RILP inhibits tumor progression in osteosarcoma via Grb10-mediated inhibition of the PI3K/AKT/mTOR pathway.

BACKGROUND: Rab-interacting lysosomal protein (RILP) contains an alpha-helical coil with an unexplored biological function in osteosarcoma. This study investigated the expression of RILP in osteosarcoma cells and tissues to determine the effect of RILP on the biological behaviors of osteosarcoma cells and the underlying mechanism. METHODS: Tumor Immune Estimation Resource (TIMER) database, The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database were used for bioinformatic analysis. Co-immunoprecipitation experiment was used to determine whether the two proteins were interacting. In functional tests, cell counting kit-8 (CCK-8) assay, colony formation assay, wound healing assay, transwell invasion assay, Immunofluorescence (IF) assay and immunohistochemical (IHC) assay were performed. RESULTS: Overexpression of RILP significantly inhibited proliferation and impaired metastasis ability of osteosarcoma cells, while silencing of RILP showed the opposite trend. RNA-seq data analysis was applied in 143B cells and pathway enrichment analysis revealed that differentially expressed genes were mainly enriched in the PI3K/AKT pathway. We further verified that overexpression of RILP restrained the PI3K/AKT/mTOR signaling pathway and induced autophagy in osteosarcoma cells, while the opposite trend was observed when PI3K pathway activator 740Y-P was used. 3-Methyladenine (3-MA), a selective autophagy inhibitor, partially attenuated the inhibitory effect of RILP on the migration and invasion ability of osteosarcoma cells, suggesting the involvement of autophagy in epithelial-mesenchymal transition regulation in osteosarcoma cells. Growth factor receptor binding protein-10 (Grb10), an adaptor protein, was confirmed as a potential target of RILP to restrain the PI3K/AKT signaling pathway. We subcutaneously injected stably overexpressing 143B osteosarcoma cells into nude mice and observed that overexpression of RILP inhibited tumor growth by inhibiting the PI3K/AKT/mTOR pathway. CONCLUSION: Our study revealed that the expression of RILP was associated with favorable prognosis of osteosarcoma and RILP inhibits proliferation, migration, and invasion and promotes autophagy in osteosarcoma cells via Grb10-mediated inhibition of the PI3K/AKT/mTOR signaling pathway. In the future, targeting RILP may be a potential strategy for osteosarcoma treatment.

GRB10 is a novel factor associated with gastric cancer proliferation and prognosis.

GRB10 and its family members GRB7 and GRB14 were important adaptor proteins. They regulated many cellular functions by interacting with various tyrosine kinase receptors and other phosphorus-containing amino acid proteins. More and more studies have shown that the abnormal expression of GRB10 is closely related to the occurrence and development of cancer. In our current research, expression data for 33 cancers from the TCGA database was downloaded for analysis. It was found that GRB10 was up-regulated in cholangiocarcinoma, colon adenocarcinoma, head and neck squamous carcinoma, renal chromophobe, clear renal carcinoma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous carcinoma, gastric adenocarcinoma and thyroid carcinoma. Especially in gastric cancer, the high GRB10 expression was closely associated with poorer overall survival. Further research showed that the knockdown of GRB10 inhibited proliferation and migration ability in gastric cancer. Also, there was a potential binding site for miR-379-5p on the 3'UTR of GRB10. Overexpression of miR-379-5p in gastric cancer cells reduced GRB10-regulated gastric cancer proliferation and migration capacity. In addition, we found that tumor growth was slower in a mice xenograft model with knock down of GRB10 expression. These findings suggested that miR-379-5p suppresses gastric cancer development by downregulating GRB10 expression. Therefore, miR-379-5p and GRB10 were expected to be potential targets for the treatment of gastric cancer.

Hypothalamic Grb10 enhances leptin signalling and promotes weight loss.

