The brain insulin receptor gene network and associations with frailty index.

OBJECTIVE: To investigate longitudinal associations between variations in the co-expression-based brain insulin receptor polygenic risk score and frailty, as well as change in frailty across follow-up. METHODS: This longitudinal study included 1605 participants from the Helsinki Birth Cohort Study. Biologically informed expression-based polygenic risk scores for the insulin receptor gene network, which measure genetic variation in the function of the insulin receptor, were calculated for the hippocampal (hePRS-IR) and the mesocorticolimbic (mePRS-IR) regions. Frailty was assessed in at baseline in 2001-2004, 2011-2013 and 2017-2018 by applying a deficit accumulation-based frailty index. Analyses were carried out by applying linear mixed models and logistical regression models adjusted for adult socioeconomic status, birthweight, smoking and their interactions with age. RESULTS: The FI levels of women were 1.19%-points (95% CI 0.12-2.26, P = 0.029) higher than in men. Both categorical and continuous hePRS-IR in women were associated with higher FI levels than in men at baseline (P < 0.05). In women with high hePRS-IR, the rate of change was steeper with increasing age compared to those with low or moderate hePRS-IR (P < 0.05). No associations were detected between mePRS-IR and frailty at baseline, nor between mePRS-IR and the increase in mean FI levels per year in either sex (P > 0.43). CONCLUSIONS: Higher variation in the function of the insulin receptor gene network in the hippocampus is associated with increasing frailty in women. This could potentially offer novel targets for future drug development aimed at frailty and ageing.

Caveolin-2 palmitoylation turnover facilitates insulin receptor substrate-1-directed lipid metabolism by insulin receptor tyrosine kinase.

Here, we show that insulin induces palmitoylation turnover of Caveolin-2 (Cav-2) in adipocytes. Acyl protein thioesterases-1 (APT1) catalyzes Cav-2 depalmitoylation, and zinc finger DHHC domain-containing protein palmitoyltransferase 21 (ZDHHC21) repalmitoylation of the depalmitoylated Cav-2 for the turnover, thereby controlling insulin receptor (IR)-Cav-2-insulin receptor substrate-1 (IRS-1)-Akt-driven signaling. Insulin-induced palmitoylation turnover of Cav-2 facilitated glucose uptake and fat storage through induction of lipogenic genes. Cav-2-, APT1-, and ZDHHC21-deficient adipocytes, however, showed increased induction of lipolytic genes and glycerol release. In addition, white adipose tissues from insulin sensitive and resistant obese patients exhibited augmented expression of LYPLA1 (APT1) and ZDHHC20 (ZDHHC20). Our study identifies the specific enzymes regulating Cav-2 palmitoylation turnover, and reveals a new mechanism by which insulin-mediated lipid metabolism is controlled in adipocytes.

Insulin Signaling Differentially Regulates the Trafficking of Insulin and Amyloid Beta Peptides at the Blood-Brain Barrier.

The blood-brain barrier (BBB) is instrumental in clearing toxic metabolites from the brain, such as amyloid-ß (Aß) peptides, and in delivering essential nutrients to the brain, like insulin. In Alzheimer's disease (AD) brain, increased Aß levels are paralleled by decreased insulin levels, which are accompanied by insulin signaling deficits at the BBB. Thus, we investigated the impact of insulin-like growth factor and insulin receptor (IGF1R and IR) signaling on Aß and insulin trafficking at the BBB. Following intravenous infusion of an IGF1R/IR kinase inhibitor (AG1024) in wild-type mice, the BBB trafficking of 125I radiolabeled Aß peptides and insulin was assessed by dynamic SPECT/CT imaging. The brain efflux of [125I]iodo-Aß42 decreased upon AG1024 treatment. Additionally, the brain influx of [125I]iodoinsulin, [125I]iodo-Aß42, [125I]iodo-Aß40, and [125I]iodo-BSA (BBB integrity marker) was decreased, increased, unchanged, and unchanged, respectively, upon AG1024 treatment. Subsequent mechanistic studies were performed using an in vitro BBB cell model. The cell uptake of [125I]iodoinsulin, [125I]iodo-Aß42, and [125I]iodo-Aß40 was decreased, increased, and unchanged, respectively, upon AG1024 treatment. Further, AG1024 reduced the phosphorylation of insulin signaling kinases (Akt and Erk) and the membrane expression of Aß and insulin trafficking receptors (LRP-1 and IR-ß). These findings reveal that insulin signaling differentially regulates the BBB trafficking of Aß peptides and insulin. Moreover, deficits in IGF1R and IR signaling, as observed in the brains of type II diabetes and AD patients, are expected to increase Aß accumulation while decreasing insulin delivery to the brain, which has been linked to the progression of cognitive decline in AD.

