The novel protective effects of loganin against 1-methyl-4-phenylpyridinium-induced neurotoxicity: Enhancement of neurotrophic signaling, activation of IGF-1R/GLP-1R, and inhibition of RhoA/ROCK pathway.

Loganin, a major iridoid glycoside obtained from fruits of Cornus officinalis, possesses anti-inflammatory, antitumor, antidiabetic, and osteoporosis prevention effects. Loganin has been linked to neuroprotection in several models of neurodegeneration, including Parkinson's disease (PD). However, mechanisms underlying the neuroprotective effects of loganin are still mostly unknown. Here, we demonstrated the protective effects of loganin against PD mimetic toxin 1-methyl-4-phenylpyridinium (MPP+ ) and the important roles of insulin-like growth factor 1 receptor (IGF-1R) and glucagon-like peptide 1 receptor (GLP-1R) in the neuroprotective mechanisms of loganin. In primary mesencephalic neuronal cultures treated with or without MPP+ , loganin up-regulated expressions of neurotrophic signals including IGF-1R, GLP-1R, p-Akt, BDNF, and tyrosine hydroxylase. Loganin protected against MPP+ -induced apoptosis by up-regulating antiapoptotic protein and down-regulating proapoptotic protein. Moreover, loganin attenuated MPP+ -induced neurite damage via up-regulation of GAP43 and down-regulation of membrane-RhoA/ROCK2/p-LIMK/p-cofilin. Loganin also attenuated MPP+ -induced reactive oxygen species (ROS) production. However, both AG1024, an IGF-1R antagonist, and exendin 9-39, a GLP-1R antagonist, attenuated the protective effects of loganin on MPP+ -induced cytotoxicity, apoptosis, neurite length decrease, and ROS production. Our results suggest that loganin attenuates MPP+ -induced apoptotic death, neurite damage, and oxidative stress through enhancement of neurotrophic signaling, activation of IGF-1R/GLP-1R, and inhibition of RhoA/ROCK pathway, providing the evidence that loganin possesses novel neuroprotective effects.

Amelioration of Diabetic Mouse Nephropathy by Catalpol Correlates with Down-Regulation of Grb10 Expression and Activation of Insulin-Like Growth Factor 1 / Insulin-Like Growth Factor 1 Receptor Signaling.

Growth factor receptor-bound protein 10 (Grb10) is an adaptor protein that can negatively regulate the insulin-like growth factor 1 receptor (IGF-1R). The IGF1-1R pathway is critical for cell growth and apoptosis and has been implicated in kidney diseases; however, it is still unknown whether Grb10 expression is up-regulated and plays a role in diabetic nephropathy. Catalpol, a major active ingredient of a traditional Chinese medicine, Rehmannia, has been reported to possess anti-inflammatory and anti-aging activities and then used to treat diabetes. Herein, we aimed to assess the therapeutic effect of catalpol on a mouse model diabetic nephropathy and the potential role of Grb10 in the pathogenesis of this diabetes-associated complication. Our results showed that catalpol treatment improved diabetes-associated impaired renal functions and ameliorated pathological changes in kidneys of diabetic mice. We also found that Grb10 expression was significantly elevated in kidneys of diabetic mice as compared with that in non-diabetic mice, while treatment with catalpol significantly abrogated the elevated Grb10 expression in diabetic kidneys. On the contrary, IGF-1 mRNA levels and IGF-1R phosphorylation were significantly higher in kidneys of catalpol-treated diabetic mice than those in non-treated diabetic mice. Our results suggest that elevated Grb10 expression may play an important role in the pathogenesis of diabetic nephropathy through suppressing IGF-1/IGF-1R signaling pathway, which might be a potential molecular target of catalpol for the treatment of this diabetic complication.

Nerve Regeneration Potential of Protocatechuic Acid in RSC96 Schwann Cells by Induction of Cellular Proliferation and Migration through IGF-IR-PI3K-Akt Signaling.