Leptin acts on hypothalamic neurons expressing agouti-related protein (AgRP) or pro-opiomelanocortin (POMC) to suppress appetite and increase energy expenditure, but the intracellular mechanisms that modulate central leptin signalling are not fully understood. Here we show that growth factor receptor-bound protein 10 (Grb10), an adaptor protein that binds to the insulin receptor and negatively regulates its signalling pathway, can interact with the leptin receptor and enhance leptin signalling. Ablation of Grb10 in AgRP neurons promotes weight gain, while overexpression of Grb10 in AgRP neurons reduces body weight in male and female mice. In parallel, deletion or overexpression of Grb10 in POMC neurons exacerbates or attenuates diet-induced obesity, respectively. Consistent with its role in leptin signalling, Grb10 in AgRP and POMC neurons enhances the anorexic and weight-reducing actions of leptin. Grb10 also exaggerates the inhibitory effects of leptin on AgRP neurons via ATP-sensitive potassium channel-mediated currents while facilitating the excitatory drive of leptin on POMC neurons through transient receptor potential channels. Our study identifies Grb10 as a potent leptin sensitizer that contributes to the maintenance of energy homeostasis by enhancing the response of AgRP and POMC neurons to leptin.

CircRNA GRB10 is a Novel Biomarker for the Accurate Diagnosis of Lumbar Degenerative Disc Disease.

Lumbar degenerative disc disease (LDDD) is frequently misdiagnosed as other spine conditions, and the accurate diagnosis is challenging. This study was conducted to explore the role of circRNA GRB10 in the accurate diagnosis of LDDD. This study included 60 cases of LDDD, 60 cases of patients with sacroiliac joint pain (SJP), 60 cases of lumbar disc herniation (LDH), 60 cases of piriformis syndrome (PS), 60 cases of entrapment neuropathy (EN) and 60 cases of healthy controls (HCs). Plasma was obtained from each patient before and after treatment. Expression of GRB10 was studied with RT-qPCR. The role of plasma GRB10 in the accurate diagnosis of LDDD was analyzed by ROC curve analysis. Compared to HCs, decreased accumulation of GRB10 RNA was only observed in LDDD group, but not in SJP, LDH, PS and EN groups. With plasma expression level of GRB10 measured before treatment as a biomarker, LDDD patients were separated from SJP, LDH, PS, EN and HC groups. After treatment, increased expression levels of GRB10 were only observed in LDDD group, but not in other groups. Therefore, GRB10 was downregulated in LDDD and may serve as a biomarker for the accurate diagnosis of LDDD.

Tissue-specific Grb10/Ddc insulator drives allelic architecture for cardiac development.

Allele-specific expression of imprinted gene clusters is governed by gametic DNA methylation at master regulators called imprinting control regions (ICRs). Non-gametic or secondary differentially methylated regions (DMRs) at promoters and exonic regions reinforce monoallelic expression but do not control an entire cluster. Here, we unveil an unconventional secondary DMR that is indispensable for tissue-specific imprinting of two previously unlinked genes, Grb10 and Ddc. Using polymorphic mice, we mapped an intronic secondary DMR at Grb10 with paternal-specific CTCF binding (CBR2.3) that forms contacts with Ddc. Deletion of paternal CBR2.3 removed a critical insulator, resulting in substantial shifting of chromatin looping and ectopic enhancer-promoter contacts. Destabilized gene architecture precipitated abnormal Grb10-Ddc expression with developmental consequences in the heart and muscle. Thus, we redefine the Grb10-Ddc imprinting domain by uncovering an unconventional intronic secondary DMR that functions as an insulator to instruct the tissue-specific, monoallelic expression of multiple genes-a feature previously ICR exclusive.

circHtra1/miR-3960/GRB10 Axis Promotes Neuronal Loss and Immune Deficiency in Traumatic Brain Injury.

Circular RNAs (circRNAs) are abundant in the brain and contribute to central nervous system diseases; however, the exact roles of circRNAs in human traumatic brain injury (TBI) have not been established. In this study, we used a competing endogenous RNA (ceRNA) chipset as well as in vitro and in vivo assays to characterize differentially expressed circRNAs in TBI. We detected 3035 differentially expressed circRNAs in the severe TBI group, 2362 in the moderate group, and 433 in the mild group. A ceRNA network was constructed. The circRNA has_circ_0020269 (circHtra1) was significantly upregulated after brain insults and was correlated with the severity of injury. circHtra1 inhibited cell proliferation and promoted apoptosis, and its knockdown reversed these effects. Further analyses revealed that circHtra1 functions as a miR-3960 sponge and increases the expression of GRB10, which is involved in NK cell infiltration after TBI. circHtra1 was identified as a target of the IGF-1/ADAR1 axis. Reduced expression of ADAR1 (involved in A-to-I editing) after brain insults upregulated circHtra1. Our results show that circHtra1 promotes neuronal loss by sponging miR-3960 and regulating GRB10 and apoptosis during brain insults. In addition, A-to-I editing could regulate circRNA expression profiles after TBI, and circHtra1 is a potential therapeutic target.

