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1.
Nat Commun ; 13(1): 173, 2022 01 10.
Article in English | MEDLINE | ID: mdl-35013311

ABSTRACT

Mechanisms of drug-tolerance remain poorly understood and have been linked to genomic but also to non-genomic processes. 5-fluorouracil (5-FU), the most widely used chemotherapy in oncology is associated with resistance. While prescribed as an inhibitor of DNA replication, 5-FU alters all RNA pathways. Here, we show that 5-FU treatment leads to the production of fluorinated ribosomes exhibiting altered translational activities. 5-FU is incorporated into ribosomal RNAs of mature ribosomes in cancer cell lines, colorectal xenografts, and human tumors. Fluorinated ribosomes appear to be functional, yet, they display a selective translational activity towards mRNAs depending on the nature of their 5'-untranslated region. As a result, we find that sustained translation of IGF-1R mRNA, which encodes one of the most potent cell survival effectors, promotes the survival of 5-FU-treated colorectal cancer cells. Altogether, our results demonstrate that "man-made" fluorinated ribosomes favor the drug-tolerant cellular phenotype by promoting translation of survival genes.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Colorectal Neoplasms/drug therapy , DNA, Neoplasm/genetics , Drug Tolerance/genetics , Fluorouracil/pharmacology , Protein Biosynthesis/drug effects , Receptor, IGF Type 1/genetics , Cell Line, Tumor , Cell Survival/drug effects , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , DNA Replication , DNA, Neoplasm/metabolism , Drug Resistance, Neoplasm/genetics , HCT116 Cells , Halogenation , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Receptor, IGF Type 1/agonists , Receptor, IGF Type 1/metabolism , Ribosomes/drug effects , Ribosomes/genetics , Ribosomes/metabolism , Xenograft Model Antitumor Assays
2.
J Neurosci ; 41(11): 2360-2372, 2021 03 17.
Article in English | MEDLINE | ID: mdl-33514676

ABSTRACT

Human fMRI studies show that insulin influences brain activity in regions that mediate reward and motivation, including the nucleus accumbens (NAc). Insulin receptors are expressed by NAc medium spiny neurons (MSNs), and studies of cultured cortical and hippocampal neurons suggest that insulin influences excitatory transmission via presynaptic and postsynaptic mechanisms. However, nothing is known about how insulin influences excitatory transmission in the NAc. Furthermore, insulin dysregulation accompanying obesity is linked to cognitive decline, depression, anxiety, and altered motivation that rely on NAc excitatory transmission. Using whole-cell patch-clamp and biochemical approaches, we determined how insulin affects NAc glutamatergic transmission in nonobese and obese male rats and the underlying mechanisms. We find that there are concentration-dependent, bidirectional effects of insulin on excitatory transmission, with insulin receptor activation increasing and IGF receptor activation decreasing NAc excitatory transmission. Increases in excitatory transmission were mediated by activation of postsynaptic insulin receptors located on MSNs. However, this effect was due to an increase in presynaptic glutamate release. This suggested feedback from MSNs to presynaptic terminals. In additional experiments, we found that insulin-induced increases in presynaptic glutamate release are mediated by opioid receptor-dependent disinhibition. Furthermore, obesity resulted in a loss of insulin receptor-mediated increases in excitatory transmission and a reduction in NAc insulin receptor surface expression, while preserving reductions in transmission mediated by IGF receptors. These results provide the first insights into how insulin influences excitatory transmission in the adult brain, and evidence for a previously unidentified form of opioid receptor-dependent disinhibition of NAc glutamatergic transmission.SIGNIFICANCE STATEMENT Data here provide the first insights into how insulin influences excitatory transmission in the adult brain, and identify previously unknown interactions between insulin receptor activation, opioids, and glutamatergic transmission. These data contribute to our fundamental understanding of insulin's influence on brain motivational systems and have implications for the use of insulin as a cognitive enhancer and for targeting of insulin receptors and IGF receptors to alter motivation.


Subject(s)
Endorphins/pharmacology , Glutamic Acid/metabolism , Insulin/pharmacology , Nucleus Accumbens/drug effects , Nucleus Accumbens/physiology , Receptor, Insulin/drug effects , Synaptic Transmission/drug effects , Animals , Diet, High-Fat , Male , Neurons/drug effects , Obesity/genetics , Patch-Clamp Techniques , Presynaptic Terminals/drug effects , Rats , Rats, Sprague-Dawley , Receptor, IGF Type 1/agonists , Receptors, Dopamine D1/drug effects , Receptors, Dopamine D2/drug effects
3.
Eur J Pharmacol ; 887: 173581, 2020 Nov 15.
Article in English | MEDLINE | ID: mdl-32949596

ABSTRACT

Glucagon-like peptide-1 (GLP-1) is an endogenous gut hormone and a key regulator in maintaining glucose homeostasis by stimulating insulin secretion. Its natural cleavage product GLP-1 (9-36), which was formerly considered a "bio-inactive" metabolite mainly due to its low affinity for GLP-1 receptor, possesses unique properties such as cardiovascular protection. Little is known about the effects and mechanisms of GLP-1 (9-36) in cerebral ischemia and reperfusion injury. Here, we report that systemic application of GLP-1 (9-36) in adult mice facilitated functional recovery and reduced infarct volume, astrogliosis, and neuronal apoptosis following middle cerebral artery occlusion and reperfusion. Interestingly, these effects were still observed in GLP-1 receptor knockout (Glp-1rKO) mice but were partially reversed in insulin-like growth factor 1 (IGF-1) receptor knockdown (Igf-1rKD) mice. Primary astrocytes were cultured and subjected to oxygen-glucose deprivation/reoxygenation (OGD/R), and enzyme-linked immunosorbent assay indicated that GLP-1 (9-36) pretreatment reduces tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 levels. This effect was not diminished in Glp-1rKO astrocytes but was reversed in Igf-1rKO astrocytes, emphasizing that the anti-inflammatory effect of GLP-1 (9-36) in astrocytes is independent of GLP-1 receptor signaling and is instead mediated by IGF-1 receptor. Immunoprecipitation experiments showed that GLP-1 (9-36) directly interacts with IGF-1 receptor in astrocytes. Western blot data indicated that GLP-1 (9-36) activates IGF-1 receptor and downstream PI3K-AKT pathway in astrocytes upon OGD/R injury, which was abrogated by preincubation with IGF-1 receptor autophosphorylation inhibitor picropodophyllin. Thus, our findings suggest that GLP-1 (9-36) improved stroke outcome by reducing inflammation in astrocytes via interaction with IGF-1 receptor.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Astrocytes/drug effects , Encephalitis/drug therapy , Encephalitis/etiology , Glucagon-Like Peptide 1/analogs & derivatives , Glucagon-Like Peptide 1/metabolism , Receptor, IGF Type 1/agonists , Stroke/complications , Stroke/drug therapy , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Behavior, Animal/drug effects , Cell Hypoxia , Cytokines/metabolism , Encephalitis/psychology , Gene Knockdown Techniques , Glucagon-Like Peptide 1/genetics , Glucagon-Like Peptide 1/pharmacology , Glucagon-Like Peptide 1/therapeutic use , Glucose/deficiency , Mice , Mice, Knockout , Primary Cell Culture , Receptor, IGF Type 1/genetics , Signal Transduction/drug effects , Stroke/psychology
4.
Sci Rep ; 9(1): 13634, 2019 09 20.
Article in English | MEDLINE | ID: mdl-31541165

