Functional adaptation of diaphragm to chronic hyperinflation in emphysematous hamsters

Costal strips of diaphragmatic muscle obtained from animals with elastase-induced emphysema generate maximum tension at significantly shorter muscle fiber lengths than muscle strips from control animals. The present study examined the consequences of alterations in the length-tension relationship assessed in vitro on the pressure generated by the diaphragm in vivo. Transdiaphragmatic pressure (Pdi) and functional residual capacity (FRC) were measured in 22 emphysematous and 22 control hamsters 4-5 mo after intratracheal injection of pancreatic elastase or saline, respectively. In 12 emphysematous and 12 control hamsters Pdi was also measured during spontaneous contractions against an occluded airway. To allow greater control over muscle excitation, Pdi was measured during bilateral tetanic (50 Hz) electrical stimulation of the phrenic nerves in 10 emphysematous and 10 control hamsters. Mean FRC in the emphysematous hamsters was 183% of the value in control hamsters (P less than 0.01). During spontaneous inspiratory efforts against a closed airway the highest Pdi generated at FRC tended to be greater in control than emphysematous hamsters. When control hamsters were inflated to a lung volume approximating the FRC of emphysematous animals, however, peak Pdi was significantly greater in emphysematous animals (70 +/- 6 and 41 +/- 8 cmH2O; P less than 0.05). With electrophrenic stimulation, the Pdi-lung volume curve was shifted toward higher lung volumes in emphysematous hamsters. Pdi at all absolute lung volumes at and above the FRC of emphysematous hamsters was significantly greater in emphysematous compared with control animals. Moreover, Pdi continued to be generated by emphysematous hamsters at levels of lung volume where Pdi of control subjects was zero.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-like growth factor I prevents mannitol-induced degradation of focal adhesion kinase and Akt

In our laboratory, we are interested in hyperosmolarity-induced apoptosis in neuronal cells. We have shown that high concentrations of glucose or mannitol induce apoptotic cell death in dorsal root ganglia in culture and in SH-SY5Y and SH-EP human neuroblastoma cells. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that has a critical role for transmitting integrin-mediated-signals. In this study, we report that hyperosmolar treatment mediates FAK dephosphorylation and cleavage, which is prevented by insulin-like growth factor I (IGF-I) treatment. Mannitol treatment of SH-EP cells transfected with vector (SH-EP/pSFFV) results in concentration- and time-dependent dephosphorylation and degradation of FAK. Dephosphorylation and degradation of FAK are tightly correlated with apoptotic morphological changes, including the disruption of actin stress fibers, the loss of focal adhesion sites, membrane blebbing, and cell detachment. Treatment of SH-EP/pSFFV cells with IGF-I or transfection of IGF-I receptor prevents these changes. Treatment of cells with pharmacologic inhibitors of the mitogen-activated protein kinase or phosphatidylinositol 3-kinase pathways does not affect mannitol-induced FAK dephosphorylation and degradation. However, phosphatidylinositol 3-kinase is necessary for IGF-I-mediated protection against FAK alteration. Mannitol treatment also results in the degradation of Akt. Mannitol induces the activation of caspases-3 and -9 in a time course similar to the dephosphorylation and degradation of FAK. Treatment of the cells with ZVAD, a general caspase inhibitor, blocks the mannitol-induced FAK and Akt degradation as well as cell detachment and apoptosis. These results suggest that one of the pathways of mannitol-mediated apoptosis is through the degradation of FAK and Akt and that IGF-I protects the cells from apoptosis by blocking the activation of caspases, which may be responsible for the loss of FAK and Akt.     

