Insulin-like Growth factor-I Treatment Research Articles

Insulinlike growth factor–I signaling in multiple myeloma: downstream elements, functional correlates, and pathway cross-talk

In multiple myeloma cells, insulinlike growth factor-I (IGF-I) activates 2 distinct signaling pathways, mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI-3K), leading to both proliferative and antiapoptotic effects. However, it is unclear through which of these cascades IGF-I regulates these different responses. The present studies identify a series of downstream targets in the PI-3K pathway, including glycogen synthase kinase-3beta, p70S6 kinase, and the 3 members of the Forkhead family of transcription factors. The contribution of the MAPK and PI-3K pathways and, where possible, individual elements to proliferation and apoptosis was evaluated by means of a series of specific kinase inhibitors. Both processes were regulated almost exclusively by the PI-3K pathway, with only minor contributions associated with the MAPK cascade. Within the PI-3K cascade, inhibition of p70S6 kinase led to significant decreases in proliferation and protection from apoptosis. Activation of p70S6 kinase could also be prevented by MAPK inhibitors, indicating regulation by both pathways. The Forkhead transcription factor FKHRL1 was observed to provide a dual effect in that phosphorylation upon IGF-I treatment resulted in a loss of ability to inhibit proliferation and induce apoptosis. The PI-3K pathway was additionally shown to exhibit cross-talk and to regulate the MAPK cascade, as inhibition of PI-3K prevented activation of Mek1/2 and other downstream MAPK elements. These results define important elements in IGF-I regulation of myeloma cell growth and provide biological correlates critical to an understanding of growth-factor modulation of proliferation and apoptosis.

Insulin-like growth factor I partly prevents axon elimination in the neonate rat optic nerve

Developmental neuronal death ensues after access of innervating neurons to target-derived neurotrophic factors is restricted. Recent evidence suggests, however, that growth factors such as those of the insulin family modulate neuronal death through autocrine/paracrine mechanisms. In rats, retinal ganglion neurons (RGNs) undergo massive death during early postnatal life. During this same period, the expression of various members of the insulin-like growth factor I (IGF-I) protein family is down regulated. To evaluate whether ocular IGF-I might modulate RGN death, we administered IGF-I in the posterior chamber of the eye of newborn rats. Optic nerve fiber number was estimated in control and IGF-I treated animals at postnatal day 5 when RGN death peaks. Intraocular IGF-I treatment at birth partly prevented optic nerve fiber elimination. Because the axon number in the optic nerve correlates to some extent with the RGN number, these results suggest that IGF-I may modulate RGN death in vivo through local interactions.

Short-Term Infusion of LongR3 Insulin-like Growth Factor (IGF)-I Decreases Hepatic IGF-I mRNA but Not IGF Binding Protein-3 mRNA Expression in Pigs

Infusion of pigs with an insulin-like growth factor-I (IGF-I) analogue (LongR3IGF-I) that does not bind to IGF-binding proteins decreases growth rate and the plasma concentration of growth hormone (GH), IGF-I, IGFBP-3, and insulin. This study was designed to determine whether the decrease is due to changes in IGF-I and IGFBP-3 gene expression. IGF-I or LongR3IGF-I (180 μg/kg/day) was infused into 55-kg finisher pigs for 4 days using Travenol infuser pumps. Plasma IGF-I concentration was measured by radioimmunoassay and plasma IGFBP-3 and IGFBP-2 were estimated by Western ligand blotting. Steady-state levels of IGF-I and IGFBP-3 mRNA were measured by RNase protection assay. Neither IGF-I nor LongR3IGF-I had a significant effect on hepatic IGF-I class 1 mRNA expression, whereas hepatic IGF-I class 2 mRNA expression was significantly reduced by both peptides. Plasma IGFBP-3 levels were unaffected by IGF-I treatment but were reduced by LongR3IGF-I treatment. The decrease in IGFBP-3 was not due to decreased gene expression in porcine liver or kidney, since neither IGF-I nor LongR3IGF-I treatment altered IGFBP-3 mRNA. This study infers a direct effect of the IGF analogue LongR3IGF-I on GH through its inhibition of plasma IGF-I concentration and class 2 IGF-I mRNA. The decrease in plasma IGFBP-3 was not accompanied by a decrease in hepatic or renal IGFBP-3 mRNA, suggesting that in this case, plasma IGFBP-3 protein levels are posttranslationally regulated or are derived from tissues other than liver or kidney.

