Insulin Receptor Tyrosine Kinase Research Articles

Phylogenetic and transcriptomic characterization of insulin and growth factor receptor tyrosine kinases in crustaceans.

Receptor tyrosine kinases (RTKs) mediate the actions of growth factors in metazoans. In decapod crustaceans, RTKs are implicated in various physiological processes, such molting and growth, limb regeneration, reproduction and sexual differentiation, and innate immunity. RTKs are organized into two main types: insulin receptors (InsRs) and growth factor receptors, which include epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR). The identities of crustacean RTK genes are incomplete. A phylogenetic analysis of the CrusTome transcriptome database, which included all major crustacean taxa, showed that RTK sequences segregated into receptor clades representing InsR (72 sequences), EGFR (228 sequences), FGFR (129 sequences), and PDGFR/VEGFR (PVR; 235 sequences). These four receptor families were distinguished by the domain organization of the extracellular N-terminal region and motif sequences in the protein kinase catalytic domain in the C-terminus or the ligand-binding domain in the N-terminus. EGFR1 formed a single monophyletic group, while the other RTK sequences were divided into subclades, designated InsR1-3, FGFR1-3, and PVR1-2. In decapods, isoforms within the RTK subclades were common. InsRs were characterized by leucine-rich repeat, furin-like cysteine-rich, and fibronectin type 3 domains in the N-terminus. EGFRs had leucine-rich repeat, furin-like cysteine-rich, and growth factor IV domains. N-terminal regions of FGFR1 had one to three immunoglobulin-like domains, whereas FGFR2 had a cadherin tandem repeat domain. PVRs had between two and five immunoglobulin-like domains. A classification nomenclature of the four RTK classes, based on phylogenetic analysis and multiple sequence alignments, is proposed.

Evaluation of Adenanthera pavonina-derived compounds against diabetes mellitus: insight into the phytochemical analysis and in silico assays.

Adenanthera pavonina is a medicinal plant with numerous potential secondary metabolites showing a significant level of antidiabetic activity. The objective of the current study was to identify potential phytochemicals from the methanolic leaf extract of Adenanthera pavonina as therapeutic agents against diabetes mellitus using GC-MS and in silico methods. The GC-MS analysis of the leaf extract revealed a total of 17 phytochemicals. Molecular docking was performed using these phytochemicals, targeting the mutated insulin receptor tyrosine kinase (5hhw), which inhibits glucose uptake by cells. Diazoprogesterone (-9.2kcal/mol), 2,4,4,7a-Tetramethyl-1-(3-oxobutyl)octahydro-1H-indene-2-carboxylic acid (-6.9kcal/mol), and 2-Naphthalenemethanol, decahydro-.alpha.,.alpha.,4a-trimethyl-8-methylene-, [2R-(2.alpha.,4a.alpha.,8a.beta.)] (-6.6kcal/mol) exhibited better binding with the target protein. The ADMET analysis was performed for the top three compounds with the best docking scores, which showed positive results with no observed toxicity in the AMES test. Furthermore, the molecular dynamics study confirmed the favorable binding of Diazoprogesterone, 2,4,4,7a-Tetramethyl-1-(3-oxobutyl)octahydro-1H-indene-2-carboxylic acid and 2-Naphthalenemethanol, decahydro-.alpha.,.alpha.,4a-trimethyl-8-methylene-, [2R-(2.alpha.,4a.alpha.,8a.beta.)] with the receptor throughout the 100ns simulation period.

Computational Screening and Experimental Validation of Inhibitor Targeting the Complex Formation of Grb14 and Insulin Receptor.

The development of drugs targeting gene products associated with insulin resistance holds the potential to enhance our understanding of type 2 diabetes mellitus (T2DM). The virtual screening, based on a three-dimensional (3D) protein structure, is a potential technique to accelerate the development of molecular target drugs. Among the targets implicated in insulin resistance, the genetic characterization and protein function of Grb14 have been clarified without contradiction. The Grb14 gene displays significant variations in T2DM, and its gene product is known to inhibit the function of the insulin receptor (IR) by directly binding to the tyrosine kinase domain. In the present study, a virtual screening, based on a 3D structure of the IR tyrosine kinase domain (IRβ) in complex with part of Grb14, was conducted to find compounds that can disrupt the complex formation between Grb14 and IRβ. First, ten compounds were selected from 154,118 compounds via hierarchical in silico structure-based drug screening, composed of grid docking-based and genetic algorithm-based programs. The experimental validations suggested that the one compound can affect the blood glucose level. The molecular dynamics simulations and co-immunoprecipitation analysis showed that the compound did not completely suppress the protein-protein interaction between Grb14 and IR, though competitively bound to IR with the tyrosine kinase pseudosubstrate region in Grb14.