PINCH-1 promotes IGF-1 receptor expression and skin cancer progression through inhibition of the GRB10-NEDD4 complex.

Background: Insulin-like growth factor 1 receptor (IGF-1R) expression and signaling play important roles in promotion of skin cancer progression. Identification of signaling pathways that regulate IGF-1R is crucial for understanding the pathogenesis and therapeutic treatment of skin cancer. Methods: Molecular, cellular and genetic approaches were used to investigate the function of PINCH-1 in regulation of IGF-1R expression and skin cell behavior. Furthermore, conditional PINCH-1 knockout mouse and carcinogen (7, 12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA))-induced skin cancer model were employed to determine the function of PINCH-1 in regulation of IGF-1R expression and skin carcinogenesis in vivo. Results: Knockdown of PINCH-1 from HaCaT keratinocytes or A431 squamous carcinoma cells diminished IGF-1R levels, suppressed cell proliferation and increased apoptosis. Re-expression of PINCH-1 in PINCH-1 knockdown cells restored IGF-1R expression, cell proliferation and survival. Furthermore, depletion of NEDD4 effectively reversed PINCH-1 deficiency-induced down-regulation of IGF-1R expression, cell proliferation and survival. Conditional knockout of PINCH-1 from keratin 5 (K5) positive keratinocytes in mice, like depletion of PINCH-1 from keratinocytes in culture, reduced the IGF-1R level. Using a mouse model of DMBA/TPA-induced skin cancer, we show that the levels of both PINCH-1 and IGF-1R were significantly increased in response to treatment with the carcinogens. Genetic ablation of PINCH-1 from the epidermis markedly reduced the IGF-1R expression and cell proliferation despite stimulation with DMBA/TPA, resulting in resistance to chemical carcinogen-induced skin cancer initiation and progression. Conclusions: Our results reveal a PINCH-1-NEDD4-IGF-1R signaling axis that is critical for promotion of skin tumorigenesis and suggest a new strategy for therapeutic control of skin cancer progression.

Treadmill training attenuate STZ-induced cognitive dysfunction in type 2 diabetic rats via modulating Grb10/IGF-R signaling.

Type 2 diabetes is a major factor contributing to cognitive decline and Alzheimer's disease (AD). Treadmill running is considered to be a critical approach for mice and rats to lower blood sugar and improve learning and memory capacity. The growth factor receptor-bound protein 10 (Grb10) has been proposed to inhibit insulin signaling and defective brain insulin signaling resulted in the cognitive deficits in patients with AD. However, the positive roles of treadmill training on diabetic- related impaired cognitive function and their molecular mechanisms remain unclear. Here, to investigate whether there was neuroprotective effects of treadmill training on impaired cognitive function caused by diabetes, the rats were injected intraperitoneally with streptozotocin at a dose of 30 mg/kg to establish diabetic model (DM). We found that higher Grb10, BACE1 and PHF10 protein levels in the hippocampus of DM rats, lower phosphorylation IGF-1Rß and IRS-1(ser307). However, 8 weeks treadmill training effectively reduced abnormal Grb10, enhanced postsynaptic density protein PSD-93, PSD-95, SYN expressions of hippocampus, restored PI3K/Akt/ERK and mTOR/AMPK signaling, thus alleviated spatial learning and memory deficit, compared with DM group. Additionally, treadmill training also increased GLUT4 transportation. Overall, our findings suggest that treadmill intervention improved cognitive impairments caused by diabetes disease partly through modulating Grb10/ PI3K/Akt/ERK as well as mTOR/AMPK signaling.

GRB10 regulates ß-cell mass by inhibiting ß-cell proliferation and stimulating ß-cell dedifferentiation.