Amniotic IGF-I supplements improve gut growth but reduce circulating IGF-I in growth-restricted fetal sheep.

Insulin-like growth factor I (IGF-I) is an important regulator of fetal growth, and circulating concentrations are reduced in intrauterine growth-restricted (IUGR) fetuses. We investigated whether IGF-I administered into amniotic fluid could ameliorate IUGR in fetal sheep. Fetuses were assigned to control (n = 9), IUGR+saline (n = 12), or IUGR+IGF-I groups (daily intra-amniotic IGF-I injections of 20 microg, n = 13). IUGR was induced by placental embolization from 114 to 120 days. Treatment was from 120 to 130 days of gestation. Embolization produced asymmetrically IUGR fetuses with decreased body weight and lighter, thinner-walled guts. Fetal plasma and amniotic IGF-I levels were reduced. During treatment, fetal plasma, but not amniotic, IGF-I levels recovered in the saline group but remained depressed in the IGF-I-treated group. IGF-I treatment restored gut weight and wall thickness to control levels and increased the number of crypt mitoses. Fetal weight was similar to that of controls, but spleen, liver, and thymic weights were reduced by 30-37%, and placentome growth was altered. Amniotic fluid IGF-I supplementation may provide the basis of future therapeutic approaches to IUGR, but the systemic effects require further investigation.

Possible mechanism for acceleration of meiotic progression of bovine follicular oocytes by growth factors in vitro

The mechanism for the accelerating effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the meiotic cell cycle of bovine oocytes cultured in vitro was investigated. Cumulus-oocyte complexes (COCs) were obtained from small (< or = 3 mm in diameter), medium (4-6 mm in diameter) or large (7-10 mm in diameter) ovarian follicles and cultured with or without a combination of EGF and IGF-I (growth factors). Growth factors significantly increased the frequency of first polar body extrusion of oocytes derived from small follicles at 16 h of culture (PB16 oocytes; with growth factors: 75%; without growth factors: 55%), but did not increase the frequency in oocytes from medium or large follicles. COCs from small follicles were cultured with individual growth factors and sampled for kinase activity. The frequencies of polar body extrusion in EGF only (67%) and EGF + IGF-I (75%) treatment groups were significantly higher than those in the control (no growth factor) group (49%), but not significantly higher than in the IGF-I only group (63%). The H1 kinase activity at 6-8 h of culture in each group increased significantly from the baseline value at 0 h of culture, and the H1 kinase activities in the EGF only, IGF-I only and EGF + IGF-I treatment groups were significantly higher than those in the control group at 8 h of culture. MAP kinase activity was significantly higher than the baseline value and significantly higher than that in the control group at 6 h of culture in the EGF treatment group only. In conclusion, EGF and IGF-I act on COCs from small follicles to accelerate the meiotic cell cycle of the oocytes. This accelerating effect may be related to increased H1 and MAP kinase activities during the early stages of maturation.

Possible mechanism for acceleration of meiotic progression of bovine follicular oocytes by growth factors in vitro

The mechanism for the accelerating effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the meiotic cell cycle of bovine oocytes cultured in vitro was investigated. Cumulus-oocyte complexes (COCs) were obtained from small (< or = 3 mm in diameter), medium (4-6 mm in diameter) or large (7-10 mm in diameter) ovarian follicles and cultured with or without a combination of EGF and IGF-I (growth factors). Growth factors significantly increased the frequency of first polar body extrusion of oocytes derived from small follicles at 16 h of culture (PB16 oocytes; with growth factors: 75%; without growth factors: 55%), but did not increase the frequency in oocytes from medium or large follicles. COCs from small follicles were cultured with individual growth factors and sampled for kinase activity. The frequencies of polar body extrusion in EGF only (67%) and EGF + IGF-I (75%) treatment groups were significantly higher than those in the control (no growth factor) group (49%), but not significantly higher than in the IGF-I only group (63%). The H1 kinase activity at 6-8 h of culture in each group increased significantly from the baseline value at 0 h of culture, and the H1 kinase activities in the EGF only, IGF-I only and EGF + IGF-I treatment groups were significantly higher than those in the control group at 8 h of culture. MAP kinase activity was significantly higher than the baseline value and significantly higher than that in the control group at 6 h of culture in the EGF treatment group only. In conclusion, EGF and IGF-I act on COCs from small follicles to accelerate the meiotic cell cycle of the oocytes. This accelerating effect may be related to increased H1 and MAP kinase activities during the early stages of maturation.