Nimbin analogs stimulate glucose uptake and glycogen storage in the insulin signalling cascade by enhancing the IRTK, PI3K and Glut-4 mechanism in myotubes

BackgroundDiabetes Mellitus is a metabolic disorder characterized by insulin dysfunction or failure of the pancreatic β-cells to produce insulin resulting in hyperglycemia. Adverse effects of hyperglycemic conditions continue to be common, reducing treatment adherence. Intensified therapies are required for the constant loss of endogenous islet reserve. AimThis study aimed to evaluate the effect of Nimbin semi-natural analogs (N2, N5, N7, and N8) from A. indica on high glucose-induced ROS and apoptosis with insulin resistance in L6 myotubes evaluated along with Wortmannin and Genistein inhibitors and the expression of key genes in the insulin signalling pathway. Materials and methodsThe analogs were screened for anti-oxidant and anti-diabetic activity using cell-free assays; The ability of analogs to suppress ROS and prevent apoptosis induced by High glucose and uptake glucose and glycogen storage in L6 myotubes was evaluated using DCFH-DA, AO-PI and 2NBDG staining. Further, the glucose uptake was performed in the presence of Insulin Receptor Tyrosine Kinase (IRTK) inhibitors, and the expression of key genes PI3K, Glut-4, GS and IRTK in the insulin signalling pathway were evaluated. Key findingsThe Nimbin analogs were not toxic to the L6 cells, and the analogs could scavenge ROS and suppress cellular damage induced due to high glucose. Enhanced glucose uptake was observed in N2, N5 and N7 compared to N8. The maximum activity of optimum concentration was found to be 100 μM. The N2, N5 and N7 showed an increase in IRTK, which is equivalent to insulin at a concentration of 100 µM. The IRTK inhibitor with Genistein (50 µM) confirmed the presence of IRTK-dependent glucose transport activation; it also supports the expression of key genes PI3K, Glut-4, GS and IRTK. As a result of PI3K activation, N2, N5, and N7 exhibited the insulin-mimetic effect by enhancing glucose uptake and glycogen conversion regulating glucose metabolism. SignificanceN2, N5 and N7 could therapeutically benefit against insulin resistance by glucose metabolism modulation, insulin secretion, β-cell stimulation, inhibition of gluconeogenic enzymes and ROS protection.

Unique ligand and kinase-independent roles of the insulin receptor in regulation of cell cycle, senescence and apoptosis

Insulin acts through the insulin receptor (IR) tyrosine kinase to exert its classical metabolic and mitogenic actions. Here, using receptors with either short or long deletion of the β-subunit or mutation of the kinase active site (K1030R), we have uncovered a second, previously unrecognized IR signaling pathway that is intracellular domain-dependent, but ligand and tyrosine kinase-independent (LYK-I). These LYK-I actions of the IR are linked to changes in phosphorylation of a network of proteins involved in the regulation of extracellular matrix organization, cell cycle, ATM signaling and cellular senescence; and result in upregulation of expression of multiple extracellular matrix-related genes and proteins, down-regulation of immune/interferon-related genes and proteins, and increased sensitivity to apoptosis. Thus, in addition to classical ligand and tyrosine kinase-dependent (LYK-D) signaling, the IR regulates a second, ligand and tyrosine kinase-independent (LYK-I) pathway, which regulates the cellular machinery involved in senescence, matrix interaction and response to extrinsic challenges.

BAIAP2L1 accelerates breast cancer progression and chemoresistance by activating AKT signaling through binding with ribosomal protein L3.