Insulin-like growth factor-I prevents apoptosis in neurons after nerve growth factor withdrawal

Insulin-like growth factor-I (IGF-I) is emerging as an important growth factor able to modulate the programmed cell death (PCD) pathway mediated by the cysteine-dependent aspartate proteases (caspases); however, little is known about the effect of IGF-I after nerve growth factor (NGF) withdrawal in neurons. To begin to understand the neuronal death-sparing effect of IGF-I under NGF-free conditions, we tested whether embryonic sensory dorsal root ganglion neurons (DRG) were able to survive in defined serum-free medium in the presence of IGF-I. We further studied the role of IGF-I signaling and caspase inhibition after NGF withdrawal. NGF withdrawal produced histological changes of apoptosis including chromatin condensation, shrinkage of the perikaryon and nucleus, retention of the plasma membrane, and deletion of single cells. Both IGF-I and Boc-aspartyl (OMe)-fluoromethylketone (BAF), a caspase inhibitor, equally reduced apoptosis after NGF withdrawal. The antiapoptotic effect of IGF-I was completely blocked by LY294002, an inhibitor of PI 3-kinase signaling, but not by the mitogen-activated protein (MAP) kinase/extracellular signal-regulated protein kinase (ERK) activated protein kinase inhibitor PD98059. Functional IGF-I receptors were extensively expressed both in rat and human DRG neurons, although they were most abundant in the neuronal growth cone. Collectively, these findings indicate that IGF-I, signaling though the PI-3 kinase pathway, is important in modulating PCD in cultured DRG neurons after NGF withdrawal, and IGF-I may be important in DRG embryogenesis. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 455–467, 1998     

Insulin-like growth factor I binding and receptor kinase in red and white muscle

IGF-I receptors were partially purified from red and white skeletal muscle by lectin-affinity chromatography and the resultant fraction was depleted of insulin receptors by insulin affinity chromatography. Equilibrium binding of 125I-IGF-I to receptor preparations from red and white muscle yielded identical Scatchard plots. The integrity of the IGF-I receptor preparation in the two fiber types was identical as determined by affinity cross-linking. The tyrosine kinase activity of the receptor from red muscle was 2-3-fold more active towards exogenous substrates in both the basal and ligand-activated states as compared to white muscle. These data show that there is IGF-I-dependent kinase activity intrinsic to IGF-I receptors from skeletal muscle, and suggest that identical cellular factors may regulate the kinase activity of insulin and IGF-I receptors in a parallel manner in vivo.     

Insulin-like growth factor I prevents the development of sensitivity to kainate neurotoxicity in cerebellar granule cells

This study reports that insulin-like growth factor I (IGF-I) prevents cerebellar granule cells from developing sensitivity to kainate neurotoxicity. Sensitivity to kainate neurotoxicity normally develops 5-6 days after switching cultures to a serum-free medium containing 25 mM K(+). Addition of either IGF-I or insulin to the serum-free medium at the time of the switch prevented the development of sensitivity to kainate, whereas brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, and nerve growth factor did not. The dose-response curves indicated IGF-I was more potent than insulin, favoring the assignment of the former as the physiological protective agent. The phosphatidylinositol 3-kinase (PI 3-K) inhibitors wortmannin (10-100 nM) and LY 294002 (0.3-1 microM) abolished the protection afforded by IGF-I. The p70 S6 kinase (p70(S6k)) inhibitor rapamycin (5-50 nM:) also abolished the protection afforded by IGF-I. The activities of both enzymes decreased in cultures switched to serum-free medium but increased when IGF-I was included; wortmannin (100 nM) lowered the activity of PI 3-K from 2 to 5 days after medium switch, whereas rapamycin (50 nM) prevented the increase observed for p70(S6k) activity over the same interval. The mitogen-activated protein kinase kinase inhibitor U 0126 and the mitogen-activated protein kinase inhibitor SB 203580 did not abolish IGF-I protection. Kainate neurotoxicity was not prevented by Joro spider toxin; therefore, the development of kainate neurotoxicity could not be explained by the formation of calcium-permeable alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors. These results indicate that IGF-I functions through a signal transduction pathway involving PI 3-K and p70(S6k) to prevent the development of sensitivity to kainate neurotoxicity in cerebellar granule cells.     

