other but for convenience of the reader they will be dealt with in separate sections. Current trends in GH–IGF‐I research are analysed and future focuses are suggested at the end of the sections.     

IGF‐I regulates the age‐dependent signaling peptide humanin

Aging is influenced by endocrine pathways including the growth hormone/insulin‐like growth factor‐1 (GH/IGF) axis. Mitochondrial function has also been linked to the aging process, but the relevant mitochondrial signals mediating the effects of mitochondria are poorly understood. Humanin is a novel signaling peptide that acts as a potent regulator of cellular stress responses and protects from a variety of in vitro and in vivo toxic and metabolic insults. The circulating levels of humanin decline with age in mice and humans. Here, we demonstrate a negative correlation between the activity of the GH‐IGF axis and the levels of humanin, as well as a positive correlation between humanin and lifespan in mouse models with altered GH/IGF‐I axis. Long‐lived, GH‐deficient Ames mice displayed elevated humanin levels, while short‐lived GH‐transgenic mice have reduced humanin levels. Furthermore, treatment with GH or IGF‐I reduced circulating humanin levels in both mice and human subjects. Our results indicate that GH and IGF are potent regulators of humanin levels and that humanin levels correlate with lifespan in mice. This suggests that humanin represents a circulating mitochondrial signal that participates in modulating the aging process, adding a coordinated mitochondrial element to the endocrine regulation of aging.     

The β3‐integrin endothelial adhesome regulates microtubule‐dependent cell migration

Integrin β3 is seen as a key anti‐angiogenic target for cancer treatment due to its expression on neovasculature, but the role it plays in the process is complex; whether it is pro‐ or anti‐angiogenic depends on the context in which it is expressed. To understand precisely β3's role in regulating integrin adhesion complexes in endothelial cells, we characterised, by mass spectrometry, the β3‐dependent adhesome. We show that depletion of β3‐integrin in this cell type leads to changes in microtubule behaviour that control cell migration. β3‐integrin regulates microtubule stability in endothelial cells through Rcc2/Anxa2‐driven control of active Rac1 localisation. Our findings reveal that angiogenic processes, both in vitro and in vivo, are more sensitive to microtubule targeting agents when β3‐integrin levels are reduced.     

Insulin‐like growth factor‐I prevents caspase‐mediated apoptosis in Schwann cells

Both neurons and glia succumb to programmed cell death (PCD) when deprived of growth factors at critical periods in development or following injury. Insulin‐like growth factor‐I (IGF‐I) prevents apoptosis in neurons in vitro. To investigate whether IGF‐I can protect Schwann cells (SC) from apoptosis, SC were harvested from postnatal day 3 rats and maintained in serum‐containing media until confluency. When cells were switched to serum‐free defined media (DM) for 12–72 h, they underwent PCD. Addition of insulin or IGF‐I prevented apoptosis. Bisbenzamide staining revealed nuclear condensation and formation of apoptotic bodies in SC grown in DM alone, but SC grown in DM plus IGF‐I had normal nuclear morphology. The phosphatidylinositol 3‐kinase (PI 3‐K) inhibitor LY294002 blocked IGF‐I–mediated protection. Caspase‐3 activity was rapidly activated upon serum withdrawal in SC, and the caspase inhibitor BAF blocked apoptosis. These results suggest that IGF‐I rescues SC from apoptosis via PI 3‐K signaling which is upstream from caspase activation. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 540–548, 1999     

Testes‐specific transgene expression in insulin‐like growth factor‐I transgenic mice