ABSTRACT

ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 2 (ST8SIA2) synthesizes polysialic acid (PSA), which is essential for brain development. Although previous studies reported that St8sia2-deficient mice that have a mixed 129 and C57BL/6 (B6) genetic background showed mild and variable phenotypes, the reasons for this remain unknown. We hypothesized that this phenotypic difference is caused by diversity in the expression or function of flanking genes of St8sia2. A genomic polymorphism and gene expression analysis in the flanking region revealed reduced expression of insulin-like growth factor 1 receptor (Igf1r) on the B6 background than on that of the 129 strain. This observation, along with the finding that administration of an IGF1R agonist during pregnancy increased litter size, suggests that the decreased expression of Igf1r associated with ST8SIA2 deficiency caused lethality. This study demonstrates the importance of gene expression level in the flanking regions of a targeted null allele having an effect on phenotype.


Subject(s)
Down-Regulation , Gene Expression Profiling/methods , Receptor, IGF Type 1/genetics , Sialyltransferases/deficiency , Animals , Female , Gene Expression Regulation , Genes, Lethal , Insulin-Like Growth Factor I/administration & dosage , Insulin-Like Growth Factor I/analogs & derivatives , Insulin-Like Growth Factor I/pharmacology , Litter Size/drug effects , Loss of Function Mutation , Male , Mice , Phenotype , Polymorphism, Single Nucleotide , Pregnancy , Receptor, IGF Type 1/agonists
5.
Exp Neurol ; 312: 72-81, 2019 02.
Article in English | MEDLINE | ID: mdl-30503192

ABSTRACT

Disruption of the blood-brain barrier results in the formation of edema and contributes to the loss of neurological function following intracerebral hemorrhage (ICH). This study examined insulin-like growth factor-1 (IGF-1) as a treatment and its mechanism of action for protecting the blood-brain barrier after ICH in mice. 171 Male CD-1 mice were subjected to ICH via collagenase or autologous blood. A dose study for recombinant human IGF-1 (rhIGF-1) was performed. Brain water content and behavioral deficits were evaluated at 24 and 72 h after the surgery, and Evans blue extravasation and hemoglobin assay were conducted at 24 h. Western blotting was performed for the mechanism study and interventions were used targeting the IGF-1R/GSK3ß/MEKK1 pathway. rhIGF-1 reduced edema and blood-brain barrier permeability, and improved neurobehavior outcomes. Western blots showed that rhIGF-1 reduced p-GSK3ß and MEKK1 expression, thereby increasing occludin and claudin-5 expression. Inhibition and knockdown of IGF-1R reversed the therapeutic benefits of rhIGF-1. The findings within suggest that stimulation of the IGF-1R is a therapeutic target for ICH which may lead to improved neurofunctional and blood-brain barrier protection.


Subject(s)
Blood-Brain Barrier/metabolism , Capillary Permeability/physiology , Cerebral Hemorrhage/metabolism , Insulin-Like Growth Factor I/administration & dosage , Animals , Blood-Brain Barrier/drug effects , Capillary Permeability/drug effects , Cerebral Hemorrhage/drug therapy , Injections, Intraventricular , Male , Mice , RNA, Small Interfering/administration & dosage , Receptor, IGF Type 1/agonists , Receptor, IGF Type 1/antagonists & inhibitors , Receptor, IGF Type 1/metabolism
6.
BMC Cancer ; 17(1): 636, 2017 Sep 07.
Article in English | MEDLINE | ID: mdl-28882129

ABSTRACT

BACKGROUND: The insulin growth factor (IGF) pathway has been proposed as a potential therapeutic target in bladder cancer. We characterized the expression of components of the IGF pathway - insulin growth factor receptors (INSR, IGF1R, IGF2R), ligands (INS, IGF1, IGF2), and binding proteins (IGFBP1-7, IGF2BP1-3) - in bladder cancer and its correlation with IGF1R activation, and the anti-proliferative efficacy of an IGF1R kinase inhibitor in this setting. METHODS: We analyzed transcriptomic data from two independent bladder cancer datasets, corresponding to 200 tumoral and five normal urothelium samples. We evaluated the activation status of the IGF pathway in bladder tumors, by assessing IGF1R phosphorylation and evaluating its correlation with mRNA levels for IGF pathway components. We finally evaluated the correlation between inhibition of proliferation by a selective inhibitor of the IGF1R kinase (AEW541), reported in 13 bladder cancer derived cell lines by the Cancer Cell Line Encyclopedia Consortium and mRNA levels for IGF pathway components. RESULTS: IGF1R expression and activation were stronger in non-muscle-invasive than in muscle-invasive bladder tumors. There was a significant inverse correlation between IGF1R phosphorylation and IGFBP5 expression in tumors. Consistent with this finding, the inhibition of bladder cell line viability by IGF1R inhibitor was also inversely correlated with IGFBP5 expression. CONCLUSION: The IGF pathway is activated and therefore a potential therapeutic target for non muscle-invasive bladder tumors and IGFBP5 could be used as a surrogate marker for predicting tumor sensitivity to anti-IGF therapy.