BAI1-associated protein 2-like 1 (BAIAP2L1), also known as insulin receptor tyrosine kinase substrate, modulates the insulin network; however, its function in breast cancer has not been explored. Immunohistochemical analysis of 140 breast cancer specimens (77 triple-negative and 63 nontriple-negative cases) indicated that BAIAP2L1 expression was higher in breast cancer tissues (56/140, 40%) than in normal breast tissues (28.3%, 15/53; p< 0.001). BAIAP2L1 expression in breast cancer was correlated with triple-negative breast cancer (p=0.0013), advanced TNM stage (p=0.001), lymph node metastasis (p=0.001), and poor patient prognosis (p=0.001). BAIAP2L1 overexpression could accelerate breast cancer proliferation, invasion, and stemness in vivo and in vitro, possibly through the activation of AKT, Snail, and cyclin D1. Treatment with the AKT inhibitor LY294002 reduced the effects of BAIAP2L1 overexpression on breast cancer cells. BAIAP2L1 may bind to the AA202-288 of ribosomal protein L3 (RPL3) within its SRC homology 3 (SH3) domain, the loss of which may abolish the transduction of the AKT signaling pathway by promoting the degradation of PIK3CA. Moreover, BAIAP2L1 overexpression may induce chemotherapy resistance, with BAIAP2L1 expression being higher in patients with advanced Miller grades than those with lower grades. Our results indicated that BAIAP2L1 promotes breast cancer progression through the AKT signaling pathway by interacting with RPL3 through its SH3 domain.

In silico screening and identification of leads against multi-targets of type II diabetes in Plectranthus vettiveroides

Generally, phytochemicals have activity on multi-targets and such molecules have been used as effective drugs against the diseases caused by multi-factorial causation like diabetes mellitus (DM). Plectranthus vettiveroides (Jacob) N.P. Singh &amp; B.D. Sharma has been used as a polychrest against various diseases including type II diabetes in tradition system of medicine. The present investigation aimed to validate anti-diabetic property of the root derived essential oil of P. vettiveroides and identification of leads with activity on six targets of DM through in silico method. A total of 78 phytochemicals determined through Gas chromatography –mass spectrometry (GC-MS) analysis of the essential oil were docked with each of the six selected targets namely 11β-hydroxysteroid dehydrogenase type 1, dipeptidyl peptidase IV, protein-tyrosine phosphatase 1B, human pancreatic alpha-amylase, human lysosomal acid-alpha-glucosidase, and phosphorylated insulin receptor tyrosine kinase. The structures of the phytochemicals were retrieved from PubChem and the targets were retrieved from Protein Data Bank (PDB). Docking was carried out in AutoDockVina and the molecules with free energy of binding ≤-6 kcal/mol were considered as hit molecule. Out of 78 phytochemicals screened, 39 have activity on all the tested targets. Based on the docking interaction, drug-likeness and absorption, distribution, metabolism, excretion and toxicity (ADMET) property the best lead molecule against each target were determined. The overall results revealed that basically majority of the hits are terpinoides and not induced any toxicity. Therefore, the oil as such can be recommended as medicament against DM, since the individual, synergistic and cumulative effect of phytochemicals may concomitantly inhibit multi-targets of DM and act as an effective drug.

Insulin receptor tyrosine kinase substrate (IRTKS) promotes the tumorigenesis of pancreatic cancer via PI3K/AKT signaling.

Pancreatic cancer (PC) is a common type of tumor, which ranks for the seventh leading cause of cancer death worldwide. Insulin receptor tyrosine kinase substrate (IRTKS) plays an important regulatory role in cell proliferation, motility and survival. In this study, we explore the effect of IRTKS on the occurrence and development of PC. The expression and clinical features of IRTKS were predicted in database, PC cell lines and samples. IRTKS overexpressed and knocked down PC cell lines were established by lentivirus. CCK-8 assay, scratch migration assay and Transwell assay were used to analyze IRTKS oncogenic functions in cell lines. Bioinformatic enrichment analysis were conducted to explore the biological functions IRTKS involved in PC and Western Bolt assay was performed to reveal the downstream signaling molecules. It is detected that IRTKS is highly expressed in PC (P < 0.05), and overexpression of IRTKS predicted worse overall survival (OS, P = 0.018). The proliferation, migration and invasion ability were significantly enhanced in IRTKS overexpressed cells and inhibited in IRTKS knocked down cells (P < 0.05). Bioinformatic enrichment analysis based on GSE46583 dataset showed that IRTKS was significantly involved in PI3K/AKT pathway. Further investigation revealed that overexpression of IRTKS upregulated the ratio of p-PI3K/PI3K and p-AKT/AKT in vitro, while silencing of IRTKS presented opposite results, and PI3K inhibitor LY294002 treatment induced the phenotypic alteration of cell lines (P < 0.05). In conclusion, IRTKS plays an important role in PC tumorigenesis via PI3K/AKT pathway phosphorylated activation, and has a potential clinical application value in prognosis for PC.