Insulin‐like growth factor‐I (IGF‐I) is a low molecular weight peptide that mediates the cell proliferating actions of growth hormone. Evidence exists indicating that IGF‐I is produced by various cell types and this growth factor has been implicated in a variety of reproductive processes. To investigate the effect of IGF‐I over‐expression on reproductive systems, we generated three independent lines of transgenic mice harbouring a human IGF‐I cDNA (hIGF‐I) under the control of a Cytomegalovirus immediate early (CMV) promoter. The CMV promoter was used in an attempt to direct expression of IGF‐I into a variety of tissues both reproductive and non‐reproductive. Yet expression of the foreign hIGF‐I gene, determined by Northern blot, was found to occur only in the testicular tissues of the male mice, apparently due to methylation of the transgene in all the tissues tested except the testes, which demonstrate transgene hypomethylation. Evaluation of the transgene expression during testicular development revealed that expression begins between 10 and 15 days of development, coinciding with the appearance of the zygotene and pachytene primary spermatocytes during early spermatogenesis, therefore indicating germ line expression of the transgene. Extensive study of the CMV‐hIGF‐I transgenic lines of mice has revealed that the effects of the transgene expression do not extend beyond the testicular tissues. No significant differences (P > 0.05) in the IGF‐I serum levels, growth rates, or testicular histology have been observed between transgenic and non‐transgenic male siblings. The ability of transgenic males to produce offspring also appears unaffected. Evaluation of the IGF binding protein (IGFBP) levels in the testicular tissues of CMV‐hIGF‐I transgenic mice by Western ligand blot revealed an increase in the concentration of testicular proteins with molecular weights corresponding to IGFBP‐2 and IGFBP‐3. These results suggest that the testicular over‐expression of IGF‐I induces increased IGFBP localization in this tissue. Inhibition of IGF activity by the IGFBPs would explain the lack of a dramatic physiological effect in the CMV‐hIGF‐I transgenic mice, despite the presence of elevated testicular IGF‐I. The observation that testis specific IGF‐I overexpression induces localization of IGFBPs in this tissue confirms the existence of a well regulated testicular IGF system and supports the convention that this growth factor plays an important role in testicular function. Mol. Reprod. Dev. 54:32–42, 1999. © 1999 Wiley‐Liss, Inc.     

Low‐carbohydrate High‐fat Diets: Regulation of Energy Balance and Body Weight Regain in Rats

The aim of the current investigations was to examine the effects of a low‐carbohydrate high‐fat diet (LC‐HFD) on body weight, body composition, growth hormone (GH), IGF‐I, and body weight regain after stopping the dietary intervention and returning the diet back to standard laboratory chow (CH). In study one, both adolescent and mature male Wistar rats were maintained on either an isocaloric LC‐HFD or CH for 16 days before having their diet switched. In study two, mature rats were maintained on either LC‐HFD or CH for 16 days to determine the effects of the LC‐HFD on fat pad weight. LC‐HFD leads to body weight loss in mature rats (P < 0.01) and lack of body weight gain in adolescent rats (P < 0.01). Despite less body weight, increased body fat was observed in rats maintained on LC‐HFD (P < 0.05). Leptin concentrations were higher (P < 0.05), and IGF‐I (P < 0.01) concentrations were reduced in the LC‐HFD rats. When the diet was returned to CH following LC‐HFD, body weight regain was above and beyond that which was lost (P < 0.01). The LC‐HFD resulted in increased body fat and had a negative effect upon both GH and IGF‐I concentrations, which might have implications for the accretion and maintenance of lean body mass (LBM), normal growth rate and overall metabolic health. Moreover, when the LC‐HFD ceases and a high‐carbohydrate diet follows, more body weight is regained as compared to when the LC‐HFD is consumed, in the absence of increased energy intake.     

IGF‐I deficient mice show reduced peripheral nerve conduction velocities and decreased axonal diameters and respond to exogenous IGF‐I treatment

Although insulin‐like growth factor‐I (IGF‐I) can act as a neurotrophic factor for peripheral neurons in vitro and in vivo following injury, the role IGF‐I plays during normal development and functioning of the peripheral nervous system is unclear. Here, we report that transgenic mice with reduced levels (two genotypes: heterozygous Igf1^+/− or homozygous insertional mutant Igf1^m/m) or totally lacking IGF‐I (homozygous Igf1^−/−) show a decrease in motor and sensory nerve conduction velocities in vivo. In addition, A‐fiber responses in isolated peroneal nerves from Igf1^+/− and Igf1^−/− mice are impaired. The nerve function impairment is most profound in Igf1^−/− mice. Histopathology of the peroneal nerves in Igf1^−/− mice demonstrates a shift to smaller axonal diameters but maintains the same total number of myelinated fibers as Igf1^+/+ mice. Comparisons of myelin thickness with axonal diameter indicate that there is no significant reduction in peripheral nerve myelination in IGF‐I–deficient mice. In addition, in Igf1^m/m mice with very low serum levels of IGF‐I, replacement therapy with exogenous recombinant hIGF‐I restores both motor and sensory nerve conduction velocities. These findings demonstrate not only that IGF‐I serves an important role in the growth and development of the peripheral nervous system, but also that systemic IGF‐I treatment can enhance nerve function in IGF‐I–deficient adult mice. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 142–152, 1999     