Insulin-like growth factor I levels in healthy children

Measurement of insulin-like growth factor I (IGF-I) levels is used during the assessment of a child for the presence of growth hormone (GH) deficiency and to monitor the efficacy of GH replacement therapy. In either case, robust normative data are required to allow IGF-I values to be expressed as standard deviation scores, enabling comparison between individuals and assessment of change over time. However, IGF-I levels in healthy children are affected by a number of parameters, including age, gender, pubertal status, height, nutrient intake, body composition, intercurrent illness and ethnicity, and the generation of such data requires the collection of samples from significant numbers of healthy children. As external quality assurance schemes for IGF-I and an international standard based on authentic recombinant IGF-I are not widely used, it is imperative for the clinician to understand the performance characteristics and limitations of the IGF-I assay used and to be aware of the source and quality of control data. It must also be recognized that IGF-I measurement is only one component of the diagnostic process and has its limitations, as tissue sensitivity to circulating serum IGF-I levels will differ between individuals.     

Insulin-like growth factor I and erythropoiesis.

The bone marrow, the primary site of hematopoiesis, is a self-renewing system consisting of a unique micro-environment that promotes the differentiation and proliferation of the various hematopoietic cell lines. While many critical factors necessary for red cell production have been identified, the regulation of erythropoiesis has not been completely elucidated. In addition to multi-lineage growth factors (e.g. interleukin 3 or 4) and lineage-specific hematopoietic growth factors (e.g. erythropoietin), several lines of evidence suggest a key role for insulin-like growth factor I (IGF-I). First, growth hormone stimulates erythropoiesis and IGF-I is known to mediate many of growth hormone's actions (somatomedin hypothesis). Second, factors in bovine serum and in serum from an anephric human with erythropoietic activity distinct from erythropoietin have been identified as IGFs. Third, IGF receptors are found on both erythrocyte precursors as well as mature erythrocytes. Fourth, in vitro IGF-I stimulates erythropoiesis in bone marrow cultures. Fifth, IGF-I administration to neonatal or hypophysectomized animals results in increased erythropoiesis in vivo. Recent studies indicate that IGF-I at physiologic concentrations stimulates erythropoiesis and that growth hormone's action is indirect, occurring via IGF-I. The physiologic source of IGF-I for the bone marrow may be delivery from the serum (an endocrine mechanism) or synthesis within the bone marrow by stromal or other cells (a paracrine mechanism). Our recent studies have shown that mouse bone marrow stromal cells secrete both IGF-I and IGF binding proteins (IGFBPs). The role of IGFBPs in erythropoiesis is not known, but they might modulate the local concentration of IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-like growth factor I levels in healthy adults

Insulin-like growth factor I (IGF-I) levels mainly reflect secretion of growth hormone (GH) in the body. The aims of this study were to compare different IGF-I assay methods in healthy individuals, test the reliability of the methods and discuss the utility of IGF-I measurement in adults. The Nichols Institute Diagnostics radioimmunoassay was used to evaluate IGF-I in two random population samples of men and women (aged 25-64 years, n = 392) taken 10 years apart, in 1985 and 1995. This method for IGF-I testing was also compared with an immunoradiometric assay (IRMA) method in 387 men and women participating in the World Health Organization MONICA (MONItoring of trends and determinants for CArdiovascular diseases) Project, Goteborg, Sweden, in 1995. Serum IGF-I decreased with increasing age in both men and women. IGF-I was higher in young women compared with young men in both cohorts, while the opposite was found in the highest age group. Age-adjusted significant correlations were found between IGF-I and smoking, fibrinogen, coffee consumption, lipoprotein (a), osteocalcin and IGF-binding protein 3. The two cohorts showed similar mean IGF-I concentrations irrespective of method. The correlation between the Nichols and the IRMA methods was high: r = 0.93 (p < 0.0001). Based on this and previous studies, population-based IGF-I measurements are robust irrespective of which commercially available method of assay is used. IGF-I levels can be used in diagnosing acromegaly as well as providing target values. IGF-I assay can be used as a complement to stimulation testing in the diagnosis of GH deficiency, and as a tool for GH dose titration.     