223-OR: ADA Presidents' Select Abstract: Novel Ligand and Kinase-Independent Signaling by the Insulin Receptor

Insulin acts through the insulin receptor (IR) tyrosine kinase and two major canonical post-receptor signaling pathways (the IRS-1/PI3K/Akt pathway and the Shc/Ras/MAP kinase pathway) to exert its classic metabolic and mitogenic actions. To create a detailed map of how different domains of the receptor might be linked to different insulin actions, we created cells in which endogenous IR and IGF1R had been genetically inactivated, after which the cells were reconstituted with either the wild-type IR, a kinase-dead mutant of IR (K1030R-IR) , a truncated IR lacking just the C-terminus of IR (∆CT-IR) and a truncated IR with the juxtamembrane domain only inside the cell (JMO-IR) . Using these cells and a multi-omics approach, we have uncovered a second novel IR signaling pathway that is dependent on the intracellular domain of IR but is ligand and tyrosinekinase-independent (LYK-I for short) . We find that the LYK-I actions of the IR are mediated by changes in a phosphorylation of a network of proteins involved in cell cycle/mitosis, ATM signaling, extracellular matrix organization, and cellular senescence. These actions result in upregulation of expression of multiple extracellular matrix-related genes and proteins, and down-regulation of immune/interferon-related and cellular senescence related genes and proteins. Functionally, LYK-I signaling results in increased cell proliferation and increased sensitivity to apoptosis. Thus, in addition to classical ligand and tyrosine kinase-dependent signaling, the IR regulates a second, novel ligand and tyrosine kinase-independent (LYK-I) pathway which regulates the cellular machinery involved in cellular senescence, extracellular matrix interaction and apoptotic sensitivity. Disclosure H.Nagao: None. W.Cai: None. H.Pan: n/a. T.M.Batista: None. B.Brandao: n/a. C.Kahn: Advisory Panel; Kaleido, Consultant; Cellarity, Flagship Pioneering, Sana Biotechnology Inc.

319-OR: Na+/K+ ATPase Activation Is a Conserved Mechanism for Gi/o Protein-Coupled Receptor Control of Pancreatic ß-Cell Electrical Excitability and Insulin Secretion

Gi/o protein-coupled receptor (Gi/o-GPCR) inhibition of pancreatic β-cell Ca2+ entry limits islet insulin secretion; however, the mechanism has not been identified. As Gi/o-GPCRs activate G protein-gated inwardly rectifying K+ (GIRK) channels, we examined their contribution to β-cell Gi/o signaling. However, Gi/o-GPCR-induced β-cell GIRK currents were not observed; this was demonstrated with a pharmacological approach and by genetic knockout of the predominant β-cell GIRK channel subunit. Thus, we increased the chemical driving force for K+ movement through GIRK channels by removing extracellular K+. Interestingly, under these conditions Gi/o-GPCR-induced β-cell membrane potential (Vm) hyperpolarization was lost even though K+ channel activation could still hyperpolarize Vm, which indicated that Gi/o-GPCRs do not hyperpolarize β-cell Vm by activating K+ channels. As extracellular K+ is required for Na+/K+ ATPase (NKA) activity, we next tested and found that mouse and human β-cell NKAs were activated by Gi/o-GPCRs. Stimulation of Gi/o-GPCRs initiated cyclical β-cell NKA activity, resulting in islet Ca2+ oscillations. Moreover, paracrine activation of β-cell NKAs by intraislet δ-cell somatostatin (SST) secretion decelerated islet Ca2+ oscillations and diminished insulin secretion, which was mediated by direct activation of β-cell Gi/o-GPCRs. Gi/o signaling induced islet Ca2+ oscillations through Src tyrosine kinase-dependent NKA phosphorylation and activation; an effect that was recapitulated by stimulating insulin receptor tyrosine kinases. Whereas, islet NKA function was completely inhibited by cAMP-dependent PKA activation. Therefore, these findings reveal that NKA-mediated hyperpolarization of β-cell Vm is the primary and conserved mechanism for Gi/o-GPCR control of electrical excitability, Ca2+ handling, and insulin secretion. Disclosure M.Dickerson: None. D.Jacobson: None. P.Dadi: None. K.E.Zaborska: None. A.Y.Nakhe: None. C.Schaub: None. J.Dobson: None. N.M.Wright: None. J.Lynch: None. C.Scott: None. Funding American Diabetes Association (1-17-IBS-024) ; Vanderbilt Integrated Training in Engineering and Diabetes Grant (T32DK101003) , National Institutes of Health Grants (DK-097392 and DK-115620) , Juvenile Diabetes Research Foundation Grant (2-SRA-2019-701-S-B) , and a Pilot and Feasibility grant through the Vanderbilt Diabetes Research and Training Center Grant (P60-DK-20593) .