Resection upregulates the IGF-I system of parenterally fed rats with jejunocolic anastomosis.

Rats maintained with parenteral nutrition following 60% jejunoileal resection plus cecectomy exhibit minimal adaptive growth in the residual jejunum but a dramatic adaptive growth in the residual colon. Coinfusion of insulin-like growth factor I (IGF-I) with parenteral nutrition induces jejunal growth but has minimal effects in the colon. Our objective was to study the role of the endogenous IGF-I system in the differential responses of jejunum and colon to resection and/or IGF-I during parenteral nutrition. We measured concentrations of immunoreactive IGF-I in plasma, jejunum, and colon, IGF-I receptor binding, and levels of IGF receptor, IGF-I, IGF binding protein (IGFBP)-3 and IGFBP-5 mRNA in residual jejunum and colon 7 days after resection and/or IGF-I treatment. IGF-I receptor number was increased (74-99%) in jejunum and colon due to resection; IGF-I mRNA was increased 5-fold in jejunum and 15-fold in colon due to resection. Resection increased circulating IGFBPs but did not alter plasma IGF-I concentration. Resection induced colonic growth in association with significantly greater colonic IGFBP-5 mRNA and significantly lower colonic immunoreactive IGF-I. IGF-I treatment had no significant effect on IGF-I mRNA or IGF-I receptor number. Concentrations of plasma and jejunal immunoreactive IGF-I were significantly increased in rats given IGF-I in association with jejunal growth. IGF-I treatment significantly increased IGFBP-5 mRNA in the jejunum, which also correlated with jejunal growth. Thus resection upregulated IGF-I receptor number and IGF-I mRNA in residual jejunum and colon, but differential adaptation of these segments correlated with differential regulation of IGFBP-5 mRNA.

Altered liver gene expression in CCl 4-cirrhotic rats is partially normalized by insulin-like growth factor-I

We have previously shown that the administration of low doses of insulin-like growth factor-I (IGF-I) to CCl 4-cirrhotic rats improves liver function and reduces fibrosis. To better understand the mechanisms behind the hepatoprotective effects of IGF-I, and to identify those genes whose expression is affected in cirrhosis and after IGF-1 treatment, we have performed differential display of mRNA analysis by means of polymerase chain reaction (PCR) in livers from control and CCl 4-cirrhotic rats treated or not with IGF-I. We have identified 16 genes that were up- or down-regulated in the cirrhotic liver. IGF-I treatment partially normalized the expression of eight of these genes, including serine proteinase inhibitors such as serpin-2 and alpha-1-antichymotripsin, alpha-1-acid glycoprotein, and alpha-2u-globulin. Additionally, we show that IGF-I enhanced the regenerative activity in the cirrhotic liver, as determined by the increased expression of the proliferating cell nuclear antigen (PCNA). Finally, IGF-I treatment partially restored the expression of growth hormone receptor (GHR) and the levels of global genomic DNA methylation, which are reduced in human and experimental cirrhosis. Taken together, our observations confirm the hepatoprotective effects of IGF-I, and suggest that this action can be exerted in part through the normalization of liver gene expression, growth hormone (GH) responsiveness and global genomic DNA methylation.

Regulation of GHRH receptor gene expression in the neonatal and adult rat pituitary.