Cross‐talk between the insulin‐like growth factor (IGF) axis and membrane integrins to regulate cell physiology

The biology of cross‐talk between activated growth factor receptors and cell‐surface integrins is an area which has attracted much interest in recent years (Schwartz and Ginsberg, 2002 ). This review discusses the relationship between the insulin‐like growth factor (IGF) axis and cell‐surface integrin receptors in the regulation of various aspects of cell physiology. Key to these interactions are signals transmitted between integrins and the IGF‐I receptor (IGF‐IR) when either or both are bound to their cognate ligands and we will review the current state of knowledge in this area. The IGF axis comprises many molecular components and we will also discuss the potential role of these species in cross‐talk with the integrin receptor. With respect to integrin ligands, we will mainly focus on the well‐characterized interactions of the two extracellular matrix (ECM) glycoproteins fibronectin (FN) and vitronectin (VN) with cell‐surface ligands, and, how this affects activity through the IGF axis. However, we will also highlight the importance of other integrin activation mechanisms and their impact on IGF activity. J. Cell. Physiol. 224: 605–611, 2010. © 2010 Wiley‐Liss, Inc.     

Osteoclast‐specific inactivation of the integrin‐linked kinase (ILK) inhibits bone resorption

Bone resorption requires the adhesion of osteoclasts to extracellular matrix (ECM) components, a process mediated by the α_vβ₃ integrin. Following engagement with the ECM, integrin receptors signal via multiple downstream effectors, including the integrin‐linked kinase (ILK). In order to characterize the physiological role of ILK in bone resorption, we generated mice with an osteoclast‐specific Ilk gene ablation by mating mice with a floxed Ilk allele with TRAP‐Cre transgenic mice. The TRAP‐Cre mice specifically excised floxed alleles in osteoclasts, as revealed by crossing them with the ROSA26R reporter strain. Osteoclast‐specific Ilk mutant mice appeared phenotypically normal, but histomorphometric analysis of the proximal tibia revealed an increase in bone volume and trabecular thickness. Osteoclast‐specific Ilk ablation was associated with an increase in osteoclastogenesis both in vitro and in vivo. However, the mutant osteoclasts displayed a decrease in resorption activity as assessed by reduced pit formation on dentin slices in vitro and decreased serum concentrations of the C‐terminal telopeptide of collagen in vivo. Interestingly, compound heterozygous mice in which one allele of Ilk and one allele of the β₃ integrin gene were inactivated (ILK^+/?; β) also had increased trabecular thickness, confirming that β₃ integrin and Ilk form part of the same genetic cascade. Our results show that ILK is important for the function, but not the differentiation, of osteoclasts. J. Cell. Biochem. 110: 960–967, 2010. © 2010 Wiley‐Liss, Inc.     

No Decrease in Free IGF‐I with Increasing Insulin in Obesity‐Related Insulin Resistance

Objective: Different facts suggest that the insulin growth factor (IGF)/ insulin growth factor‐binding protein (IGFBP) system may be regulated by factors other than growth hormone. It has been proposed that, in healthy subjects, free IGF‐I plays a role in glucose metabolism. The role of free IGF‐I in glucose homeostasis in insulin resistance is poorly understood. This study was undertaken to evaluate the effects of acute changes in plasma glucose and insulin levels on free IGF‐I and IGFBP‐1 in obese and non‐obese subjects. Research Methods and Procedures: Nineteen lean and 24 obese subjects were investigated. A frequently sampled intravenous glucose tolerance test was performed. Free IGF‐I and IGFBP‐1 were determined at 0, 19, 22, 50, 100, and 180 minutes. Results: Basal free IGF‐I levels tended to be higher and IGFBP‐1 lower in obese than in lean subjects. IGFBP‐1 levels inversely correlated with basal insulin concentration. To determine the effects of insulin on the availability of free IGF‐I and IGFBP‐1, changes in their plasma concentrations were measured during a frequently sampled intravenous glucose tolerance test. After insulin administration, a significant suppression of free IGF‐I at 22% was observed in lean subjects. In contrast, plasma‐free IGF‐I levels remained essentially unchanged in the obese group. The differences between both groups were statistically significant at 100 minutes (p < 0.01) and 180 minutes (p < 0.05). Serum IGFBP‐1 was suppressed to a similar extent in both groups. Discussion: These data suggest that the concentrations of free IGF‐I and IGFBP‐1 are differentially regulated by obesity. Obesity‐related insulin resistance leads to unsuppressed free IGF‐I levels.     