Decreased functional ß-cell mass is the hallmark of diabetes, but the cause of this metabolic defect remains elusive. Here, we show that the levels of the growth factor receptor-bound protein 10 (GRB10), a negative regulator of insulin and mTORC1 signaling, are markedly induced in islets of diabetic mice and high glucose-treated insulinoma cell line INS-1 cells. ß-cell-specific knockout of Grb10 in mice increased ß-cell mass and improved ß-cell function. Grb10-deficient ß-cells exhibit enhanced mTORC1 signaling and reduced ß-cell dedifferentiation, which could be blocked by rapamycin. On the contrary, Grb10 overexpression induced ß-cell dedifferentiation in MIN6 cells. Our study identifies GRB10 as a critical regulator of ß-cell dedifferentiation and ß-cell mass, which exerts its effect by inhibiting mTORC1 signaling.

Circ_0009910 shuttled by exosomes regulates proliferation, cell cycle and apoptosis of acute myeloid leukemia cells by regulating miR-5195-3p/GRB10 axis.

The exosomes are involved in intercellular communication via RNA trafficking in human diseases. Hsa_circ_0009910 (circ_0009910) is a novel leukemia-related circular RNA. However, the mechanism of circ_0009910 in acute myeloid leukemia (AML) cell-to-cell communication remained obscure. Expression of circ_0009910, miRNA (miR)-5195-3p and growth factor receptor-bound protein 10 (GRB10) was detected by quantitative real-time polymerase chain reaction and Western blotting. A stable cell coculture model was established and functional experiment was performed using Cell Counting Kit-8 assay, flow cytometry, and Western blotting. The interaction among circ_0009910, miR-5195-3p and GRB10 was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation. As a result, circ_0009910 was upregulated in AML bone marrows and cells (HL-60 and MOLM-13), even higher in AML cells-derived exosomes. Functionally, blocking circ_0009910 via small interfering RNA (siRNA) suppressed cell proliferation and cell cycle progression, but facilitated apoptosis rate of HL-60 and MOLM-13 cells, accompanied with lower B-cell lymphoma 2 (Bcl-2) level and higher Bcl-2-associated X protein (Bax) level. circ_0009910 shuttled via exosomes negatively regulated miR-5195-3p expression by target binding. Furthermore, circ_0009910 knockdown via exosomes and miR-5195-3p overexpression via mimic resulted in similar results of circ_0009910 siRNA in proliferation, apoptosis and cell cycle progression of AML cells. Meanwhile, the role of circ_0009910 knockdown in AML cells was partially reversed by miR-5195-3p deletion, and restoring GRB10 could abrogate miR-5195-3p effect as well. Notably, GRB10 was a downstream target of miR-5195-3p. circ_0009910-containing exosomes mediated proliferation, apoptosis and cell cycle progression of AML cells partially through miR-5195-3p/GRB10 axis.

GRB10 sustains AR activity by interacting with PP2A in prostate cancer cells.

Prostate cancer (PCa) progression is driven by androgen receptor (AR) signaling. Unfortunately, androgen-deprivation therapy and the use of even more potent AR pathway inhibitors (ARPIs) cannot bring about a cure. ARPI resistance (ie, castration-resistant PCa, CRPC) will inevitably develop. Previously, we demonstrated that GRB10 is an AR transcriptionally repressed gene that functionally contributes to CRPC development and ARPI resistance. GRB10 expression is elevated prior to CRPC development in our patient-derived xenograft models and is significantly upregulated in clinical CRPC samples. Here, we analyzed transcriptomic data from GRB10 knockdown in PCa cells and found that AR signaling is downregulated. While the mRNA expression of AR target genes decreased upon GRB10 knockdown, AR expression was not affected at the mRNA or protein level. We further found that phosphorylation of AR serine 81 (S81), which is critical for AR transcriptional activity, is decreased by GRB10 knockdown and increased by its overexpression. Luciferase assay using GRB10-knockdown cells also indicate reduced AR activity. Immunoprecipitation coupled with mass spectrometry revealed an interaction between GRB10 and the PP2A complex, which is a known phosphatase of AR. Further validations and analyses showed that GRB10 binds to the PP2Ac catalytic subunit with its PH domain. Mechanistically, GRB10 knockdown increased PP2Ac protein stability, which in turn decreased AR S81 phosphorylation and reduced AR activity. Our findings indicate a reciprocal feedback between GRB10 and AR signaling, implying the importance of GRB10 in PCa progression.