PREDICTION OF TOXICITY, PHARMACOKINETICS OF SELECTED PHYTOCHEMICALS OF LEAF OF DRUMSTICK (Moringa Sp.) AND MOLECULAR DOCKING STUDIES ON TWO RECEPTORS AS INSULIN TYROSINE KINASE FOR ANTIDIABETIC POTENTIAL

The medicinal plant, Moringa oleifera Lam is a common tree and the leaf of this plant contains several phytochemicals that have potent anti-diabetic properties. The objective of the present study was to predict toxicity, pharmacokinetics and receptor-ligand binding energy and interaction through molecular docking for selected phytocompounds against mutated protein A and B insulin receptor tyrosine kinase (PDB IDs: 3ekk and 3ekn). In silico study especially molecular docking and pharmacokinetics, bioavailability, drug-likeness, and medicinal chemistry prediction was performed by using PyRx tool (Version 0.8) and Swiss ADME online tool. The molecular interaction was visualized in the molecular graphics laboratory (MGL) tool (Version 1.5.6). About 6 selected phytoconstituents and 2 nos. of synthetic medicines were taken for present prediction. Present in silico study especially molecular docking revealed that favourable binding energy was obtained for Serpentine and Niazirin for A receptor and Anthraquinone for B receptor when compared to synthetic medicines viz. Glibenclamide and Metformin. The pharmacokinetics, bioavailability and drug-likeness and medicinal chemistry to know lead-likeness prediction Serpentine and Niazirincan be suitable drug candidates, which may be potent antidiabetic compound. In conclusion, the binding was obtained near the active site, which may be due to competitive inhibition. Moreover, in future research this predictive data should be validated with further toxicological and pharmacological assay for confirmation of antidiabetic potential.

Functionally selective signaling and broad metabolic benefits by novel insulin receptor partial agonists

Insulin analogs have been developed to treat diabetes with focus primarily on improving the time action profile without affecting ligand-receptor interaction or functional selectivity. As a result, inherent liabilities (e.g. hypoglycemia) of injectable insulin continue to limit the true therapeutic potential of related agents. Insulin dimers were synthesized to investigate whether partial agonism of the insulin receptor (IR) tyrosine kinase is achievable, and to explore the potential for tissue-selective systemic insulin pharmacology. The insulin dimers induced distinct IR conformational changes compared to native monomeric insulin and substrate phosphorylation assays demonstrated partial agonism. Structurally distinct dimers with differences in conjugation sites and linkers were prepared to deliver desirable IR partial agonist (IRPA). Systemic infusions of a B29-B29 dimer in vivo revealed sharp differences compared to native insulin. Suppression of hepatic glucose production and lipolysis were like that attained with regular insulin, albeit with a distinctly shallower dose-response. In contrast, there was highly attenuated stimulation of glucose uptake into muscle. Mechanistic studies indicated that IRPAs exploit tissue differences in receptor density and have additional distinctions pertaining to drug clearance and distribution. The hepato-adipose selective action of IRPAs is a potentially safer approach for treatment of diabetes.