Peripheral nerve injuries, caused by accidental trauma, acute compression or surgery, often result in temporary or life-long neuronal dysfunctions and inflict great economic or social burdens on the patients. Nerve cell proliferation is an essential process to restore injured nerves of adults. Schwann cells play a crucial role in endogenous repair of peripheral nerves due to their ability to proliferate, migrate and provide trophic support to axons via expression of various neurotrophic factors, such as the nerve growth factor (NGF), especially after nerve injury. Protocatechuic acid (PCA) is a dihydroxybenzoic acid, a type of phenolic acid, isolated from the kernels of Alpinia oxyphylla Miq (AOF), a traditional Chinese herbal medicine the fruits of which are widely used as a tonic, aphrodisiac, anti-salivation and anti-diarrheatic. This study investigated the molecular mechanisms by which PCA induces Schwann cell proliferation by activating IGF-IR-PI3K-Akt pathway. Treatment with PCA induces phosphorylation of the insulin-like growth factor-I (IGF-I)-mediated phosphatidylinositol 3 kinase/serine - threonine kinase (PI3K/Akt) pathway, and activates expression of cell nuclear antigen (PCNA) in a dose-dependent manner. Cell cycle analysis after 18 h of treatment showed that proliferation of the RSC96 cells was enhanced by PCA treatment. The PCA induced proliferation was accompanied by modulation in the expressions of cell cycle proteins cyclin D1, cyclin E and cyclin A. Knockdown of PI3K using small interfering RNA (siRNA) and inhibition of IGF-IR receptor resulted in the reduction in cell survival proteins. The results collectively showed that PCA treatment promoted cell proliferation and cell survival via IGF-I signaling.

Insulin receptor substrate signaling suppresses neonatal autophagy in the heart.

The induction of autophagy in the mammalian heart during the perinatal period is an essential adaptation required to survive early neonatal starvation; however, the mechanisms that mediate autophagy suppression once feeding is established are not known. Insulin signaling in the heart is transduced via insulin and IGF-1 receptors (IGF-1Rs). We disrupted insulin and IGF-1R signaling by generating mice with combined cardiomyocyte-specific deletion of Irs1 and Irs2. Here we show that loss of IRS signaling prevented the physiological suppression of autophagy that normally parallels the postnatal increase in circulating insulin. This resulted in unrestrained autophagy in cardiomyocytes, which contributed to myocyte loss, heart failure, and premature death. This process was ameliorated either by activation of mTOR with aa supplementation or by genetic suppression of autophagic activation. Loss of IRS1 and IRS2 signaling also increased apoptosis and precipitated mitochondrial dysfunction, which were not reduced when autophagic flux was normalized. Together, these data indicate that in addition to prosurvival signaling, insulin action in early life mediates the physiological postnatal suppression of autophagy, thereby linking nutrient sensing to postnatal cardiac development.

Alleviation of body weight loss by dietary methionine is independent of insulin-like growth factor-I in protein-starved young chickens.

It is well known that in protein-starved chickens, small amounts of amino acid supplement, especially methionine, reduces nitrogen excretion and thereby improves nitrogen balance. On the other hand, excess intake of methionine causes growth depression and the growth-depressive effect of excess methionine can be alleviated by consumption of dietary glycine. Insulin-like growth factor-I (IGF-I) is one of various growth-promoting factors relating to the efficiency of animal production and is known to be very sensitive to changes in nutritional status. In the present study, the interactive effect of glycine on nitrogen sparing effect of methionine in protein-starved chickens was examined. In addition, the relation of IGF-I and its specific binding protein to the nitrogen sparing effect of supplemented methionine was also investigated. Two-days refeeding of methionine supplemented to protein-free diet could promptly alleviate body weight loss in protein-starved chickens, and the alleviation of body weight loss by methionine was not improved by glycine supplements. Moreover, such acute alleviation of body weight loss by dietary methionine was independent of the change in plasma IGF-I concentration.

Estrogen signaling in hypothalamic circuits controlling reproduction.