The growth hormone releasing hormone (GHRH) receptor gene is essential for normal growth, and its expression is developmentally regulated. The factors that control GHRH receptor expression in the neonatal pituitary are not well understood. This study focuses on the regulation of GHRH receptor gene expression by thyroid hormone, glucocorticoids, insulin-like growth factor-I (IGF-I) and IGF-II in rat pituitary cell cultures. In newborn pituitaries, both T3 and hydrocortisone (24 h) caused a dose-dependent increase in GHRH receptor mRNA abundance, reaching levels 4.8-fold (P<0.001) and 6.5-fold (P<0.001) over corresponding controls. T3 and hydrocortisone also stimulated GHRH receptor expression in adult (70 day) pituitary cell cultures, consistent with our earlier findings. IGF-I treatment suppressed the inductive effects of T3 (P<0.02) and hydrocortisone (P<0.03) on GHRH receptor expression in adult pituitaries but not in neonatal pituitaries. Unlike IGF-I, IGF-II treatment had no effect on T3-induced or hydrocortisone-induced GHRH receptor expression in either neonates or adults. Taken together, these results indicate that (1) thyroid hormone and hydrocortisone act directly at the neonatal pituitary as potent stimulators of GHRH receptor gene expression, (2) IGF-I, but not IGF-II, acts at the pituitary to suppress GHRH receptor mRNA expression and (3) the effects of IGF-I on GHRH receptor gene expression are developmentally determined.

Exacerbated inflammatory response induced by insulin-like growth factor I treatment in rats with ischemic acute renal failure.

In agreement with recent studies showing a deleterious effect of growth hormone treatment in critically ill patients, preliminary data showed that insulin-like growth factor I (IGF-I) administration increased the mortality rate of rats with ischemic acute renal failure (ARF). The present study was designed to investigate the mechanism responsible for this unexpected effect. Male rats with ischemic ARF were given subcutaneous IGF-I, 50 microg/100 g at 0, 8, and 16 h after reperfusion (ARF+IGF-I, n = 5) or were untreated (ARF, n = 5). A group of 5 sham-operated rats were used as controls. Rats were killed 48 h after declamping, and the following studies were performed: in serum, creatinine and urea nitrogen; and in kidneys, histologic damage score, cellular proliferation by bromodeoxyuridine labeling, apoptosis by morphologic criteria, macrophage infiltration by immunohistochemistry using a specific antibody against ED-1, neutrophil infiltration by naphthol AS-D chloroacetate esterase staining, and levels of IGF-I and IGF-I receptor mRNA by RNase protection assay. ARF and ARF+IGF-I groups had a severe and similar degree of renal failure. Kidney damage was histologically more evident in ARF+IGF-I (1.9 +/- 0.1) than in ARF (1.3 +/- 0.2) rats, and the number of neutrophils/mm(2) of tissue was significantly greater in ARF+IGF-I than in ARF rats at the corticomedullary junction (52.3 +/- 5.2 versus 37.2 +/- 4.1) as well as at the renal medulla (172.5 +/- 30.0 versus 42.1 +/- 9.6). No other differences between the groups were found. It is concluded that IGF-I treatment enhanced the inflammatory response in rats with ischemic ARF. Cell toxicity derived from increased neutrophil accumulation might play a key role in the greater mortality risk of critically ill patients that are treated with growth hormone.

Insulin-like Growth Factor-I Induces Renal Cell Hypertrophy via a Calcineurin-dependent Mechanism

Insulin-like growth factor-I (IGF-I) may play an important role in the development of renal hypertrophy. In this study we determined the effect of IGF-I on cultured mesangial cells (MCs) and examined activation of key signaling pathways. IGF-I induced hypertrophy as determined by an increase in cell size and an increase in protein to DNA ratio and increased accumulation of extracellular matrix (ECM) proteins. IGF-I also activated both Erk1/Erk2 MAPK and phosphatidylinositol 3-kinase (PI3K) in MCs. Inhibition of either MAPK or PI3K, however, had no effect on IGF-I-induced hypertrophy or ECM production. Next, we examined the effect of IGF-I on activation of the calcium-dependent phosphatase calcineurin. IGF-I treatment stimulated calcineurin activity and increased the protein levels of calcineurin and the calcineurin binding protein, calmodulin. Cyclosporin A, an inhibitor of calcineurin, blocked both IGF-I-mediated hypertrophy and up-regulation of ECM. In addition, calcineurin resulted in sustained Akt activation, indicating possible cross-talk with other signaling pathways. Finally, IGF-I treatment resulted in the calcineurindependent nuclear localization of NFATc1. Therefore, IGF-I induces hypertrophy and increases ECM accumulation in MCs. IGF-I-mediated hypertrophy is associated with activation of Erk1/Erk2 MAPK and PI3K but does not require either of these pathways. Instead, IGF-I mediates hypertrophy via a calcineurin-dependent pathway.