Subject(s)
Gene Expression Regulation, Neoplastic , Insulin-Like Growth Factor Binding Protein 5/genetics , Receptor, IGF Type 1/agonists , Receptor, IGF Type 1/antagonists & inhibitors , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/metabolism , Aged , Aged, 80 and over , Carrier Proteins , Cell Line, Tumor , Cell Proliferation , Female , Humans , Insulin-Like Growth Factor Binding Protein 5/metabolism , Ligands , Male , Middle Aged , Neoplasm Grading , Neoplasm Staging , Protein Binding , RNA, Messenger/genetics , Signal Transduction , Urinary Bladder Neoplasms/pathology
7.
Breast Cancer Res ; 19(1): 14, 2017 02 07.
Article in English | MEDLINE | ID: mdl-28173837

ABSTRACT

BACKGROUND: The insulin-like growth factor 1 (IGF1) signaling axis plays a major role in tumorigenesis. In a previous experiment, we chronically treated mice with several agonists of the IGF1 receptor (IGF1R). We found that chronic treatment with insulin analogues with high affinity towards the IGF1R (IGF1 and X10) decreased the mammary gland tumor latency time in a p53R270H/+WAPCre mouse model. Frequent injections with insulin analogues that only mildly activated the IGF1R in vivo (glargine and insulin) did not significantly decrease the tumor latency time in this mouse model. METHODS: Here, we performed next-generation RNA sequencing (40 million, 100 bp reads) on 50 mammary gland tumors to unravel the underlying mechanisms of IGF1R-promoted tumorigenesis. Mutational profiling of the individual tumors was performed to screen for treatment-specific mutations. The transcriptomic data were used to construct a support vector machine (SVM) classifier so that the phenotypic characteristics of tumors exposed to the different insulin analogue treatments could be predicted. For translational purposes, we ran the same classifiers on transcriptomic (micro-array) data of insulin analogue-exposed human breast cancer cell lines. Genome-scale metabolic modeling was performed with iMAT. RESULTS: We found that chronic X10 and IGF1 treatment resulted in tumors with an increased and sustained proliferative and invasive transcriptomic profile. Furthermore, a Warburg-like effect with increased glycolysis was observed in tumors of the X10/IGF1 groups and, to a lesser extent, also in glargine-induced tumors. A metabolic flux analysis revealed that this enhanced glycolysis programming in X10/IGF1 tumors was associated with increased biomass production programs. Although none of the treatments induced genetic instability or enhanced mutagenesis, mutations in Ezh2 and Hras were enriched in X10/IGF1 treatment tumors. CONCLUSIONS: Overall, these data suggest that the decreased mammary gland tumor latency time caused by chronic IGF1R activation is related to modulation of tumor progression rather than increased tumor initiation.


Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Glucose/metabolism , Receptor, IGF Type 1/metabolism , Animals , Biomarkers , Breast Neoplasms/genetics , Breast Neoplasms/mortality , Cell Line, Tumor , Cell Movement , Enhancer of Zeste Homolog 2 Protein/genetics , Epithelial-Mesenchymal Transition/genetics , Female , Gene Expression Profiling , Glycolysis , Insulin/metabolism , Insulin-Like Growth Factor I/metabolism , Insulin-Like Growth Factor I/pharmacology , Mice , Mice, Transgenic , Mutation , Prognosis , Receptor, IGF Type 1/agonists , Signal Transduction , Transcriptome , Tumor Burden , ras Proteins/genetics
8.
Cardiovasc Diabetol ; 15(1): 161, 2016 12 01.
Article in English | MEDLINE | ID: mdl-27905925

ABSTRACT

BACKGROUND: Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) is a major contributor to the development of atherosclerotic process. In a previous work, we demonstrated that the insulin receptor isoform A (IRA) and its association with the insulin-like growth factor-I receptor (IGF-IR) confer a proliferative advantage to VSMCs. However, the role of IR and IGF-IR in VSMC migration remains poorly understood. METHODS: Wound healing assays were performed in VSMCs bearing IR (IRLoxP+/+ VSMCs), or not (IR-/- VSMCs), expressing IRA (IRA VSMCs) or expressing IRB (IRB VSMCs). To study the role of IR isoforms and IGF-IR in experimental atherosclerosis, we used ApoE-/- mice at 8, 12, 18 and 24 weeks of age. Finally, we analyzed the mRNA expression of total IR, IRB isoform, IGF-IR and IGFs by qRT-PCR in the medial layer of human aortas. RESULTS: IGF-I strongly induced migration of the four cell lines through IGF-IR. In contrast, insulin and IGF-II only caused a significant increase of IRA VSMC migration which might be favored by the formation of IRA/IGF-IR receptors. Additionally, a specific IGF-IR inhibitor, picropodophyllin, completely abolished insulin- and IGF-II-induced migration in IRB, but not in IRA VSMCs. A significant increase of IRA and IGF-IR, and VSMC migration were observed in fibrous plaques from 24-week-old ApoE-/- mice. Finally, we observed a marked increase of IGF-IR, IGF-I and IGF-II in media from fatty streaks as compared with both healthy aortas and fibrolipidic lesions, favoring the ability of medial VSMCs to migrate into the intima. CONCLUSIONS: Our data suggest that overexpression of IGF-IR or IRA isoform, as homodimers or as part of IRA/IGF-IR hybrid receptors, confers a stronger migratory capability to VSMCs as might occur in early stages of atherosclerotic process.