It is well known that many of the actions of 17ß-estradiol (E2) in the central nervous system are mediated via intracellular receptor/transcription factors that interact with steroid response elements on target genes. However, there is compelling evidence for membrane steroid receptors for estrogen in hypothalamic and other brain neurons. Yet, it is not well understood how estrogen signals via membrane receptors and how these signals impact not only membrane excitability but also gene transcription in neurons that modulate GnRH neuronal excitability. Indeed, it has been known for some time that E2 can rapidly alter neuronal activity within seconds, indicating that some cellular effects can occur via membrane delimited events. In addition, E2 can affect second messenger systems including calcium mobilization and a plethora of kinases to alter cell signaling. Therefore, this review will consider our current knowledge of rapid membrane-initiated and intracellular signaling by E2 in hypothalamic neurons critical for reproductive function.

Long-acting insulin analogues elicit atypical signalling events mediated by the insulin receptor and insulin-like growth factor-I receptor.

AIMS/HYPOTHESIS: Insulin analogues were developed to improve the pharmacological properties of injected insulin and to better mimic endogenous insulin output. However, certain insulin analogues have been suggested to display IGF-I-like biological activities. Furthermore, several recent epidemiological studies have suggested a potential increase in cancer risk for treatment of diabetes patients with long-acting analogue insulin glargine (A21Gly,B31Arg,B32Arg human insulin). Additional studies, however, reported no increased cancer risk. The purpose of the present study was to identify the receptor(s) and signal transduction pathways responsible for the biological actions of insulin glargine and insulin detemir (B29Lys[ε-tetradecanoyl],desB30 human insulin). METHODS: The colon cancer-derived cell line HCT116 was treated with increasing doses of insulin glargine, insulin detemir, regular insulin or IGF-I, and receptor activation was evaluated by immunoprecipitation assays. IGF-I receptor (IGF-IR) internalisation following insulin glargine treatment was assessed by confocal microscopy. Activation of the Akt and extracellular signal-regulated kinase pathways was evaluated by western blots. The anti-apoptotic effect of the analogues was measured by poly-(ADP ribose) polymerase antibody and annexin assays. RESULTS: We found evidence for dual activation of the insulin receptor and IGF-IR by the analogues. Dose-dependency experiments showed that insulin glargine was able to phosphorylate the IGF-IR at fivefold lower doses than those required to activate the insulin receptor. We also showed that insulin glargine can lead to prolonged activation of the receptors and therefore promote abnormal signalling. Confocal imaging experiments showed that insulin glargine, but not regular insulin induced IGF-IR internalisation similarly to IGF-I. Finally, both analogues displayed IGF-I-like anti-apoptotic activities and stimulated cell cycle progression. CONCLUSIONS/INTERPRETATION: Our data indicate that insulin glargine and insulin detemir display atypical signalling activities that differ from those elicited by regular insulin and involve activation of the anti-apoptotic IGF-IR.

[Safety of insulin analogues: what to evaluate, how to do it, and how to interpret the results]. / Seguridad de los análogos de insulina: qué evaluar, cómo hacerlo y cómo interpretar los resultados.

The widespread use of insulin analogues is based not only on the pharmacokinetics of these preparations, which is much closer to the physiology of insulin secretion under normal conditions, but also on their safety and effectiveness. The publication of a possible association between the use of a long-acting insulin analogue (glargine) and breast cancer has caused uneasiness among the medical community regarding the safety of these analogues. The mechanism of increased tumor activity of insulin analogues is explained by the fact that they act through insulin receptors (IR) and insulin-like growth factor-1 (IGF-1R), stimulating cell growth and inhibiting apoptosis. There are two major mechanisms: an increase in the binding time of insulin to IR and increased activation of IGF-1R. Therefore, to evaluate the safety of an analogue, the slower dissociation rate from its insulin receptor must be excluded, as well as the increased affinity for the IGF-1 receptor. This is equivalent to an index of mitogenic/metabolic activity of less than 1. These aspects can only be evaluated through study of cell lines and animal testing, which are reductionist models that cannot always be extrapolated to humans. To date, there are no data to question the safety of insulin analogues in general. However, the results of observational studies and some in vitro studies, suggesting a potential risk of mitogenicity with the administration of glargine, have caused some alarm among the medical community. Until now, there are no data to refute or confirm this risk and, therefore, evaluation of the existing data is crucial to obtain objective information.