Tumor Necrosis Factor‐α Stimulates Cell Proliferation in Adipose Tissue‐Derived Stromal‐Vascular Cell Culture: Promotion of Adipose Tissue Expansion by Paracrine Growth Factors

Objective: Elevated levels of tumor necrosis factor‐α (TNF‐α) protein and mRNA have been reported in adipose tissue from obese humans and rodents. However, TNF‐α has catabolic and antiadipogenic effects on adipocytes. Addressing this paradox, we tested the hypothesis that paracrine levels of TNF‐α, alone or together with insulin‐like growth factor‐I (IGF‐I), support preadipocyte development. Research Methods and Procedures: Cultured stromal‐vascular cells from rat inguinal fat depots were exposed to serum‐free media containing insulin and 0.2 nM TNF‐α, 2.0 nM TNF‐α, or 0.2 nM TNF‐α + 1.0 nM IGF‐I at different times during 7 days of culture. Results: TNF‐α inhibited adipocyte differentiation as indicated by a reduction in both immunocytochemical reactivity for the preadipocyte‐specific antigen (AD3; early differentiation marker) and glycerol‐3‐phosphate dehydrogenase activity (late differentiation marker). Early exposure (Days 1 through 3 of culture) to 0.2 nM TNF‐α did not have a long term effect on inhibiting differentiation. Continuous exposure to 0.2 nM TNF‐α from Days 1 through 7 of culture resulted in a 75% increase in cell number from control. There was a synergistic effect of 0.2 nM TNF‐α + 1 nM IGF‐I on increasing cell number by Day 7 of culture to levels greater than those observed with either treatment applied alone. Discussion: These data suggest that paracrine levels (0.2 nM) of TNF‐α alone or in combination with IGF‐I may support adipose tissue development by increasing the total number of stromal‐vascular and/or uncommitted cells within the tissue. These cells may then be recruited to become preadipocytes or may alternatively serve as infrastructure to support adipose tissue growth.     

Growth hormone and gene expression of in vitro‐matured rhesus macaque oocytes

Growth hormone (GH) in rhesus macaque in vitro oocyte maturation (IVM) has been shown to increase cumulus expansion and development of embryos to the 9–16 cell stage in response to 100 ng/ml recombinant human GH (r‐hGH) supplementation during IVM. Although developmental endpoints for metaphase II (MII) oocytes and embryos are limited in the macaque, gene expression analysis can provide a mechanism to explore GH action on IVM. In addition, gene expression analysis may allow molecular events associated with improved cytoplasmic maturation to be detected. In this study, gene expression of specific mRNAs in MII oocytes and cumulus cells that have or have not been exposed to r‐hGH during IVM was compared. In addition, mRNA expression was compared between in vitro and in vivo‐matured metaphase II (MII) oocytes and germinal vesicle (GV)‐stage oocytes. Only 2 of 17 genes, insulin‐like growth factor 2 (IGF2) and steroidogenic acute regulator (STAR), showed increased mRNA expression in MII oocytes from the 100 ng/ml r‐hGH treatment group compared with other IVM treatment groups, implicating insulin‐like growth factor (IGF) and steroidogenesis pathways in the oocyte response to GH. The importance of IGF2 is notable, as expression of IGF1 was not detected in macaque GV‐stage or MII oocytes or cumulus cells. Mol. Reprod. Dev. 77: 353–362, 2010. © 2009 Wiley‐Liss, Inc.     

Differential effects of the trophic factors BDNF,NT‐4, GDNF,and IGF‐I on the isthmo‐optic nucleus in chick embryos