Recombinant Chromosome 7 Driven by Maternal Chromosome 7 Pericentric Inversion in a Girl with Features of Silver-Russell Syndrome.

Maternal uniparental disomy of chromosome 7 is present in 5-10% of patients with Silver-Russell syndrome (SRS), and duplication of 7p including GRB10 (Growth Factor Receptor-Bound Protein 10), an imprinted gene that affects pre-and postnatal growth retardation, has been associated with the SRS phenotype. Here, we report on a 17 year old girl referred to array-CGH analysis for short stature, psychomotor delay, and relative macrocephaly. Array-CGH analysis showed two copy number variants (CNVs): a ~12.7 Mb gain in 7p13-p11.2, involving GRB10 and an ~9 Mb loss in 7q11.21-q11.23. FISH experiments performed on the proband's mother showed a chromosome 7 pericentric inversion that might have mediated the complex rearrangement harbored by the daughter. Indeed, we found that segmental duplications, of which chromosome 7 is highly enriched, mapped at the breakpoints of both the mother's inversion and the daughter's CNVs. We postulate that pairing of highly homologous sequences might have perturbed the correct meiotic chromosome segregation, leading to unbalanced outcomes and acting as the putative meiotic mechanism that was causative of the proband's rearrangement. Comparison of the girl's phenotype to those of patients with similar CNVs supports the presence of 7p in a locus associated with features of SRS syndrome.

miR-504 modulates the stemness and mesenchymal transition of glioma stem cells and their interaction with microglia via delivery by extracellular vesicles.

Glioblastoma (GBM) is a highly aggressive tumor with poor prognosis. A small subpopulation of glioma stem cells (GSCs) has been implicated in radiation resistance and tumor recurrence. In this study we analyzed the expression of miRNAs associated with the functions of GSCs using miRNA microarray analysis of these cells compared with human neural stem cells. These analyses identified gene clusters associated with glioma cell invasiveness, axonal guidance, and TGF-ß signaling. miR-504 was significantly downregulated in GSCs compared with NSCs, its expression was lower in GBM compared with normal brain specimens and further decreased in the mesenchymal glioma subtype. Overexpression of miR-504 in GSCs inhibited their self-renewal, migration and the expression of mesenchymal markers. The inhibitory effect of miR-504 was mediated by targeting Grb10 expression which acts as an oncogene in GSCs and GBM. Overexpression of exogenous miR-504 resulted also in its delivery to cocultured microglia by GSC-secreted extracellular vesicles (EVs) and in the abrogation of the GSC-induced polarization of microglia to M2 subtype. Finally, miR-504 overexpression prolonged the survival of mice harboring GSC-derived xenografts and decreased tumor growth. In summary, we identified miRNAs and potential target networks that play a role in the stemness and mesenchymal transition of GSCs and the miR-504/Grb10 pathway as an important regulator of this process. Overexpression of miR-504 exerted antitumor effects in GSCs as well as bystander effects on the polarization of microglia via delivery by EVs.

Mice lacking paternal expression of imprinted Grb10 are risk-takers.

The imprinted genes Grb10 and Nesp influence impulsive behavior on a delay discounting task in an opposite manner. A recently developed theory suggests that this pattern of behavior may be representative of predicted effects of imprinted genes on tolerance to risk. Here we examine whether mice lacking paternal expression of Grb10 show abnormal behavior across a number of measures indicative of risk-taking. Although Grb10+/p mice show no difference from wild type (WT) littermates in their willingness to explore a novel environment, their behavior on an explicit test of risk-taking, namely the Predator Odor Risk-Taking task, is indicative of an increased willingness to take risks. Follow-up tests suggest that this risk-taking is not simply because of a general decrease in fear, or a general increase in motivation for a food reward, but reflects a change in the trade-off between cost and reward. These data, coupled with previous work on the impulsive behavior of Grb10+/p mice in the delayed reinforcement task, and taken together with our work on mice lacking maternal Nesp, suggest that maternally and paternally expressed imprinted genes oppositely influence risk-taking behavior as predicted.