Type 1 insulin-like growth factor receptor signaling is essential for the development of the hippocampal formation and dentate gyrus.

Type 1 insulin-like growth factor receptor (IGF1R) signaling in neuronal development was studied in mutant mice with blunted igf1r gene expression in nestin-expressing neuronal precursors. At birth [postnatal (P) day 0] brain weights were reduced to 37% and 56% of controls in mice homozygous (nes-igf1r(-/-)) and heterozygous (nes-igf1r(-/Wt)) for the null mutation, respectively, and this brain growth retardation persisted postnatally. Stereological analysis demonstrated that the volumes of the hippocampal formation, CA fields 1-3, dentate gyrus (DG), and DG granule cell layer (GCL) were decreased by 44-54% at P0 and further by 65-69% at P90 in nes-igf1r(-/Wt) mice. In nes-igf1r(-/-) mice, volumes were 29-31% of controls at P0 and, in the two mice that survived to P90, 6-19% of controls, although the hilus could not be identified. Neuron density did not differ among the mice at any age studied; therefore, decreased volumes were due to reduced cell number. In postnatal nes-igf1r(-/Wt) mice, the percentage of apoptotic cells, as judged by activated caspase-3 immunostaining, was increased by 3.5-5.3-fold. The total number of proliferating DG progenitors (labeled by BrdU incorporation and Ki67 staining) was reduced by approximately 50%, but the percentage of these cells was similar to the percentages in littermate controls. These findings suggest that 1) the postnatal reduction in DG size is due predominantly to cell death, pointing to the importance of the IGF1R in regulating postnatal apoptosis, 2) surviving DG progenitors remain capable of proliferation despite reduced IGF1R expression, and 3) IGF1R signaling is necessary for normal embryonic brain development.

Heterogeneity of receptor function in colon carcinoma cells determined by cross-talk between type I insulin-like growth factor receptor and epidermal growth factor receptor.

This study identifies a novel cross-talk paradigm between the type I insulin-like growth factor receptor (IGF1R) and epidermal growth factor receptor (EGFR) in colon cancer cells. IGF1R activation by ligand exposure in growth factor-deprived cells induces Akt activation in the FET, CBS, and GEO colon cancer cell lines. Investigation of IGF1R-mediated signaling pathways using small interfering RNA approaches indicated that, as expected, phosphatidylinositol 3'-kinase (PI3K) was activated by IGF1R. Mitogen-activated protein kinase (MAPK) activity as reflected by phospho-extracellular signal-regulated kinase (ERK) induction was not significantly activated until later times following release of these cells from growth factor deprivation stress. The appearance of phospho-ERK was proximal to EGFR activation. Treatment of cells with the PI3K inhibitor LY294002 before release from stress resulted in a concentration-dependent loss of EGFR activation, whereas treatment with the MAPK inhibitor PD98059 did not block EGFR activation, indicating that EGFR activation was downstream of the IGF1R/PI3K pathway. PD98059 inhibition of MAPK was associated with a concentration-dependent reduction in EGFR-mediated phospho-ERK. EGFR inhibitor blocked induction of phospho-ERK, showing that MAPK activity was a consequence of EGFR-mediated signaling. On the other hand, a small-molecule IGF1R inhibitor, PQIP, blocked Akt phosphorylation. The divergent signaling functions of IGF1R and EGFR suggested the potential for synergism by a combination of therapy directed at the two receptors. Combination treatment with PQIP and EGFR inhibitor Tarceva resulted in synergistic effects as indicated by combination index analysis in all three cell lines tested.

Rosiglitazone attenuates insulin-like growth factor 1 receptor survival signaling in PC-3 cells.