The isthmo‐optic nucleus (ION) of chick embryos is a model system for the study of retrograde trophic signaling in developing CNS neurons. The role of brain‐derived neurotrophic factor (BDNF) is well established in this system. Recent work has implicated neurotrophin‐4 (NT‐4), glial cell line–derived neurotrophic factor (GDNF), and insulin‐like growth factor I (IGF‐I) as additional trophic factors for ION neurons. Here it was examined in vitro and in vivo whether these factors are target‐derived trophic factors for the ION in 13‐ to 16‐day‐old chick embryos. Unlike BDNF, neither GDNF, NT‐4, nor IGF‐I increased the survival of ION neurons in dissociated cultures identified by retrograde labeling with the fluorescent tracer DiI. BDNF and IGF‐I promoted neurite outgrowth from ION explants, whereas GDNF and NT‐4 had no effect. Injections of NT‐4, but not GDNF, in the retina decreased the survival of ION neurons and accelerated cell death in the ION. NT‐4–like immunoreactivity was present in the retina and the ION. Exogenous, radiolabeled NT‐4, but not GDNF or IGF‐I, was retrogradely transported from the retina to the ION. NT‐4 transport was significantly reduced by coinjection of excess cold nerve growth factor (NGF), indicating that the majority of NT‐4 bound to p75 neurotrophin receptors during axonal transport. Binding of NT‐4 to chick p75 receptors was confirmed in L‐cells, which express chick p75 receptors. These data indicate that GDNF has no direct trophic effects on ION neurons. IGF‐I may be an afferent trophic factor for the ION, and NT‐4 may act as an antagonist to BDNF, either by competing with BDNF for p75 and/or trkB binding or by signaling cell death via p75. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 289–303, 2000     

Amyloid β‐induced astrogliosis is mediated by β1‐integrin via NADPH oxidase 2 in Alzheimer's disease

Astrogliosis is a hallmark of Alzheimer′s disease (AD) and may constitute a primary pathogenic component of that disorder. Elucidation of signaling cascades inducing astrogliosis should help characterizing the function of astrocytes and identifying novel molecular targets to modulate AD progression. Here, we describe a novel mechanism by which soluble amyloid‐β modulates β1‐integrin activity and triggers NADPH oxidase (NOX)‐dependent astrogliosis in vitro and in vivo. Amyloid‐β oligomers activate a PI3K/classical PKC/Rac1/NOX pathway which is initiated by β1‐integrin in cultured astrocytes. This mechanism promotes β1‐integrin maturation, upregulation of NOX2 and of the glial fibrillary acidic protein (GFAP) in astrocytes in vitro and in hippocampal astrocytes in vivo. Notably, immunochemical analysis of the hippocampi of a triple‐transgenic AD mouse model shows increased levels of GFAP, NOX2, and β1‐integrin in reactive astrocytes which correlates with the amyloid β‐oligomer load. Finally, analysis of these proteins in postmortem frontal cortex from different stages of AD (II to V/VI) and matched controls confirmed elevated expression of NOX2 and β1‐integrin in that cortical region and specifically in reactive astrocytes, which was most prominent at advanced AD stages. Importantly, protein levels of NOX2 and β1‐integrin were significantly associated with increased amyloid‐β load in human samples. These data strongly suggest that astrogliosis in AD is caused by direct interaction of amyloid β oligomers with β1‐integrin which in turn leads to enhancing β1‐integrin and NOX2 activity via NOX‐dependent mechanisms. These observations may be relevant to AD pathophysiology.     

Reductions in serum IGF‐1 during aging impair health span

In lower or simple species, such as worms and flies, disruption of the insulin‐like growth factor (IGF)‐1 and the insulin signaling pathways has been shown to increase lifespan. In rodents, however, growth hormone (GH) regulates IGF‐1 levels in serum and tissues and can modulate lifespan via/or independent of IGF‐1. Rodent models, where the GH/IGF‐1 axis was ablated congenitally, show increased lifespan. However, in contrast to rodents where serum IGF‐1 levels are high throughout life, in humans, serum IGF‐1 peaks during puberty and declines thereafter during aging. Thus, animal models with congenital disruption of the GH/IGF‐1 axis are unable to clearly distinguish between developmental and age‐related effects of GH/IGF‐1 on health. To overcome this caveat, we developed an inducible liver IGF‐1‐deficient (iLID) mouse that allows temporal control of serum IGF‐1. Deletion of liver Igf ‐1 gene at one year of age reduced serum IGF‐1 by 70% and dramatically impaired health span of the iLID mice. Reductions in serum IGF‐1 were coupled with increased GH levels and increased basal STAT5B phosphorylation in livers of iLID mice. These changes were associated with increased liver weight, increased liver inflammation, increased oxidative stress in liver and muscle, and increased incidence of hepatic tumors. Lastly, despite elevations in serum GH, low levels of serum IGF‐1 from 1 year of age compromised skeletal integrity and accelerated bone loss. We conclude that an intact GH/IGF‐1 axis is essential to maintain health span and that elevated GH, even late in life, associates with increased pathology.