Subject(s)
Atherosclerosis/metabolism , Cell Movement , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Receptor Cross-Talk , Receptor, IGF Type 1/metabolism , Receptor, Insulin/metabolism , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Apolipoproteins E/genetics , Apolipoproteins E/metabolism , Atherosclerosis/genetics , Atherosclerosis/pathology , Cell Line , Cell Movement/drug effects , Diet, Western , Disease Models, Animal , Gene Expression Regulation , Humans , Insulin/pharmacology , Insulin-Like Growth Factor I/pharmacology , Insulin-Like Growth Factor II/pharmacology , Male , Mice, Inbred C57BL , Mice, Knockout , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/pathology , Protein Isoforms , Receptor Cross-Talk/drug effects , Receptor, IGF Type 1/agonists , Receptor, IGF Type 1/genetics , Receptor, Insulin/agonists , Receptor, Insulin/genetics , Receptors, Somatomedin/genetics , Receptors, Somatomedin/metabolism , Signal Transduction , Time Factors
9.
Endocrinology ; 157(1): 61-9, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26556536

ABSTRACT

In comparison with young females, middle-aged female rats sustain greater cerebral infarction and worse functional recovery after stroke. These poorer stroke outcomes in middle-aged females are associated with an age-related reduction in IGF-I levels. Poststroke IGF-I treatment decreases infarct volume in older females and lowers the expression of cytokines in the ischemic hemisphere. IGF-I also reduces transfer of Evans blue dye to the brain, suggesting that this peptide may also promote blood-brain barrier function. To test the hypothesis that IGF-I may act at the blood-brain barrier in ischemic stroke, 2 approaches were used. In the first approach, middle-aged female rats were subjected to middle cerebral artery occlusion and treated with IGF-I after reperfusion. Mononuclear cells from the ischemic hemisphere were stained for CD4 or triple-labeled for CD4/CD25/FoxP3 and subjected to flow analyses. Both cohorts of cells were significantly reduced in IGF-I-treated animals compared with those in vehicle controls. Reduced trafficking of immune cells to the ischemic site suggests that blood-brain barrier integrity is better maintained in IGF-I-treated animals. The second approach directly tested the effect of IGF-I on barrier function of aging endothelial cells. Accordingly, brain microvascular endothelial cells from middle-aged female rats were cultured ex vivo and subjected to ischemic conditions (oxygen-glucose deprivation). IGF-I treatment significantly reduced the transfer of fluorescently labeled BSA across the endothelial monolayer as well as cellular internalization of fluorescein isothiocyanate-BSA compared with those in vehicle-treated cultures, Collectively, these data support the hypothesis that IGF-I improves blood-brain barrier function in middle-aged females.


Subject(s)
Aging , Blood-Brain Barrier/drug effects , Brain Ischemia/drug therapy , Endothelium, Vascular/drug effects , Insulin-Like Growth Factor I/therapeutic use , Receptor, IGF Type 1/agonists , Signal Transduction/drug effects , Animals , Blood-Brain Barrier/immunology , Blood-Brain Barrier/metabolism , Blood-Brain Barrier/pathology , Brain Ischemia/immunology , Brain Ischemia/metabolism , Brain Ischemia/pathology , Capillary Permeability/drug effects , Cell Hypoxia/drug effects , Cells, Cultured , Cerebrum/drug effects , Cerebrum/immunology , Cerebrum/metabolism , Cerebrum/pathology , Drug Implants , Endothelium, Vascular/immunology , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Female , Humans , Hypoglycemia/etiology , Insulin-Like Growth Factor I/administration & dosage , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor I/pharmacology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/pathology , Microvessels/drug effects , Microvessels/immunology , Microvessels/metabolism , Microvessels/pathology , Nerve Tissue Proteins/agonists , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/metabolism , Rats, Sprague-Dawley , Receptor, IGF Type 1/metabolism , Recombinant Proteins/administration & dosage , Recombinant Proteins/pharmacology , Recombinant Proteins/therapeutic use , Stroke/drug therapy , Stroke/immunology , Stroke/metabolism , Stroke/pathology
10.
Endocrinology ; 157(1): 336-45, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26469138

ABSTRACT

IGF-1 receptor (IGF-1R) signaling is implicated in cardiac hypertrophy and longevity. However, the role of IGF-1R in age-related cardiac remodeling is only partially understood. We therefore sought to determine whether the deletion of the IGF-1R in cardiomyocytes might delay the development of aging-associated myocardial pathologies by examining 2-year-old male cardiomyocyte-specific IGF-1R knockout (CIGF1RKO) mice. Aging was associated with the induction of IGF-1R expression in hearts. Cardiomyocytes hypertrophied with age in wild-type (WT) mice. In contrast, the cardiac hypertrophic response associated with aging was blunted in CIGF1RKO mice. Concomitantly, fibrosis was reduced in aged CIGF1RKO compared with aged WT hearts. Expression of proinflammatory cytokines such as IL-1α, IL-1ß, IL-6, and receptor activator of nuclear factor-κB ligand was increased in aged WT hearts, but this increase was attenuated in aged CIGF1RKO hearts. Phosphorylation of Akt was increased in aged WT, but not in aged CIGF1RKO, hearts. In cultured cardiomyocytes, IGF-1 induced senescence as demonstrated by increased senescence-associated ß-galactosidase staining, and a phosphoinositide 3-kinase inhibitor inhibited this effect. Furthermore, inhibition of phosphoinositide 3-kinase significantly prevented the increase in IL-1α, IL-1ß, receptor activator of nuclear factor-κB ligand, and p21 protein expression by IGF-1. These data reveal an essential role for the IGF-1-IGF-1R-Akt pathway in mediating cardiomyocyte senescence.


Subject(s)
Aging , Cardiomegaly/metabolism , Heart Ventricles/metabolism , Myocytes, Cardiac/metabolism , Receptor, IGF Type 1/metabolism , Ventricular Remodeling , Animals , Biomarkers/metabolism , Cardiomegaly/immunology , Cardiomegaly/pathology , Cardiomegaly/prevention & control , Cells, Cultured , Cellular Senescence/drug effects , Cytokines/antagonists & inhibitors , Cytokines/genetics , Cytokines/metabolism , Enzyme Inhibitors/pharmacology , Fibrosis , Gene Expression Regulation, Developmental/drug effects , Heart Ventricles/drug effects , Heart Ventricles/immunology , Heart Ventricles/pathology , Insulin-Like Growth Factor I/metabolism , Male , Mice, Inbred C57BL , Mice, Knockout , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/immunology , Myocytes, Cardiac/pathology , Phosphatidylinositol 3-Kinase/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Protein Processing, Post-Translational/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Receptor, IGF Type 1/agonists , Receptor, IGF Type 1/genetics , Signal Transduction/drug effects , Ventricular Remodeling/drug effects
11.
J Biol Chem ; 291(9): 4547-60, 2016 Feb 26.
Article in English | MEDLINE | ID: mdl-26702053