Alterations in Hypothalamic Insulin‐Like Growth Factor‐I and its Associations with Gonadotropin Releasing Hormone Neurones During Reproductive Development and Ageing

Insulin-like growth factor-I (IGF-I) is thought to play a role in the onset of reproductive ability at puberty and the control of reproductive function throughout adult life. It is believed that these effects are mediated at least in part by the activation of gonadotropin releasing hormone (GnRH) neurones by IGF-I, but the interactions of IGF-I with GnRH neurones in vivo are largely unknown. We first examined the anatomical relationship between GnRH and IGF-I cells in neuroendocrine regions. Using double-label immunocytochemistry, we observed that in the preoptic area-anterior hypothalamus (POA-AH), the site of GnRH perikarya, the majority (78%) of GnRH cell bodies expressed IGF-I immunoreactivity. IGF-I immunoreactivity was also high in the median eminence, the site of GnRH release, and GnRH neuroterminals were seen to interweave among IGF-I-immunopositive cells. Due to this substantial overlap of GnRH and IGF-I immunoreactive elements, we then tested the hypothesis that changes in IGF-I may regulate the GnRH system. Animals were examined at the two important reproductive life transitions: puberty and reproductive senescence. IGF-I mRNA levels were measured in POA-AH and medial basal hypothalamus-median eminence (MBH-ME) and effects of IGF-I treatment on GnRH mRNA levels were quantified by RNase protection assay. Although IGF-I treatment did not alter GnRH gene expression, there were significant alterations in hypothalamic IGF-I gene expression at both puberty and reproductive senescence. During puberty, IGF-I mRNA levels in the MBH-ME of rats increased from the juvenile stage (P25) to the day of vaginal opening (P35), and from the day of vaginal opening to young adulthood (P45) in the POA-AH. During reproductive ageing, IGF-I mRNA levels were significantly lower in middle-aged than young rats, particularly in the MBH-ME. At all ages, IGF-I expression was greater in the MBH-ME than in the POA-AH. These experiments demonstrate that: (i) the majority of adult GnRH neurones are immunopositive for the IGF-I protein; (ii) hypothalamic IGF-I levels increase at the onset of reproductive function and decrease at reproductive senescence in a regionally specific manner; and (iii) despite the presence of IGF-I in GnRH perikarya, IGF-I does not affect GnRH gene expression, suggesting that IGF-I may act at the level of GnRH release rather than gene expression.

Polyamine- and insulin-like growth factor-I-mediated proliferation of porcine uterine endometrial cells: a potential role for spermidine/spermine N(1)-acetyltransferase during peri-implantation.

Insulin-like growth factor-I (IGF-I) and the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) are progesterone-regulated genes with maximal expression at peri-implantation in the porcine uterine endometrium. However, while IGF-I stimulates cell proliferation, SSAT, by acetylating the naturally occurring polyamines (PA) spermine (SPM) and spermidine (SPD), typically functions as a cell growth inhibitor. The present study examined the functional relationships of IGF-I, SSAT, and PA in the control of endometrial cell proliferation. Northern blot analysis indicated that SSAT mRNA levels change with distinct pregnancy stages, in contrast to those for the PA biosynthetic enzyme ornithine decarboxylase (ODC). Primary cultures of luminal and glandular epithelial (LE, GE) and stromal (ST) cells isolated from Day 12 pregnant pig endometrium had IGF-I mRNA levels for ST > LE > GE cells. The mRNA levels for SSAT and ODC were transiently diminished by IGF-I treatment, but only in GE cells. By contrast, SPM and SPD increased SSAT mRNA levels in GE and ST cells, but increased ODC mRNA levels only in GE cells. IGF-I, putrescine (PUT), and SPM individually increased cellular DNA synthesis as measured by tritiated thymidine incorporation in GE and ST cells, while SPD had an effect only in ST cells. IGF-I enhanced the proliferative effect of each PA in GE cells, but only of SPD in ST cells. The mitogen-activated protein kinase inhibitor, PD98059, inhibited the induction by SPM of GE cell DNA synthesis but not that of IGF-I. Wortmannin, a phosphatidylinositol-3-kinase inhibitor had no effect on either IGF-I or SPM induction of GE cell DNA synthesis. The relative concentrations of SPM, SPD, and PUT in uterine luminal fluids differed, with the levels for each PA higher at pregnancy Day 12 than at 11.5. These results suggest that IGF-I and PA act through distinct signaling pathways to mediate cell-type-specific growth of early pregnancy pig uterine endometrium. Further, SSAT, through its control of intracellular PA levels, likely plays a modulatory role in the establishment of an optimal uterine environment for successful embryo attachment.