Long-term effects of clenbuterol on diaphragm morphology and contractile properties in emphysematous hamsters

The aim of thepresent study was to investigate the effect of chronic long-termclenbuterol treatment (1 mg/kg subcutaneously twice a day for 12 wk) ondiaphragm morphology and function in emphysematous (EH) and normalhamsters (NH). Clenbuterol increased body weight, diaphragm weight, andskeletal muscle weight in both EH and NH to a similarextent. In the diaphragm, clenbuterol significantly increased myosin heavy chain type I, IIa, and IIx muscle fiber cross-sectional areas by ~35-55% in both EH and NH. Thisresponse to clenbuterol treatment was not significantly differentbetween EH and NH diaphragm. In EH, twitch force(P_t), maximal tetanic force, andforce-frequency curve were significantly reduced compared with NH. InEH, clenbuterol increased P_t by~10%, restoring P_t to NH level.A similar improvement was observed in the force-frequency characteristics. Clenbuterol did not alter contractile properties inNH. In conclusion, long-term clenbuterol treatment resulted in anincreased size of all diaphragm muscle fiber types in both NH and EH.Clenbuterol completely abolished the reduced force generation inducedby emphysema.

     

Insulin-like growth factor I stimulates elastin synthesis by bovine pulmonary arterial smooth muscle cells

Insulin-like growth factor I stimulates mitogenesis in smooth muscle cells, and upregulates elastin synthesis in embryonic aortic tissue. Increased smooth muscle elastin synthesis may play an important role in vascular remodeling in chronic pulmonary hypertension. Therefore, we studied the effect of IGF-I on elastin and total protein synthesis by pulmonary arterial smooth muscle cells in vitro. Tropoelastin synthesis was measured by enzyme immunoassay, and total protein synthesis was measured by [3H]-leucine incorporation. In addition, the steady-state levels of tropoelastin mRNA were determined by slot blot hybridization. Incubation of confluent cultures with various concentrations of IGF-I resulted in a dose-dependent stimulation of elastin synthesis, with a 2.4-fold increase over control levels at 1000 ng/ml of IGF. The increase in elastin synthesis was reflected by a stimulation of the steady-state levels of tropoelastin mRNA. We conclude that IGF-I has potent elastogenic effects on vascular smooth muscle cells, and speculate that it may contribute to vascular wall remodeling in chronic hypertension.     

Insulin-like growth factor I and impaired glucose tolerance

The effects of circulating insulin-like growth factor I (IGF-I) on glucose metabolism are well recognized. IGF-I is also important in maintaining beta-cell mass and regulating endogenous growth hormone (GH) levels. Low IGF-I levels could explain links between small birth size and the risk of developing type 2 diabetes mellitus in short, obese adults. In a recent prospective study, childhood insulin secretion was related to IGF-I levels and statural growth, whereas insulin sensitivity was related to early post-natal weight gain. Common genetic polymorphisms in the IGF1 gene have been linked to small birth size, post-natal growth and future diabetes risk, but these results have been inconsistent. Recent adult studies have demonstrated that lower baseline IGF-I levels predict the subsequent development of impaired glucose tolerance (IGT), type 2 diabetes and cardiovascular disease. Administration of low-dose GH therapy, at a dose that minimizes the lipolytic effects of GH and has the ability to increase IGF-I levels, enhances insulin sensitivity in young healthy adults and in GH-deficient adults and increases insulin secretion in individuals with IGT. Whether the administration of low-dose GH, recombinant IGF-I or combined IGF-I/IGF-binding protein 3 therapy prevents future development of IGT or type 2 diabetes in high-risk normoglycaemic and GH-deficient individuals merits further long-term studies.     

Insulin-like growth factor I and insulin-like growth factor binding protein 5 in Crohn's disease

Insulin-like growth factor (IGF)-I and its binding protein IGF binding protein 5 (IGFBP-5) were highly expressed in inflamed and fibrotic intestine in experimental Crohn's disease. IGF-I induced proliferation and increased collagen synthesis by smooth muscle cells and fibroblasts/myofibroblasts in vitro. Here we studied IGF-I and IGFBP-5 in Crohn's disease tissue. Tissue was collected from patients undergoing intestinal resection for Crohn's disease. IGF-I and IGFBP-5 mRNAs were quantitated by RNase protection assay and Northern blot analysis, respectively. In situ hybridization was performed to localize mRNA expression, and Western immunoblot was performed to quantitate protein expression. IGF-I and IGFBP-5 mRNAs were increased in inflamed/fibrotic intestine compared with normal-appearing intestine. IGF-I mRNA was expressed in multiple cell types in the lamina propria and fibroblast-like cells of the submucosa and muscularis externa. IGFBP-5 mRNA was highly expressed in smooth muscle of the muscularis mucosae and muscularis externa as well as fibroblast-like cells throughout the bowel wall. Tissue IGFBP-5 protein correlated with collagen type I (r = 0.82). These findings are consistent with a mechanism whereby IGF-I acts on smooth muscle and fibroblasts/myofibroblasts to increase collagen synthesis and cellular proliferation; its effects may be modulated by locally expressed IGFBP-5.     