The Antitumor Activity of hAMSCs Secretome in HT-29 Colon Cancer Cells Through Downregulation of EGFR/c-Src/IRTKS Expression and p38/ERK1/2 Phosphorylation.

Colon cancer is considered as one of the main causes of mortality worldwide. Identifying a novel and more effective platform with fewer side effects is still progress. In various cancer types, Epidermal growth factor receptor (EGFR) and c-Src (a key mediator in EGFR signaling pathway) are the key targets for cancer therapy. Moreover, insulin receptor tyrosine kinase substrate (IRTKS or BAI1-associated protein 2-like 1: BAIAP2L1) is a member of the subfamily of inverse BAR (I-BAR) domain proteins, which mediates cell morphology and movement through regulation of actin polymerization. In this study, we employed a co-culture system using Transwell six-well plates. After 72 h, hAMSCs-treated HT-29 cells, EGFR, c-Src, IRTKS, p38, and ERK1/2 expression were analyzed using quantitative real time PCR (qRT-PCR) and western blot methods. The significant reduction in tumor cell growth and motility through downregulation of EGFR/c-Src/IRTKS expression and p38/ERK1/2 phosphorylation in HT-29 cells was demonstrated based on 2D and 3D cell culture models. The induction of cellular apoptosis was also found. Our results support the idea that the hAMSCS secretome has therapeutic effects on cancer cells. However, further experiments will be required to identify the exact molecular mechanisms.

A Review on Metformin: Clinical Significance and Side Effects

Metformin is a biguanide that has been used extensively worldwide for the treatment of type II diabetes mellitus. It improves glycaemic control by enhancing insulin sensitivity in liver and muscle. An advantage of metformin treatment is the tendency of weight reduction and the absence of significant hypoglycaemia; blood glucose levels are reduced only to normal as it does not stimulate insulin secretion. Metformin also has a beneficial effect on several cardiovascular risk factors including dyslipidemia, elevated plasminogen activator inhibitor 1 levels, other fibrinolytic abnormalities, hyperinsulinemia, and insulin resistance. Metformin enhances muscle and adipocyte insulin receptor number and/or affinity, increases insulin receptor tyrosine kinase activity, stimulates glucose transport and glycogen synthesis, and reduces both hepatic gluconeogenesis and glycogenolysis. The disadvantages are confined to the gastro-intestinal side effects and the potential risk of vitamin B 12 and folic acid deficiency during long-term use. These side effects can be minimized by slow titration and consumption with meals. The under lying mechanism for gastrointestinal intolerance are proposed to be stimulation of intestinal secretion of serotonin, alteration in incretin and metabolism of glucose, and malabsorption of bile salts. Lactic acidosis is rare contraindication associated with metformin. Most reported cases of lactic acidosis occur in patients with contraindications, particularly impaired renal function. Metformin has a good safety profile, efficacy and comparatively reduced cost. Its ability to improve insulin sensitivity and the cardiovascular risk profile of type II diabetic patients has enhanced its clinical use as first-line therapy.

Lack of Brain Insulin Receptor Substrate-1 Causes Growth Retardation, With Decreased Expression of Growth Hormone-Releasing Hormone in the Hypothalamus.

Insulin receptor substrate-1 (Irs1) is one of the major substrates for insulin receptor and insulin-like growth factor-1 (IGF-1) receptor tyrosine kinases. Systemic Irs1-deficient mice show growth retardation, with resistance to insulin and IGF-1, although the underlying mechanisms remain poorly understood. For this study, we generated mice with brain-specific deletion of Irs1 (NIrs1KO mice). The NIrs1KO mice exhibited lower body weights, shorter bodies and bone lengths, and decreased bone density. Moreover, the NIrs1KO mice exhibited increased insulin sensitivity and glucose utilization in the skeletal muscle. Although the ability of the pituitary to secrete growth hormone (GH) remained intact, the amount of hypothalamic growth hormone-releasing hormone (GHRH) was significantly decreased and, accordingly, the pituitary GH mRNA expression levels were impaired in these mice. Plasma GH and IGF-1 levels were also lower in the NIrs1KO mice. The expression levels of GHRH protein in the median eminence, where Irs1 antibody staining is observed, were markedly decreased in the NIrs1KO mice. In vitro, neurite elongation after IGF-1 stimulation was significantly impaired by Irs1 downregulation in the cultured N-38 hypothalamic neurons. In conclusion, brain Irs1 plays important roles in the regulation of neurite outgrowth of GHRH neurons, somatic growth, and glucose homeostasis.