Growth hormone as a neuronal rescue factor during recovery from CNS injury.

There is growing evidence to suggest that growth hormone plays a role in the growth and development of the CNS. Specifically, growth hormone has been implicated in promoting brain growth, myelination, neuronal arborisation, glial differentiation and cognitive function. Here we investigate if growth hormone has a role in the recovery from an unilateral hypoxic-ischaemic brain injury. Using moderate (15 min hypoxia) and severe (60 min hypoxia) models of hypoxic-ischaemia in juvenile rats and standard immunohistochemical techniques, we found intense growth hormone-like immunoreactivity present within regions of cell loss by 3 days (P<0.05). Growth hormone-like immunoreactivity was observed on injured neurones, myelinated axons, glial cells within and surrounding infarcted tissue and on the choroid plexus plus ependymal cells within the injured hemisphere. The pattern of immunoreactivity suggests that (a) growth hormone (or a growth hormone-like substance) is transported via the cerebrospinal fluid and (b) that growth hormone (or a growth hormone-like substance) is acting in a neurotrophic manner specifically targeted to injured neurones and glia. To test this hypothesis we treated a moderate hypoxic-ischaemic brain injury with 20 microg of rat growth hormone by intracerebroventricular infusion starting 2 h after injury (n=12/group). After 3 days the animals were killed and the extent of neuronal loss quantified. Growth hormone treatment reduced neuronal loss in the frontoparietal cortex (P<0.001), hippocampus (P<0.01) and dorsolateral thalamus (P<0.01) but not in the striatum. This spatial distribution of the neuroprotection conveyed by growth hormone correlates with the spatial distribution of the constitutive neural growth hormone receptor, but not with the neuroprotection offered by insulin-like growth factor-I treatment in this model. These results suggest that some of the neuroprotective effects of growth hormone are mediated directly through the growth hormone receptor and do not involve insulin-like growth factor-I induction.In summary, we have found that a growth hormone-like factor increased in the brain in the days after injury. In addition, treatment with growth hormone soon after an hypoxic-ischaemic injury reduced the extent of neuronal loss. These results further suggest that a neural growth hormone axis is activated during recovery from injury and that this may act to restrict the extent of neuronal death.

IGF-I treatment improves the functional properties of fast- and slow-twitch skeletal muscles from dystrophic mice.

Although insulin-like growth factor-I (IGF-I) has been proposed for use by patients suffering from muscle wasting conditions, few studies have investigated the functional properties of dystrophic skeletal muscle following IGF-I treatment. 129P1 ReJ-Lama2(dy) (129 ReJ dy/dy) dystrophic mice suffer from a deficiency in the structural protein, laminin, and exhibit severe muscle wasting and weakness. We tested the hypothesis that 4 weeks of IGF-I treatment ( approximately 2 mg/kg body mass, 50 g/h via mini-osmotic pump, subcutaneously) would increase the mass and force producing capacity of skeletal muscles from dystrophic mice. IGF-I treatment increased the mass of the extensor digitorum longus (EDL) and soleus muscles of dystrophic mice by 20 and 29%, respectively, compared with untreated dystrophic mice (administered saline-vehicle only). Absolute maximum force (P(o)) of the EDL and soleus muscle was increased by 40 and 32%, respectively, following IGF-I treatment. Specific P(o) (sP(o)) was increased by 23% in the EDL muscles of treated compared with untreated mice, but in the soleus muscle sP(o) was unchanged. IGF-I treatment increased the proportion of type IIB and type IIA fibres and decreased the proportion of type I fibres in the EDL muscles of dystrophic mice. In the soleus muscles of dystrophic mice, IGF-I treatment increased the proportion of type IIA fibres and decreased the proportion of type I fibres. Average fibre cross-sectional area was increased in the EDL and soleus muscles of treated compared with untreated mice. We conclude that IGF-I treatment ameliorates muscle wasting and improves the functional properties of skeletal muscles of dystrophic mice. The findings have important implications for the role of IGF-I in ameliorating muscle wasting associated with the muscular dystrophies.