Novel visualized quantitative epigenetic imprinted gene biomarkers diagnose the malignancy of ten cancer types.

BACKGROUND: Epigenetic alterations are involved in most cancers, but its application in cancer diagnosis is still limited. More practical and intuitive methods to detect the aberrant expressions from clinical samples using highly sensitive biomarkers are needed. In this study, we developed a novel approach in identifying, visualizing, and quantifying the biallelic and multiallelic expressions of an imprinted gene panel associated with cancer status. We evaluated the normal and aberrant expressions measured using the imprinted gene panel to formulate diagnostic models which could accurately distinguish the imprinting differences of normal and benign cases from cancerous tissues for each of the ten cancer types. RESULTS: The Quantitative Chromogenic Imprinted Gene In Situ Hybridization (QCIGISH) method developed from a 1013-case study which provides a visual and quantitative analysis of non-coding RNA allelic expressions identified the guanine nucleotide-binding protein, alpha-stimulating complex locus (GNAS), growth factor receptor-bound protein (GRB10), and small nuclear ribonucleoprotein polypeptide N (SNRPN) out of five tested imprinted genes as efficient epigenetic biomarkers for the early-stage detection of ten cancer types. A binary algorithm developed for cancer diagnosis showed that elevated biallelic expression (BAE), multiallelic expression (MAE), and total expression (TE) measurements for the imprinted gene panel were associated with cell carcinogenesis, with the formulated diagnostic models achieving consistently high sensitivities (91-98%) and specificities (86-98%) across the different cancer types. CONCLUSIONS: The QCIGISH method provides an innovative way to visually assess and quantitatively analyze individual cells for cancer potential extending from hyperplasia and dysplasia until carcinoma in situ and invasion, which effectively supplements standard clinical cytologic and histopathologic diagnosis for early cancer detection. In addition, the diagnostic models developed from the BAE, MAE, and TE measurements of the imprinted gene panel GNAS, GRB10, and SNRPN could provide important predictive information which are useful in early-stage cancer detection and personalized cancer management.

Lifestyle mediates the role of nutrient-sensing pathways in cognitive aging: cellular and epidemiological evidence.

Aging induces cellular and molecular changes including modification of stem cell pools. In particular, alterations in aging neural stem cells (NSCs) are linked to age-related cognitive decline which can be modulated by lifestyle. Nutrient-sensing pathways provide a molecular basis for the link between lifestyle and cognitive decline. Adopting a back-translation strategy using stem cell biology to inform epidemiological analyses, here we show associations between cellular readouts of NSC maintenance and expression levels of nutrient-sensing genes following NSC exposure to aging human serum as well as morphological and gene expression alterations following repeated passaging. Epidemiological analyses on the identified genes showed associations between polymorphisms in SIRT1 and ABTB1 and cognitive performance as well as interactions between SIRT1 genotype and physical activity and between GRB10 genotype and adherence to a Mediterranean diet. Our study contributes to the understanding of neural stem cell molecular mechanisms underlying human cognitive aging and hints at lifestyle modifiable factors.

Detailed analysis of paternal knockout Grb10 mice suggests effects on stability of social behavior, rather than social dominance.

Imprinted genes are highly expressed in monoaminergic regions of the midbrain and their functions in this area are thought to have an impact on mammalian social behaviors. One such imprinted gene is Grb10, of which the paternal allele is generally recognized as mediating social dominance behavior. However, there has been no detailed study of social dominance in Grb10 +/p mice. Moreover, the original study examined tube-test behavior in isolated mice 10 months of age. Isolation testing favors more territorial and aggressive behaviors, and does not address social dominance strategies employed in group housing contexts. Furthermore, isolation stress impacts midbrain function and dominance related behavior, often through alterations in monoaminergic signaling. Thus, we undertook a systematic study of Grb10 +/p social rank and dominance behavior within the cage group, using a number of convergent behavioral tests. We examined both male and female mice to account for sex differences and tested cohorts aged 2, 6 and 10 months to examine any developments related to age. We found group-housed Grb10 +/p mice do not show evidence of enhanced social dominance, but cages containing Grb10 +/p and wild-type mice lacked the normal correlation between three different measures of social rank. Moreover, a separate study indicated isolation stress induced inconsistent changes in tube test behavior. Taken together, these data suggest future research on Grb10 +/p mice should focus on the stability of social behaviors, rather than dominance per se.