PPARgamma, a member of the peroxisome proliferator-activated receptor family, is overexpressed in prostate cancer. Natural and synthetic ligands of PPARgamma via genomic and nongenomic actions promote cell cycle arrest and apoptosis of several prostate cancer cells, in vitro. Insulin-like growth factor 1 (IGF-1) inhibits the adriamycin-induced apoptosis of PC-3 human prostate cancer cells. Therefore, we have analyzed the ability of two PPARgamma ligands,15dPGJ2 and rosiglitazone, a natural and a synthetic PPARgamma ligand, respectively, to increase the adriamycin-induced cytotoxicity of PC-3 cells and to suppress the IGF-1 survival effect on adriamycin-induced apoptosis of PC-3 cells. Our data revealed that both the PPARgamma ligands increased the adriamycin-induced cytostasis of PC-3 cells, however, only rosiglitazone added to the adriamycin-induced apoptosis of PC-3 cells. In addition, rosiglitazone attenuated the type I IGF receptor (IGF-1R) survival signaling on adriamycin-induced apoptosis of PC-3 cells via its nongenomic action on ERK1/2 and AKT phosphorylation. Because the IGF-1R signaling is probably the most important host tissue (bone) metastasis microenvironment-related survival signaling for prostate cancer cells, we conclude that rosiglitazone effects on IGF-1R-mediated activation of ERK1/2 and AKT could have clinical implications for the management of androgen ablation-refractory and chemotherapy-resistant advanced prostate cancer with bone metastasis.

A variable degree of intrauterine and postnatal growth retardation in a family with a missense mutation in the insulin-like growth factor I receptor.

CONTEXT: The type 1 IGF-I receptor (IGF1R) mediates the biological functions of IGF-I. Binding of IGF-I to the IGF1R results in autophosphorylation of the intracellular beta-subunit and activation of intracellular signaling. OBJECTIVE: The objective of this study was to evaluate the functional characteristics of a novel IGF1R mutation and describe the phenotypic features of two patients with this mutation. DESIGN: The study was performed in a university hospital. PATIENTS: We describe a 35-yr-old female with mild intrauterine growth failure, progressive postnatal growth retardation, severe failure to thrive, and microcephaly. Her daughter was born with severe intrauterine growth retardation and also showed postnatal failure to thrive and microcephaly. RESULTS: We found a heterozygous G3148-->A nucleotide substitution in the IGF1R gene, changing a negatively charged glutamic acid at position 1050 into a positively charged lysine residue (E1050K). E1050 is a conserved residue in the intracellular kinase domain. Dermal fibroblasts of the mother showed normal binding of iodinated IGF-I, but autophosphorylation and activation of downstream signaling cascades upon challenging with IGF-I was markedly reduced. Consequently, the maximal [(3)H]thymidine incorporation upon challenge with a dose range of IGF-I was reduced compared with a panel of control cells (3.65 +/- 1.79-fold vs. 6.75 +/- 4.7-fold stimulation; P < 0.01). These data suggest that the mutation results in the inactivation of one copy of the IGF1R gene. CONCLUSIONS: These two patients support the key role for IGF-I in intrauterine and postnatal growth. The different phenotypes of these and earlier described patients may be associated with variability in IGF-I signaling. The degree of intrauterine growth retardation may be partially determined by the presence or absence of maternal IGF-I resistance.

Genistein inhibits insulin-like growth factor-I receptor signaling in HT-29 human colon cancer cells: a possible mechanism of the growth inhibitory effect of Genistein.