IGF-I differentially regulates Bcl-xL and Bax and confers myocardial protection in the rat heart.

Bcl-2 family proteins play a crucial role in the cytoprotective action of insulin-like growth factor-I (IGF-I) by regulating cell death signaling at the mitochondrial level. The present study examined the effect of IGF-I on the expression of Bcl-2 family proteins in the rat heart mitochondria in relation to myocardial protection against ischemia-reperfusion injury. Systemic IGF-I (1 mg) treatment in the rat increased Bcl-xL and attenuated Bax 12-24 h later in the heart mitochondria fraction. Permeability transition and cytochrome c release occurred in a Ca(2+) concentration-dependent manner in the vehicle-treated mitochondria. This was significantly inhibited by the IGF-I-pretreatment. Moreover, ATP synthesis was significantly greater in the IGF-I-pretreated mitochondria. IGF-I pretreatment 24 h before 25 min of global ischemia in the isolated rat heart model significantly improved recovery of isovolumic left ventricular function and inhibited creatine kinase release during reperfusion. This was associated with a significantly less number of terminal transferase labeling-positive myocytes and nonmyocytes 2 h after reperfusion. These results suggest that IGF-1 differentially regulates Bcl-xL and Bax in heart mitochondria, which may be causally related to myocardial protection against ischemia-reperfusion injury.

Insulin-like growth factor-I and Bcl-X(L) inhibit c-jun N-terminal kinase activation and rescue Schwann cells from apoptosis.

We previously reported that Schwann cells undergo apoptosis after serum withdrawal. Insulin-like growth factor-I, via phosphatidylinositol-3 kinase, inhibits caspase activation and rescues Schwann cells from serum withdrawal-induced apoptosis. In this study, we examined the role of c-jun N-terminal protein kinase (JNK) in Schwann cell apoptosis induced by serum withdrawal. Activation of both JNK1 and JNK2 was detected 1 h after serum withdrawal with the maximal level detected at 2 h. A dominant negative JNK mutant, JNK (APF), blocked JNK activation induced by serum withdrawal and Schwann cell apoptosis, suggesting JNK activation participates in Schwann cell apoptosis. Serum withdrawal-induced JNK activity was caspase dependent and inhibited by a caspase 3 inhibitor, Ac-DEVD-CHO. Because insulin-like growth factor-I and Bcl-X(L) are both Schwann cell survival factors, we tested their effects on JNK activation during apoptosis. Insulin-like growth factor-I treatment decreased both JNK1 and JNK2 activity induced by serum withdrawal. LY294002, a phosphatidylinositol-3 kinase inhibitor, blocked insulin-like growth factor-I inhibition on JNK activation, suggesting that phosphatidylinositol-3 kinase mediates the effects of insulin-like growth factor-I. Overexpression of Bcl-X(L) also resulted in less Schwann cell death and inhibition of JNK activation after serum withdrawal. Collectively, these results suggest JNK activation is involved in Schwann cell apoptosis induced by serum withdrawal. Insulin-like growth factor-I and Bcl family proteins rescue Schwann cells, at least in part, by inhibition of JNK activity.