ABSTRACT

The ubiquitous phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates many cellular functions. However, the mechanism by which G protein-coupled receptors (GPCRs) signal to activate PI3K is poorly understood. We have used ovarian granulosa cells as a model to investigate this pathway, based on evidence that the GPCR agonist follicle-stimulating hormone (FSH) promotes the protein kinase A (PKA)-dependent phosphorylation of insulin receptor substrate 1 (IRS1) on tyrosine residues that activate PI3K. We report that in the absence of FSH, granulosa cells secrete a subthreshold concentration of insulin-like growth factor-1 (IGF-1) that primes the IGF-1 receptor (IGF-1R) but fails to promote tyrosine phosphorylation of IRS1. FSH via PKA acts to sensitize IRS1 to the tyrosine kinase activity of the IGF-1R by activating protein phosphatase 1 (PP1) to promote dephosphorylation of inhibitory Ser/Thr residues on IRS1, including Ser(789). Knockdown of PP1ß blocks the ability of FSH to activate PI3K in the presence of endogenous IGF-1. Activation of PI3K thus requires both PKA-mediated relief of IRS1 inhibition and IGF-1R-dependent tyrosine phosphorylation of IRS1. Treatment with FSH and increasing concentrations of exogenous IGF-1 triggers synergistic IRS1 tyrosine phosphorylation at PI3K-activating residues that persists downstream through protein kinase B (AKT) and FOXO1 (forkhead box protein O1) to drive synergistic expression of genes that underlies follicle maturation. Based on the ability of GPCR agonists to synergize with IGFs to enhance gene expression in other cell types, PP1 activation to relieve IRS1 inhibition may be a more general mechanism by which GPCRs act with the IGF-1R to activate PI3K/AKT.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Follicle Stimulating Hormone/metabolism , Granulosa Cells/metabolism , Insulin Receptor Substrate Proteins/metabolism , Insulin-Like Growth Factor I/metabolism , Phosphatidylinositol 3-Kinase/metabolism , Protein Phosphatase 1/metabolism , Animals , Cells, Cultured , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Enzyme Activation , Female , Granulosa Cells/cytology , Humans , Insulin Receptor Substrate Proteins/agonists , Insulin Receptor Substrate Proteins/antagonists & inhibitors , Insulin-Like Growth Factor I/genetics , Mutation , Phosphatidylinositol 3-Kinase/chemistry , Phosphorylation , Protein Phosphatase 1/antagonists & inhibitors , Protein Phosphatase 1/chemistry , Protein Phosphatase 1/genetics , Protein Processing, Post-Translational , RNA Interference , Rats, Sprague-Dawley , Receptor, IGF Type 1/agonists , Receptor, IGF Type 1/metabolism , Recombinant Proteins/metabolism , Signal Transduction , Tyrosine/metabolism
12.
Diabetes ; 64(12): 4148-57, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26384384

ABSTRACT

Insulin-like growth factor 2 (IGF2), produced and secreted by adult ß-cells, functions as an autocrine activator of the ß-cell insulin-like growth factor 1 receptor signaling pathway. Whether this autocrine activity of IGF2 plays a physiological role in ß-cell and whole-body physiology is not known. Here, we studied mice with ß-cell-specific inactivation of Igf2 (ßIGF2KO mice) and assessed ß-cell mass and function in aging, pregnancy, and acute induction of insulin resistance. We showed that glucose-stimulated insulin secretion (GSIS) was markedly reduced in old female ßIGF2KO mice; glucose tolerance was, however, normal because of increased insulin sensitivity. While on a high-fat diet, both male and female ßIGF2KO mice displayed lower GSIS compared with control mice, but reduced ß-cell mass was observed only in female ßIGF2KO mice. During pregnancy, there was no increase in ß-cell proliferation and mass in ßIGF2KO mice. Finally, ß-cell mass expansion in response to acute induction of insulin resistance was lower in ßIGF2KO mice than in control mice. Thus, the autocrine action of IGF2 regulates adult ß-cell mass and function to preserve in vivo GSIS in aging and to adapt ß-cell mass in response to metabolic stress, pregnancy hormones, and acute induction of insulin resistance.


Subject(s)
Aging , Insulin Resistance , Insulin-Like Growth Factor II/metabolism , Insulin-Secreting Cells/metabolism , Insulin/metabolism , Receptor, IGF Type 1/agonists , Signal Transduction , Allostasis , Animals , Apoptosis , Cell Proliferation , Crosses, Genetic , Diet, High-Fat/adverse effects , Female , Gene Expression Regulation, Developmental , Glucose Intolerance/etiology , Glucose Intolerance/metabolism , Glucose Intolerance/pathology , Insulin Secretion , Insulin-Like Growth Factor II/genetics , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/pathology , Male , Mice, Knockout , Mice, Transgenic , Pregnancy , Receptor, IGF Type 1/genetics , Receptor, IGF Type 1/metabolism , Sex Characteristics , Tissue Culture Techniques
13.
Arch Physiol Biochem ; 121(1): 32-9, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25897878

ABSTRACT

BACKGROUND: We have previously shown that both insulin and IGF1 lead to increased proliferation of keratinocytes. However, whereas insulin supports keratinocytes differentiation, IGF1 inhibits this process. The aim of the present study was to examine the proliferative and differentiative effects of insulin analogues (glargine, detemir, lispro and aspart) in primary keratinocytes in comparison with insulin and IGF1. METHODS: Primary keratinocytes cultures were produced from newborn BALB/c mice skin. Proliferation rates were assessed by [(3)H]-thymidine incorporation and XTT assays and differentiation was evaluated by Western blots analysis. Insulin receptor and IGF1 receptor phosphorylation was assessed by immunoprecipitation assays. RESULTS: Treatment with glargine or detemir resulted in an insulin-like effect on the differentiation process whereas lispro and aspart treatment led to an IGF1-like effect. In addition, treatment of keratinocytes with aspart led to a rapid phosphorylation of the IGF1 receptor. CONCLUSIONS: Our study provides evidence that insulin analogues elicit atypical actions in the skin.