Genistein, a soy isoflavone, has attracted much attention for its chemopreventive properties. Overexpression and constitutive activation of receptor tyrosine kinases are frequent events in human cancer. Because genistein has previously been reported to decrease HT-29 cell growth, the present study compared the effects of genistein with daidzein on the protein levels of the members of the ErbB receptor family and insulin-like growth factor-I (IGF-I) receptor (IGF-IR). HT-29 cells were cultured in serum-free medium, with 0, 25, 50, or 100 micromol/L genistein, daidzein, and/or 10 nmol/L IGF-I. DNA synthesis was estimated by 5-bromo-2'-deoxyuridine incorporation. Apoptotic cells were analyzed by annexin-V staining followed by flow cytometry. Genistein inhibited viable HT-29 cell numbers, in a dose-dependent manner, whereas daidzein had no effect on cell growth. The decrease in cell growth caused by genistein was due to decreased DNA synthesis and apoptosis induction. Immunoblot analysis showed that neither genistein nor daidzein decreased the protein levels of either of the epidermal growth factor receptors, ErbB2 or ErbB3. Genistein did, however, decrease the IGF-IR protein levels, whereas daidzein had no effect. Genistein did not change the protein levels of insulin-receptor substrate-1 (IRS-1), the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K), or Akt. Immunoprecipitation/western blot analyses revealed that genistein decreased IGF-I-stimulated phosphorylation of IGF-IR and IRS-1, recruitment of p85 to IGF-IR, and phosphorylation of Akt. These results suggest that inhibition of cell proliferation and induction of apoptosis by genistein are mediated, at least in part, by its ability to inhibit IGF-IR signaling and the PI3K/Akt pathway.

Genetic blockade of the insulin-like growth factor-I receptor: a promising strategy for human pancreatic cancer.

Pancreatic cancer is one of the most lethal malignant tumors. Insulin-like growth factor (IGF)-I receptor (IGF-Ir) signaling is required for maintenance of growth and tumorigenicity of many tumors, but this pathway has not been well studied in pancreatic cancer. We have shown previously successful therapy in colorectal and lung cancer xenograft models using recombinant adenoviruses expressing dominant negative IGF-I receptors. In this study, we sought to better dissect the mechanism of action of this virus and determine whether IGF-Ir targeted adenoviruses represent potentially effective therapeutics for human pancreatic cancer cells. Truncated IGF-I receptors (IGF-Ir/dn; 482 and 950 amino acids long, respectively, IGF-Ir/482st and IGF-Ir/950st) that function as dominant negative inhibitor were cloned into recombinant adenoviruses and used to treat human pancreatic cancer cells. We assessed the effect of IGF-Ir/dn on signaling blockade, growth, stress response, chemotherapy, radiation-induced apoptosis, and in vivo therapeutic efficacy in xenografts. IGF-Ir/dn expression suppressed tumorigenicity both in vitro and in vivo and up-regulated stressor-induced apoptosis. It effectively blocked both IGF-I and IGF-II-induced activation of Akt-1. IGF-Ir/dn expression increased radiation and chemotherapy-induced apoptosis, and the combination therapy of IGF-Ir/dn with chemotherapy was very effective against tumors in mice. In an i.p. model, IGF-Ir/dn therapy reduced dissemination and prolonged survival times. Moreover, IGF-Ir/482st was more effective than IGF-Ir/950st because of its bystander effect. The antitumor activity of IGF-Ir/dn is mediated through inhibition of Akt-1 and enhances the efficacy of chemotherapy. Adenovirus-IGF-Ir/482st may be a useful anticancer therapeutic for pancreatic cancer.

Immunoglobulin activation of T cell chemoattractant expression in fibroblasts from patients with Graves' disease is mediated through the insulin-like growth factor I receptor pathway.

Graves' disease (GD) is associated with T cell infiltration, but the mechanism for lymphocyte trafficking has remained uncertain. We reported previously that fibroblasts from patients with GD express IL-16, a CD4-specific chemoattractant, and RANTES, a C-C chemokine, in response to GD-specific IgG (GD-IgG). We unexpectedly found that these responses result from a functional interaction between GD-IgG and the insulin-like growth factor (IGF)-I receptor (IGF-IR). IGF-I and the IGF-IR-specific IGF-I analog, des(1-3), mimic the effects of GD-IgG. Neither GD-IgG nor IGF-I activates chemoattractant expression in control fibroblasts from donors without GD. Interrupting IGF-IR function with specific receptor-blocking Abs or by transiently transfecting fibroblasts with a dominant negative mutant IGF-IR completely attenuates signaling provoked by GD-IgG. Moreover, GD-IgG displaces specific (125)I-labeled IGF-I binding to fibroblasts and attenuates IGF-IR detection by flow cytometry. These findings identify a novel disease mechanism involving a functional GD-IgG/IGF-IR bridge, which potentially explains T cell infiltration in GD. Interrupting this pathway may constitute a specific therapeutic strategy.