Protein Kinase C α-mediated Negative Feedback Regulation Is Responsible for the Termination of Insulin-like Growth Factor I-induced Activation of Nuclear Phospholipase C β1 in Swiss 3T3 Cells

Previous studies from several independent laboratories have demonstrated the existence of an autonomous phosphoinositide (PI) cycle within the nucleus, where it is involved in both cell proliferation and differentiation. Stimulation of Swiss 3T3 cells with insulin-like growth factor-I (IGF-I) has been shown to induce a transient and rapid increase in the activity of nuclear-localized phospholipase C (PLC) beta1, which in turn leads to the production of inositol trisphosphate and diacylglycerol in the nucleus. Nuclear diacylglycerol provides the driving force for the nuclear translocation of protein kinase C (PKC) alpha. Here, we report that treatment of Swiss 3T3 cells with Go6976, a selective inhibitor of PKC alpha, caused a sustained elevation of IGF-I-stimulated nuclear PLC activity. A time course study revealed an inverse relationship between nuclear PKC activity and the activity of nuclear PLC in IGF-I-treated cells. A time-dependent association between PKC alpha and PLC beta1 in the nucleus was also observed following IGF-I treatment. Two-dimensional phosphopeptide mapping and site-directed mutagenesis demonstrated that PKC promoted phosphorylation of PLC beta1 at serine 887 in the nucleus of IGF-I-treated cells. Overexpression of either a PLC beta1 mutant in which the PKC phosphorylation site Ser(887) was replaced by alanine, or a dominant-negative PKC alpha, resulted in a sustained activation of nuclear PLC following IGF-I stimulation. These results indicate that a negative feedback regulation of PLC beta1 by PKC alpha plays a critical role in the termination of the IGF-I-dependent signal that activates the nuclear PI cycle.

Insulin-like Growth Factor-I Stimulates Dephosphorylation of IκB through the Serine Phosphatase Calcineurin (Protein Phosphatase 2B)

Astrocytes represent the most abundant cell type of the adult nervous system. Under normal conditions, astrocytes participate in neuronal feeding and detoxification. However, following brain injury, local increases in inflammatory cytokines trigger a reactive phenotype in astrocytes during which these cells produce their own inflammatory cytokines and neurotoxic free radicals. Indeed, progression of this inflammatory reaction is responsible for most neurological damage associated with brain trauma. Insulin-like growth factor-I (IGF-I) protects neurons against a variety of brain pathologies associated with glial overproduction of proinflammatory cytokines. Here, we demonstrate that in astrocyte cultures IGF-I regulates NFkappaB, a transcription factor known to play a key role in the inflammatory reaction. IGF-I induces a site-specific dephosphorylation of IkappaBalpha (phospho-Ser(32)) in astrocytes. Moreover, IGF-I-mediated dephosphorylation of IkappaBalpha protects this molecule from tumor necrosis factor alpha (TNFalpha)-stimulated degradation; therefore, IGF-I also inhibits the nuclear translocation of NFkappaB (p65) induced by TNFalpha exposure. Finally, we show that dephosphorylation of IkappaBalpha by IGF-I pathways requires activation of calcineurin. Activation of this phosphatase is independent of phosphatidylinositol 3-kinase and mitogen-activated protein kinase. Thus, these data suggest that the therapeutic benefits associated with IGF-I treatment of brain injury are derived from both its positive effects on neuronal survival and inhibition of the glial inflammatory reaction.

IGF-I treatment attenuates renal abnormalities induced by neonatal ACE inhibition.

An intact renin-angiotensin system (RAS) during nephrogenesis is essential for normal renal development. We have shown previously that neonatal inhibition of the RAS, either with ANG II type 1-receptor blockade or angiotensin-converting enzyme (ACE) inhibition, induces irreversible renal abnormalities. The aim of the present study was to investigate whether an interrupted RAS can be compensated for by exogenous administration of another important renal growth-promoting factor, the insulin-like growth factor-I (IGF-I). Rats were treated daily with either the ACE inhibitor enalapril (10 mg/kg), recombinant human IGF-I (3 mg/kg), or the combination enalapril + IGF-I from perinatal day 3 to 13. Urinary concentrating ability, renal function, and renal morphology were assessed at adult age. The gene expression and localization of IGF-I, its receptor, and the growth hormone receptor (GHR) were investigated during ongoing ACE inhibition. The present study demonstrates normalized renal function and histology in enalapril + IGF-I-treated animals. Ongoing ACE inhibition suppressed the medullary IGF-I mRNA expression and altered the local distribution of both IGF-I and GHR. Thus the present study provides evidence for an interaction between the RAS and GH/IGF-I axis in renal development.