Subject(s)
Insulin Aspart/pharmacology , Insulin Detemir/pharmacology , Insulin Glargine/pharmacology , Insulin Lispro/pharmacology , Insulin-Like Growth Factor I/pharmacology , Keratinocytes/drug effects , Animals , Animals, Newborn , Cell Differentiation , Cell Proliferation , Gene Expression , Keratinocytes/cytology , Keratinocytes/metabolism , Mice , Mice, Inbred BALB C , Phosphorylation/drug effects , Primary Cell Culture , Receptor, IGF Type 1/agonists , Receptor, IGF Type 1/genetics , Receptor, IGF Type 1/metabolism , Skin/cytology , Skin/drug effects , Skin/metabolism
14.
Cell Signal ; 27(7): 1297-304, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25817573

ABSTRACT

Neuropeptide Y binds to G-protein coupled receptors whose action results in inhibition of adenylyl cyclase activity. Using HEK293 cells stably expressing the native neuropeptide Y Y1 receptors, we found that the NPY agonist elicits a transient phosphorylation of the extracellular signal-regulated kinases (ERK1/2). We first show that ERK1/2 activation following Y1 receptor stimulation is dependent on heterotrimeric Gi/o since it is completely inhibited by pre-treatment with pertussis toxin. In addition, ERK1/2 activation is internalization-independent since mutant Y1 receptors unable to recruit ß-arrestins, can still activate ERK signaling to the same extent as wild-type receptors. We next show that this activation of the MAPK pathway is inhibited by the MEK inhibitor U0126, is not dependent on calcium signaling at the Y1 receptor (no effect upon inhibition of phospholipase C, protein kinase C or protein kinase D) but instead dependent on Gß/γ and associated signaling pathways that activate PI3-kinase. Although inhibition of the epidermal-growth factor receptor tyrosine kinase did not influence NPY-induced ERK1/2 activation, we show that the inhibition of insulin growth factor receptor IGFR by AG1024 completely blocks activation of ERK1/2 by the Y1 receptor. This Gß/γ-PI3K-AG1024-sensitive pathway does not involve activation of IGFR through the release of a soluble ligand by metalloproteinases since it is not affected by the metalloproteinase inhibitor marimastat. Finally, we found that a similar pathway, sensitive to wortmannin-AG1024 but insensitive to marimastat, is implicated in activation of ERK signaling in HEK293 cells by endogenously expressed GPCRs coupled to Gq-protein (muscarinic M3 receptors) or coupled to Gs-protein (endothelin ETB receptors). Our analysis is the first to show that ß-arrestin recruitment to the NPY Y1 receptor is not necessary for MAPK activation by this receptor but that transactivation of the IGFR receptor is required.


Subject(s)
Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Receptor, IGF Type 1/metabolism , Receptor, IGF Type 2/metabolism , Receptors, Neuropeptide Y/metabolism , Butadienes/pharmacology , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , HEK293 Cells , Humans , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Neuropeptide Y/pharmacology , Nitriles/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Receptor, IGF Type 1/agonists , Receptor, IGF Type 1/genetics , Receptor, IGF Type 2/antagonists & inhibitors , Receptor, IGF Type 2/genetics , Receptors, Neuropeptide Y/genetics , Signal Transduction/drug effects , Transcriptional Activation , Tyrphostins/pharmacology
15.
J Biol Chem ; 290(1): 467-77, 2015 Jan 02.
Article in English | MEDLINE | ID: mdl-25391655

ABSTRACT

Ginsenoside Rg5 is a compound newly synthesized during the steaming process of ginseng; however, its biological activity has not been elucidated with regard to endothelial function. We found that Rg5 stimulated in vitro angiogenesis of human endothelial cells, consistent with increased neovascularization and blood perfusion in a mouse hind limb ischemia model. Rg5 also evoked vasorelaxation in aortic rings isolated from wild type and high cholesterol-fed ApoE(-/-) mice but not from endothelial nitric-oxide synthase (eNOS) knock-out mice. Angiogenic activity of Rg5 was highly associated with a specific increase in insulin-like growth factor-1 receptor (IGF-1R) phosphorylation and subsequent activation of multiple angiogenic signals, including ERK, FAK, Akt/eNOS/NO, and Gi-mediated phospholipase C/Ca(2+)/eNOS dimerization pathways. The vasodilative activity of Rg5 was mediated by the eNOS/NO/cGMP axis. IGF-1R knockdown suppressed Rg5-induced angiogenesis and vasorelaxation by inhibiting key angiogenic signaling and NO/cGMP pathways. In silico docking analysis showed that Rg5 bound with high affinity to IGF-1R at the same binding site of IGF. Rg5 blocked binding of IGF-1 to its receptor with an IC50 of ∼90 nmol/liter. However, Rg5 did not induce vascular inflammation and permeability. These data suggest that Rg5 plays a novel role as an IGF-1R agonist, promoting therapeutic angiogenesis and improving hypertension without adverse effects in the vasculature.


Subject(s)
Angiogenesis Inducing Agents/pharmacology , Ginsenosides/pharmacology , Hindlimb/blood supply , Ischemia/drug therapy , Receptor, IGF Type 1/agonists , Vasodilation/drug effects , Animals , Aorta/drug effects , Aorta/metabolism , Apolipoproteins E/deficiency , Apolipoproteins E/genetics , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Focal Adhesion Kinase 1/genetics , Focal Adhesion Kinase 1/metabolism , Gene Expression Regulation , Hindlimb/drug effects , Hindlimb/pathology , Humans , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor I/metabolism , Ischemia/genetics , Ischemia/metabolism , Ischemia/pathology , Mice , Neovascularization, Physiologic , Nitric Oxide Synthase Type III/genetics , Nitric Oxide Synthase Type III/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptor, IGF Type 1/antagonists & inhibitors , Receptor, IGF Type 1/genetics , Receptor, IGF Type 1/metabolism , Signal Transduction , Tissue Culture Techniques , Type C Phospholipases/genetics , Type C Phospholipases/metabolism
16.
Diabetes Obes Metab ; 16 Suppl 1: 16-20, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25200291

ABSTRACT

Insulin and insulin-like growth factors (IGFs) are important regulators of growth and metabolism. In both vertebrates and invertebrates, insulin/IGFs are made available to various organs, including the brain, through two routes: the circulating systemic insulin/IGFs act on distant organs via endocrine signalling, whereas insulin/IGF ligands released by local tissues act in a paracrine or autocrine fashion. Although the mechanisms governing the secretion and action of systemic insulin/IGF have been the focus of extensive investigation, the significance of locally derived insulin/IGF has only more recently come to the fore. Local insulin/IGF signalling is particularly important for the development and homeostasis of the central nervous system, which is insulated from the systemic environment by the blood-brain barrier. Local insulin/IGF signalling from glial cells, the blood-brain barrier and the cerebrospinal fluid has emerged as a potent regulator of neurogenesis. This review will address the main sources of local insulin/IGF and how they affect neurogenesis during development. In addition, we describe how local insulin/IGF signalling couples neural stem cell proliferation with systemic energy state in Drosophila and in mammals.