Insulin-like growth factor I receptors in retinal rod outer segments

We have previously reported that the GDP-bound alpha-subunit of the GTP-binding protein transducin, present in outer segments of retinal rod cells (ROS), serves as a high affinity in vitro substrate (Km = 1 microM) for the insulin receptor kinase. The present study demonstrates that transducin also serves as in vitro substrate for an endogenous IGF-I receptor kinase isolated from ROS membranes. The presence of insulin-like growth factor I (IGF-I) receptors in ROS is evident from the high affinity and specific binding of 125I-IGF-I to ROS membranes (Kd = 3 nM) which contain 110 fmol of IGF-I binding sites/mg of membrane protein. Furthermore, cross-linking of 125I-IGF-I labels the 135-kDa alpha-subunit of this receptor. 125I-Insulin binding capacity to ROS membranes is less than 5% that of IGF-I. The IGF-I-stimulated tyrosine kinase activity in solubilized and partially purified receptors from ROS autophosphorylates its own 95-kDa beta-subunits as well as other substrates like transducin. Insulin, which is 200-fold less potent than IGF-I in competing for 125I-IGF-I binding, is only 5-fold less potent than IGF-I in stimulating the receptor kinase activity. This suggests that insulin is much more potent than IGF-I in coupling ligand binding with kinase activation. The previously reported presence of IGF-I in the vitreous, together with our present studies, strongly suggest that the IGF-I receptor kinase, through phosphorylation of endogenous proteins like transducin, could play a role in mediating transmembrane signal transduction in ROS.     

Insulin-like growth factor I mediates high-fat diet-induced adipogenesis in Osborne-Mendel rats

To determine if high-fat (HF) diet-induced changes in adipose tissue cellularity are associated with the presence of paracrine growth factor(s) that alter preadipocyte proliferation, Osborne-Mendel rats were fed either a HF (76% energy) or a low-fat (LF, 12% energy) diet for 85 days. HF-fed rats had greater (P < 0.05) fat pad size, total fat cell number, number of small (30-70 microm) and large (80-140 microm) adipocytes, and percentage of 100- to 140-microm adipocytes compared with LF-fed rats. Preadipocytes in primary cell culture treated with inguinal adipose tissue conditioned medium (ATCM) prepared from HF-fed rats had greater (P < 0.05) proliferation compared with cultures treated with ATCM from LF-fed rats. Proliferative capacity of ATCM prepared from HF-fed rats was attenuated after the stripping of the medium of insulin-like growth factor I using an immunomagnetic bead separation system. These data are consistent with the concept that insulin-like growth factor I is involved in the paracrine regulation of adipogenesis.     

Insulin-like growth factor I supports differentiation of cultured osteoblast-like cells

Rat calvaria cells grown in culture for one week had properties of osteoblasts: a high content in alkaline phosphatase and a marked cyclic AMP response to parathyroid hormone (PTH). In short-term experiments, insulin-like growth factor I (IGF I) stimulated the incorporation of [14C] glucose into glycogen. When IGF I was present in the medium during 6 days the cell number increased slightly and there was a substantial, disproportionate rise in alkaline phosphatase activity of the cultures. Thus, IGF I stimulates growth, and in addition, and in contrast to other growth factors, mainly enhances differentiation of osteoblasts.     

Insulin-like growth factor type I selectively binds to G-quadruplex structures

Background

G-quadruplex has been viewed as a promising therapeutic target in oncology due to its potentially important roles in physiological and pathological processes. Emerging evidence suggests that the biological functions of G-quadruplexes are closely related to the binding of some proteins. Insulin-like growth factor type I (IGF-1), as a significant modulator of cell growth and development, may serve as a quadruplex-binding protein.