Insulin-like growth factor I receptor antagonism augments response to chemoradiation therapy in colon cancer cells.

BACKGROUND: Colorectal cancer remains one of the most prevalent malignancies in the United States. Improvement in local disease control is seen when 5-fluorouracil (5-FU) is used in combination with pelvic irradiation for rectal adenocarcinoma. The frequent overexpression of insulin-like growth factor I receptor (IGF-I-R) in rectal adenocarcinoma suggests that inhibition of the signal transduction pathway may be a novel approach to enhance tumor response. This investigation seeks to define the role of IGF-I-R antagonism, using monoclonal antibody alpha-IR3, in augmenting cytotoxicity to adjuvant chemoradiation therapy for adenocarcinoma of the rectum. MATERIALS AND METHODS: SW 480 colon cancer cells were cultured to semiconfluent conditions with dose titrations performed for 5-FU to determine that the IC(50) (inhibitory concentration of 50% of the cells) was 0.5 microg/ml. The IC(50) for 5-FU was reassessed in the presence of IGF-I. Experimental groups included colon cancer cells combined with 5-FU; 6-MeV external beam radiation (100-500 cGy); and alpha-IR-3. RESULTS: The addition of 100 ng/ml IGF-I 1 h prior to 5-FU or radiation significantly blunted the expected cytotoxicity, resulting in a 10-fold increase in the IC(50) (from 0.5 to 5 microg/ml). Receptor antagonism using the monoclonal antibody alpha-IR-3 (100-400 ng/ml) produced a dose-dependent increase in cytotoxicity compared with 5-FU alone. The addition of radiation produced synergistic amplification of this response. CONCLUSIONS: IGF-I-R activation blocks the expected cytotoxic effects of 5-FU and external beam radiation. Receptor antagonism increased the cytotoxic response of chemoradiation therapy. These data suggest the utility of inhibiting IGF-I-R signal transduction in the treatment of rectal adenocarcinoma.

Mitogenicity and transforming activity of the insulin-like growth factor-I receptor with mutations in the tyrosine kinase domain.

We have investigated the effect of mutations in tyrosines 1131, 1135, and 1136 of the human insulin-like growth factor-I receptor (IGF-IR) on the growth and transformation of mammalian cells. We have used for this purpose R- cells, which are 3T3-like fibroblasts derived from mouse embryos with a targeted disruption of the IGF-IR genes. These cells have no IGF-IR, do not grow in serum-free medium supplemented with the growth factors that sustain the growth of 3T3 cells, and cannot be transformed by simian virus 40 large tumor antigen or other oncogenes. The R- cells were transfected with plasmids expressing: 1) a wild type human IGF-IR cDNA; 2) a receptor with a triple mutation in the above mentioned tyrosines; and 3) receptors with single tyrosine mutations. Cells expressing the wild type or the single tyrosine mutants Y1 (Y1131F) and Y2 (Y1135F) grew in serum-free medium supplemented solely with IGF-I. Cells expressing the triple tyrosine mutant YF or the single mutant Y3 (Y1136F) failed to grow in response to IGF-I only. All mutants, though, failed to form colonies in soft agar, indicating that a fully functional IGF-IR is more critical for anchorage-independent growth than for monolayer growth. The triple mutant expression plasmid also functioned as a dominant negative, inhibiting the growth of wild type cells transformed by the simian virus tumor antigen.

Signal transduction by a chimeric insulin-like growth factor-1 (IGF-1) receptor having the carboxyl-terminal domain of the insulin receptor.