Subject(s)
Feedback, Physiological , Insulin-Like Growth Factor II/metabolism , Insulin-Like Growth Factor I/metabolism , Insulin/metabolism , Models, Neurological , Neurogenesis , Signal Transduction , Animals , Antigens, CD/metabolism , Central Nervous System/growth & development , Central Nervous System/metabolism , Homeostasis , Humans , Insulin Secretion , Nerve Tissue Proteins/agonists , Nerve Tissue Proteins/metabolism , Neurons/cytology , Neurons/metabolism , Receptor, IGF Type 1/agonists , Receptor, IGF Type 1/metabolism , Receptor, Insulin/agonists , Receptor, Insulin/metabolism
17.
Growth Horm IGF Res ; 24(5): 157-63, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25002025

ABSTRACT

It is virtually undisputed that IGF-I promotes cell growth and survival. However, the presence of several IGF-I isoforms, vast numbers of intracellular signaling components, and multiple receptors results in a complex and highly regulated system by which IGF-I actions are mediated. IGF-I has long been recognized as one of the critical factors for coordinating muscle growth, enhancing muscle repair, and increasing muscle mass and strength. How to optimize this panoply of pathways to drive anabolic processes in muscle as opposed to aberrant growth in other tissues is an area that deserves focus. This review will address how advances in the bioavailability, potency, and tissue response of IGF-I can provide new potential directions for skeletal muscle therapeutics.


Subject(s)
Insulin-Like Growth Factor I/therapeutic use , Muscle, Skeletal , Muscular Diseases/drug therapy , Animals , Humans , Insulin-Like Growth Factor I/chemistry , Muscle, Skeletal/drug effects , Muscle, Skeletal/pathology , Muscle, Skeletal/physiology , Protein Isoforms , Receptor, IGF Type 1/agonists , Regeneration/drug effects , Satellite Cells, Skeletal Muscle/drug effects , Satellite Cells, Skeletal Muscle/physiology
18.
PLoS Pathog ; 10(6): e1004165, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24967908

ABSTRACT

Host arginase 1 (arg1) expression is a significant contributor to the pathogenesis of progressive visceral leishmaniasis (VL), a neglected tropical disease caused by the intracellular protozoan Leishmania donovani. Previously we found that parasite-induced arg1 expression in macrophages was dependent on STAT6 activation. Arg1 expression was amplified by, but did not require, IL-4, and required de novo synthesis of unknown protein(s). To further explore the mechanisms involved in arg1 regulation in VL, we screened a panel of kinase inhibitors and found that inhibitors of growth factor signaling reduced arg1 expression in splenic macrophages from hamsters with VL. Analysis of growth factors and their signaling pathways revealed that the Fibroblast Growth Factor Receptor 1 (FGFR-1) and Insulin-like Growth Factor 1 Receptor (IGF-1R) and a number of downstream signaling proteins were activated in splenic macrophages isolated from hamsters infected with L. donovani. Recombinant FGF-2 and IGF-1 increased the expression of arg1 in L. donovani infected hamster macrophages, and this induction was augmented by IL-4. Inhibition of FGFR-1 and IGF-1R decreased arg1 expression and restricted L. donovani replication in both in vitro and ex vivo models of infection. Inhibition of the downstream signaling molecules JAK and AKT also reduced the expression of arg1 in infected macrophages. STAT6 was activated in infected macrophages exposed to either FGF-2 or IGF-1, and STAT6 was critical to the FGFR-1- and IGF-1R-mediated expression of arg1. The converse was also true as inhibition of FGFR-1 and IGF-1R reduced the activation of STAT6 in infected macrophages. Collectively, these data indicate that the FGFR/IGF-1R and IL-4 signaling pathways converge at STAT6 to promote pathologic arg1 expression and intracellular parasite survival in VL. Targeted interruption of these pathological processes offers an approach to restrain this relentlessly progressive disease.


Subject(s)
Arginase/metabolism , Leishmaniasis, Visceral/immunology , Receptor, Fibroblast Growth Factor, Type 1/agonists , Receptor, IGF Type 1/agonists , STAT6 Transcription Factor/metabolism , Signal Transduction , Th2 Cells/immunology , Animals , Arginase/genetics , Cell Line , Cells, Cultured , Disease Progression , Enzyme Induction/drug effects , Host-Parasite Interactions/drug effects , Interleukin-4/metabolism , Leishmania donovani/growth & development , Leishmania donovani/immunology , Leishmania donovani/pathogenicity , Leishmania donovani/physiology , Leishmaniasis, Visceral/metabolism , Leishmaniasis, Visceral/parasitology , Leishmaniasis, Visceral/physiopathology , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Macrophages/parasitology , Mesocricetus , Protein Kinase Inhibitors/pharmacology , RNA Interference , Receptor, Fibroblast Growth Factor, Type 1/antagonists & inhibitors , Receptor, Fibroblast Growth Factor, Type 1/genetics , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Receptor, IGF Type 1/antagonists & inhibitors , Receptor, IGF Type 1/genetics , Receptor, IGF Type 1/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , STAT6 Transcription Factor/agonists , STAT6 Transcription Factor/antagonists & inhibitors , STAT6 Transcription Factor/genetics , Signal Transduction/drug effects , Th2 Cells/drug effects , Th2 Cells/metabolism , Th2 Cells/parasitology
19.
J Clin Endocrinol Metab ; 99(7): E1183-90, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24758182