Direct visualization of epithelial microvilli biogenesis

Microvilli are actin-bundle-supported surface protrusions that play essential roles in diverse epithelial functions. To develop our understanding of microvilli biogenesis, we used live imaging to directly visualize protrusion growth at early stages of epithelial differentiation. Time-lapse data revealed that specific factors, including epidermal growth factor pathway substrate 8 (EPS8) and insulin-receptor tyrosine kinase substrate (IRTKS) (also known as BAIAP2L1), appear in diffraction-limited puncta at the cell surface and mark future sites of microvillus growth. New core actin bundles elongate from these puncta in parallel with the arrival of ezrin and subsequent plasma membrane encapsulation. In addition to de novo growth, we also observed that new microvilli emerge from pre-existing protrusions. Moreover, we found that nascent microvilli can also collapse, characterized first by loss of membrane wrapping and ezrin enrichment, followed by a sharp decrease in distal tip EPS8 and IRTKS levels, and ultimately disassembly of the core actin bundle itself. These studies are the first to offer a temporally resolved microvillus growth mechanism and highlight factors that participate in this process; they also provide important insights on the growth of apical specializations that will likely apply to diverse epithelial contexts.

Probiotics-rich emulsion improves insulin signalling in Palmitate/Oleate-challenged human hepatocarcinoma cells through the modulation of Fetuin-A/TLR4-JNK-NF-κB pathway

BackgroundFetuin-A, also known as α2-Heremans-Schmid glycoprotein (AHSG), is an abundant plasmatic serum protein synthesized predominantly in liver and adipose tissue. This glycoprotein is known to negatively regulate insulin signaling through the inhibition of insulin receptor (IR) autophosphorylation and tyrosine kinase activity, which participates in insulin resistance (IR) and metabolic syndrome development. Recent studies demonstrated that IR and associated metabolic disorders, are closely related to the gut microbiota and modulating it by probiotics could be effective in metabolic diseases management. ObjectiveIn this present work we aimed to evaluate the effects of a probiotics-rich emulsion on reducing the IR induced by free fatty acids accumulation in human hepatocarcinoma cell line, and to elucidate the implicated molecular pathways, with a specific emphasis on the hepatokin Fetuin-A-related axis. ResultsHere we showed, that probiotics improve HepG2 viability, protect against apoptosis under normal and IR conditions. Moreover, the emulsion was successful in attenuating oxidative stress as well as improving mitochondrial metabolism and dynamics. Interestingly, application of the probiotics to lipotoxic HepG2 cells resulted in significant reduction of Fetuin-A/TLR4/JNK/NF-κB pathway activation, which suggests a protective effect against inflammation, obesity as well as liver related insulin resistant. ConclusionOverall, the presented data reports clearly on the potent potential of probiotics formulated in an emulsion vehicle to enhance metabolic functions of affected IR HepG2 cells, and suggest the possibility of using such preparations as insulin sensitizing therapy, playing at the same time protective role for the development of liver related insulin resistant.

Serum fetuin-A and Ser312 phosphorylated fetuin-A responses and markers of insulin sensitivity after a single bout of moderate intensity exercise.