Insulin-like growth factor-1 receptors (IGF-1R) and insulin receptors (IR) are closely related tyrosine kinases. However, the IR plays a major role in metabolism control, whereas the IGF-1R is mainly involved in growth and differentiation. With these observations in mind, we wished to define the regions of IR and IGF-1R responsible for generation of biological specificity. We constructed a chimeric IGF-1R in which the carboxyl-terminal domain was replaced by that of IR. This receptor (IGF/CTIR) was expressed in NIH3T3 cells, and we compared its biological activity with that of wild-type receptors. The IGF/CTIR was fully functional regarding kinase activity and biological properties. Comparison of insulin and IGF-1 effects on IR and IGF-1R cells, respectively, indicated that the IR is more efficient in stimulating glycogen synthesis and p44mapk activity than is the IGF-1R. Interestingly, in IGF/CTIR16 cells expressing only 250,000 receptors glycogen synthesis was better stimulated than in IGF-1R cells with 600,000 receptors. Similarly, p44mapk activation was slightly higher in IGF/CTIR16 cells than in IGF-1R cells. These results suggest that the carboxyl-terminal domain of IR is more tightly coupled to the stimulation of glycogen synthesis and to the p44mapk pathway than is that of IGF-1R. We propose that this domain plays a crucial role in the transmission of biological effects and could account, at least in part, for receptor specificity.

Human growth hormone and human aging.

In humans, both aging and GH deficiency are associated with reduced protein synthesis, decreased lean body and bone mass, and increased percent body fat. In healthy individuals, spontaneous and stimulated GH secretion, as well as circulating IGF-I and IGFBP-3 levels, are significantly decreased with advancing age. The extent to which these age-related changes in GH and IGF-I contribute to alterations in body composition and function remains to be elucidated. GH treatment of GH-deficient adults or old men with reduced IGF-I levels with exogenous GH increases plasma IGF-I, nitrogen retention, and lean body mass, decreases percent body fat, and exerts little effect on bone mineral density. Short-term adverse effects of GH therapy have been minimized by using low-dose regimens, but it is still uncertain whether long-term GH supplementation in adult life increases the risk of metabolic abnormalities or malignancy. Administration of GHRH, which has been shown to maintain the pattern of pulsatile GH secretion in old men, may represent another possible physiological approach to therapy. It may be justifiable initially to limit use of GH to certain elderly patients such as those suffering from catabolic illnesses, malnourishment, burns, cachexia, etc. A great deal more research will be necessary to determine whether normalization of GH and IGF-I levels in healthy older persons will lead to improvements in their physical and psychological functional capacity and quality of life.

Basic fibroblast growth factor modulates insulin-like growth factor-I, its receptor, and its binding proteins in hypothalamic cell cultures.

Interactions between different growth factors may be important in the regulation of cell growth and differentiation in the nervous system. For instance, basic fibroblast growth factor (bFGF) regulates neuroblast division through a mechanism probably involving insulin-like growth factor-I (IGF-I). In this regard, we previously found that simultaneous addition of both factors produces an additive effect on survival and differentiation of hypothalamic neuronal and glial cells in culture. To further analyze these interactions, we explored the influence of bFGF on IGF-I, its membrane receptor, and its binding proteins in hypothalamic cells. We also tested the effects of IGF-I on its own receptor and binding proteins (IGFBPs) to determine the specificity of bFGF's actions. Treatment of neuronal and glial cultures with bFGF produced an increase in IGF-I receptors, without changing their affinity, together with an increase in the apparent M(r) of the receptor. On the other hand, IGF-I elicited a down-regulation of its own receptor in both neurons and glia, without modifying its affinity. Treatment with bFGF also produced a marked differential effect on the IGFBPs secreted by the cells. While IGFBP levels in neuronal cultures were greatly increased by bFGF, their production by glial cells was inhibited. On the other hand, IGF-I increased the amount of IGFBPs in both types of cells. Finally, addition of bFGF to the cultures elicited a dose-dependent increase in the release of IGF-I to the medium, but only a moderate increase in cellular IGF-I content, in both neurons and glia. We conclude that bFGF strongly modulates IGF-I, its receptors, and its binding proteins in the two major cell types of the hypothalamus. These findings reinforce the possibility that IGF-I and/or its receptors and binding proteins are involved in the trophic effects of bFGF on developing brain cells.