ABSTRACT

CONTEXT: Graves' orbitopathy (GO) is caused by expansion of the orbital contents by excess adipogenesis and overproduction of hyaluronan (HA). Immunosuppressive and antiinflammatory treatments of GO are not always effective and can have side effects, whereas targeting GO-associated tissue remodeling might be a more logical therapeutic strategy. Previously we reported that signaling cascades through IGF1 receptor and thyrotropin receptor within orbital preadipocytes/fibroblasts drove adipogenesis and HA production. Our current study combined the stimulation of IGF1 receptor and thyrotropin receptor increase of HA accumulation, which we hypothesize is by activation of phosphatidylinositol 3-kinase (PI3K)-1A/PI3K1B, respectively. The central aim of this study was to investigate whether PI3K/mammalian target of rapamycin complex 1 (mTORC1) inhibitors affected adipogenesis and/or HA production within orbital preadipocyte/fibroblasts. METHODS: Human orbital preadipocytes were treated with/without inhibitors, LY294002 (PI3K1A/mTORC1), AS-605240 (PI3K1B), or PI103 (PI3K1A/mTORC1) in serum-free medium for 24 hours or cultured in adipogenic medium for 15 days. Quantitative PCR was used to measure hyaluronan synthases (HAS2) transcripts and the terminal adipogenesis differentiation marker lipoprotein lipase. HA accumulation in the medium was measured by an ELISA. RESULTS: Unlike AS-605240, both LY294002 (10 µM) and PI-103 (5 µM) significantly decreased HAS2 transcripts/HA accumulation and adipogenesis. Because PI-103 and LY294002 are dual PI3K/mTOR inhibitors, we investigated the inhibition of mTORC1 (rapamycin 100 nM), which significantly decreased adipogenesis but had no effect on HAS2 transcripts/HA, implicating PI3K-1A in the latter. CONCLUSIONS: The combined inhibition of PI3K1A and mTORC1 signaling in vitro decreased both HA accumulation and adipogenesis. Because PI3K and mTOR inhibitors are clinically used to treat other conditions, they have the potential to be repositioned to be used as an alternative nonimmunosuppressive therapy of GO.


Subject(s)
Drug Discovery , Graves Ophthalmopathy/therapy , Molecular Targeted Therapy , Adipogenesis/drug effects , Adipogenesis/genetics , Cells, Cultured , Chromones/pharmacology , Gene Expression Regulation/drug effects , Graves Ophthalmopathy/genetics , Graves Ophthalmopathy/metabolism , Humans , Hyaluronic Acid/genetics , Hyaluronic Acid/metabolism , Morpholines/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Quinoxalines/pharmacology , Receptor, IGF Type 1/agonists , Receptor, IGF Type 1/metabolism , Receptors, Thyrotropin/agonists , Receptors, Thyrotropin/metabolism , Signal Transduction/genetics , Thiazolidinediones/pharmacology
20.
Endocrinology ; 155(5): 1827-37, 2014 May.
Article in English | MEDLINE | ID: mdl-24617524

ABSTRACT

This study investigated potential mechanisms by which age and IGF-I receptor (IGF-Ir) signaling in the neuroendocrine hypothalamus affect estradiol-positive feedback effects on GnRH neuronal activation and on kisspeptin and N-methyl-D-aspartate (NMDA)-induced LH release and on the abundance of NMDA receptor subunits Nr1 and Nr2b and Kiss1r transcript and protein in the hypothalamus of young and middle-aged female rats. We infused vehicle, IGF-I, or JB-1, a selective antagonist of IGF-Ir, into the third ventricle of ovariectomized female rats primed with estradiol or vehicle and injected with vehicle, kisspeptin (3 or 30 nmol/kg), or NMDA (15 or 30 mg/kg). Regardless of dose, NMDA and kisspeptin resulted in significantly more LH release, GnRH/c-Fos colabeling, and c-Fos immunoreative cells in young than in middle-aged females. Estradiol priming significantly increased Kiss1r, Nr1, and Nr2b receptor transcript and protein abundance in young but not middle-aged female hypothalamus. JB-1 attenuated kisspeptin and NMDA-induced LH release, numbers of GnRH/c-Fos and c-Fos cells, and Kiss1r, Nr1, and Nr2b transcript and protein abundance in young females to levels observed in middle-aged females. IGF-I significantly enhanced NMDA and kisspeptin-induced LH release in middle-aged females without increasing numbers of GnRH/c-Fos or c-Fos immunoreactive cells. IGF-I infusion in middle-aged females also increased Kiss1r, Nr1, and Nr2b protein and transcript to levels that were equivalent to young estradiol-primed females. These findings indicate that age-related changes in estradiol-regulated responsiveness to excitatory input from glutamate and kisspeptin reflect reduced IGF-Ir signaling.


Subject(s)
Aging , Insulin-Like Growth Factor I/metabolism , Kisspeptins/metabolism , Luteinizing Hormone/metabolism , Receptor, IGF Type 1/agonists , Receptors, N-Methyl-D-Aspartate/agonists , Synaptic Transmission , Animals , Female , Gene Expression Regulation, Developmental/drug effects , Hypothalamo-Hypophyseal System/growth & development , Hypothalamo-Hypophyseal System/metabolism , Hypothalamus/cytology , Hypothalamus/drug effects , Hypothalamus/growth & development , Hypothalamus/metabolism , Infusions, Intraventricular , Insulin-Like Growth Factor I/administration & dosage , Insulin-Like Growth Factor I/analogs & derivatives , Insulin-Like Growth Factor I/antagonists & inhibitors , N-Methylaspartate/metabolism , Nerve Tissue Proteins/agonists , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neuroendocrine Cells/cytology , Neuroendocrine Cells/drug effects , Neuroendocrine Cells/metabolism , Oligopeptides/administration & dosage , Oligopeptides/pharmacology , Rats , Rats, Sprague-Dawley , Receptor, IGF Type 1/antagonists & inhibitors , Receptor, IGF Type 1/metabolism , Receptors, G-Protein-Coupled/biosynthesis , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Receptors, Kisspeptin-1 , Receptors, N-Methyl-D-Aspartate/genetics , Receptors, N-Methyl-D-Aspartate/metabolism , Signal Transduction/drug effects , Synaptic Transmission/drug effects
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