Role of Grb10 in mTORC1-dependent regulation of insulin signaling and action in human skeletal muscle cells.

Growth factor receptor-bound protein 10 (Grb10) is an adaptor protein that binds to the insulin receptor, upon which insulin signaling and action are thought to be inhibited. Grb10 is also a substrate for the mechanistic target of rapamycin complex 1 (mTORC1) that mediates its feedback inhibition on phosphatidylinositide 3-kinase (PI3K)/Akt signaling. To characterize the function of Grb10 and its regulation by mTORC1 in human muscle, primary skeletal muscle cells were isolated from healthy lean young men and then induced to differentiate into myotubes. Knockdown of Grb10 enhanced insulin-induced PI3K/Akt signaling and glucose uptake in myotubes, reinforcing the notion underlying its function as a negative regulator of insulin action in human muscle. The increased insulin responsiveness in Grb10-silenced myotubes was associated with a higher abundance of the insulin receptor. Furthermore, insulin and amino acids independently and additively stimulated phosphorylation of Grb10 at Ser476. However, acute inhibition of mTORC1 with rapamycin blocked Grb10 Ser476 phosphorylation and repressed a negative-feedback loop on PI3K/Akt signaling that increased myotube responsiveness to insulin. Chronic rapamycin treatment reduced Grb10 protein abundance in conjunction with increased insulin receptor protein levels. Based on these findings, we propose that mTORC1 controls PI3K/Akt signaling through modulation of insulin receptor abundance by Grb10. These findings have potential implications for obesity-linked insulin resistance, as well as clinical use of mTORC1 inhibitors.

Distinct Effects of Carrageenan and High-Fat Consumption on the Mechanisms of Insulin Resistance in Nonobese and Obese Models of Type 2 Diabetes.

Exposure to low concentration of the common food additive carrageenan (10 mg/L) for only six days led to glucose intolerance and insulin resistance in the C57BL/6J mouse. Longer exposure produced fasting hyperglycemia but with no increase in weight, in contrast to the HFD. Glucose intolerance was attributable to carrageenan-induced inflammation and to increased expression of GRB10. Both HFD and carrageenan increased p(Ser32)-IκBα and p(Ser307)-IRS1, and the increases were greater following the combined exposure. The effects of carrageenan were inhibited by the combination of the free radical inhibitor Tempol and BCL10 siRNA, which had no impact on the HFD-mediated increase. In contrast, the PKC inhibitor sotrastaurin blocked the HFD-induced increases, without an effect on the carrageenan-mediated effects. HFD had no impact on the expression of GRB10. Both carrageenan and high fat increased hepatic infiltration by F4/80-positive macrophages. Serum galectin-3 and galectin-3 binding to the insulin receptor increased by carrageenan and by HFD. Tyrosine phosphorylation of the insulin receptor declined following either exposure and was further reduced by their combination. Carrageenan reduced the activity of the enzyme N-acetylgalactosamine-4-sulfatase (ARSB; arylsulfatase B), which was unchanged following HFD. Dietary exposure to both high fat and carrageenan can impair insulin signaling through both similar and distinct mechanisms.

Contribution of GRB10 to the prenatal phenotype in Silver-Russell syndrome? Lessons from 7p12 copy number variations.

The growth factor binding protein 10 (GRB10) has been suggested as a candidate gene for Silver-Russell syndrome because of its localization in 7p12, its imprinting status, data from mice models and its putative role in growth. Based on a new patient with normal growth carrying a GRB10 deletion affecting the paternal allele and data from the literature, we conclude that the heterogeneous clinical findings in patients with copy number variations (CNVs) of GRB10 gene depend on the size and the gene content of the CNV. However, evidence from mouse and human cases indicate a growth suppressing role of GRB10 in prenatal development. As a result, an increase of active maternal GRB10 copies, e.g. by maternal uniparental disomy of chromosome 7 or duplications of the region results in intrauterine growth retardation. In contrast, a defective GRB10 copy might result in prenatal overgrowth, whereas the paternal GRB10 allele is not required for proper prenatal growth.