Fetuin‐A (Fet‐A), secreted by the liver and adipose tissue, inhibits insulin receptor tyrosine kinase activity and modulates insulin action. Numerous studies have shown association of elevated serum Fet‐A concentrations with obesity, non‐alcoholic fatty liver disease, and type 2 diabetes. Both moderate body weight loss (5%–10%) and significant body weight loss have been shown to decrease serum Fet‐A and improve insulin sensitivity. Currently, there are no studies examining the effects of a single bout of exercise on serum Fet‐A or Ser312‐pFet‐A (pFet‐A) responses. We hypothesized that a single bout of moderate‐intensity exercise will lower serum Fet‐A and that these changes will be associated with an improvement in insulin sensitivity. Thirty‐one individuals with obesity and 11 individuals with normal body weight were recruited. Participants underwent a single bout of treadmill walking, expending 500 kcal at 60%–70% VO2max. Oral glucose tolerance tests (OGTT) were administered before the single bout of exercise (Pre Ex) and 24 h after exercise (24h Post Ex). In individuals with obesity, we observed a transient elevation of serum Fet‐A concentrations, but not pFet‐A, immediately after exercise (Post Ex). Further, a single bout of exercise decreased glucoseAUC, insulinAUC, and insulin resistance index in individuals with obesity. Consistent with this improvement in insulin sensitivity, we observed that Fet‐AAUC, pFet‐AAUC, 2 h pFet‐A, and 2 h pFet‐A/Fet‐A were significantly lower following a single bout of exercise. Further, reductions in serum Fet‐AAUC 24h Post Ex were correlated with a reduction in insulin resistance index. Together, this suggests that alterations in serum Fet‐A following a single bout of moderate‐intensity endurance exercise may play a role in the improvement of insulin sensitivity.Clinical Trial RegistrationNCT03478046; https://clinicaltrials.gov/ct2/show/NCT03478046.

Insulin Receptor Substrate p53 Ameliorates High-Glucose-Induced Activation of NF-κB and Impaired Mobility of HUVECs.

Diabetes-related macrovascular and microvascular complications lead to poor prognosis. Insulin receptor substrate p53 (IRSp53) is known to act as a substrate for the insulin receptor tyrosine kinase, but its role in endothelial dysfunction remains unclear. Human umbilical vein endothelial cells (HUVECs) treated with D-glucose at different concentrations and a streptozocin-induced rat diabetes mellitus (DM) model were used to investigate the effects of hyperglycemia on the expression levels of IRSp53 and galectin-3 (gal-3) and the inflammatory state and mobility of HUVECs. Thereafter, IRSp53-overexpressing HUVECs and IRSp53-knockdown HUVECs were established using IRSp53-overexpressing lentivirus or IRSp53-siRNA to explore the role of IRSp53 in the HUVEC inflammatory state and HUVEC mobility. D-glucose at high concentration (HG) and hyperglycemia were found to induce downregulation of IRSp53 and upregulation of gal-3 in vitro and in vivo. Treatment with HG resulted in activation of NF-κB in HUVECs and impaired HUVEC mobility. Insulin restored HG-induced changes in the expression levels of IRSp53 and gal-3 in HUVECs and protected the cells from NF-κB activation and impaired mobility. Overexpression of IRSp53 inhibited the activation of NF-κB in HUVECs and strengthened HUVEC migration. Knockdown of IRSp53 facilitated the activation of NF-κB in HUVECs and decreased HUVEC migration. However, neither overexpression nor knockdown of IRSp53 altered the effects of insulin on HG-induced detrimental changes in HUVECs. HG and hyperglycemia resulted in downregulation of IRSp53 in vitro and in vivo. IRSp53 is concluded to inhibit the activation of NF-κB in HUVECs and to strengthen HUVEC migration.

The Insulin Receptor Adaptor IRS2 is an APC/C Substrate That Promotes Cell Cycle Protein Expression and a Robust Spindle Assembly Checkpoint

Insulin receptor substrate 2 (IRS2) is an essential adaptor that mediates signaling downstream of the insulin receptor and other receptor tyrosine kinases. Transduction through IRS2-dependent pathways is important for coordinating metabolic homeostasis, and dysregulation of IRS2 causes systemic insulin signaling defects. Despite the importance of maintaining proper IRS2 abundance, little is known about what factors mediate its protein stability. We conducted an unbiased proteomic screen to uncover novel substrates of the Anaphase Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that controls the abundance of key cell cycle regulators. We found that IRS2 levels are regulated by APC/C activity and that IRS2 is a direct APC/C target in G1 Consistent with the APC/C's role in degrading cell cycle regulators, quantitative proteomic analysis of IRS2-null cells revealed a deficiency in proteins involved in cell cycle progression. We further show that cells lacking IRS2 display a weakened spindle assembly checkpoint in cells treated with microtubule inhibitors. Together, these findings reveal a new pathway for IRS2 turnover and indicate that IRS2 is a component of the cell cycle control system in addition to acting as an essential metabolic regulator.