Creation of Knock-In Alleles of Insulin Receptor Tagged by Fluorescent Proteins mCherry or EYFP in Fruit Fly Drosophila melanogaster.

The insulin/insulin-like growth factor-like signaling (IIS) pathway is highly conserved across metazoans and regulates numerous physiological functions, including development, metabolism, fecundity, and lifespan. The insulin receptor (InR), a crucial membrane receptor in the IIS pathway, is known to be ubiquitously expressed in various tissues, albeit at generally low levels, and its subcellular localization remains incompletely characterized. In this study, we employed CRISPR-mediated mutagenesis in the fruit fly Drosophila to create knock-in alleles of InR tagged with fluorescent proteins (InR::mCherry or InR::EYFP). By inserting the coding sequence of the fluorescent proteins mCherry or EYFP near the end of the coding sequence of the endogenous InR gene, we could trace the natural InR protein through their fluorescence. As an example, we investigated epithelial cells of the male accessory gland (AG), an internal reproductive organ, and identified two distinct patterns of InR::mCherry localization. In young AG, InR::mCherry accumulated on the basal plasma membrane between cells, whereas in mature AG, it exhibited intracellular localization as multiple puncta, indicating endocytic recycling of InR during cell growth. In the AG senescence accelerated by the mutation of Diuretic hormone 31 (Dh31), the presence of InR::mCherry puncta was more pronounced compared to the wild type. These findings raise expectations for the utility of the newly created InR::mCherry/EYFP alleles for studying the precise expression levels and subcellular localization of InR. Furthermore, this fluorescently tagged allele approach can be extended to investigate other membrane receptors with low abundance, facilitating the direct examination of their true expression and localization.

Extracellular glucose and dysfunctional insulin receptor signaling independently upregulate arterial smooth muscle TMEM16A expression.

Diabetes alters the function of ion channels responsible for regulating arterial smooth muscle membrane potential, resulting in vasoconstriction. Our prior research demonstrated an elevation of TMEM16A in diabetic arteries. Here, we explored the mechanisms involved in Transmembrane protein 16A (TMEM16A) gene expression. Our data indicate that a Snail-mediated repressor complex regulates arterial TMEM16A gene transcription. Snail expression was reduced in diabetic arteries while TMEM16A expression was upregulated. The TMEM16A promoter contained three canonical E-box sites. Electrophoretic mobility and super shift assays revealed that the -154 nt E-box was the binding site of the Snail repressor complex and binding of the repressor complex decreased in diabetic arteries. High glucose induced a biphasic contractile response in pressurized nondiabetic mouse hindlimb arteries incubated ex vivo. Hindlimb arteries incubated in high glucose also showed decreased phospho-protein kinase D1 and TMEM16A expression. In hindlimb arteries from nondiabetic mice, administration of a bolus dose of glucose activated protein kinase D1 signaling to induce Snail degradation. In both in vivo and ex vivo conditions, Snail expression exhibited an inverse relationship with the expression of protein kinase D1 and TMEM16A. In diabetic mouse arteries, phospho-protein kinase D1 increased while Akt2 and pGSK3ß levels declined. These results indicate that in nondiabetic mice, high glucose triggers a transient deactivation of the Snail repressor complex to increase arterial TMEM16A expression independently of insulin signaling. Conversely, insulin resistance activates GSK3ß signaling and enhances arterial TMEM16A channel expression. These data have uncovered the Snail-mediated regulation of arterial TMEM16A expression and its dysfunction during diabetes.NEW & NOTEWORTHY The calcium-activated chloride channel, TMEM16A, is upregulated in the diabetic vasculature to cause increased vasoconstriction. In this paper, we have uncovered that the TMEM16A gene expression is controlled by a Snail-mediated repressor complex that uncouples with both insulin-dependent and -independent pathways to allow for upregulated arterial protein expression thereby causing vasoconstriction. The paper highlights the effect of short- and long-term glucose-induced dysfunction of an ion channel expression as a causative factor in diabetic vascular disease.

Infection with SARS-CoV-2 can cause pancreatic impairment.

Evidence suggests associations between COVID-19 patients or vaccines and glycometabolic dysfunction and an even higher risk of the occurrence of diabetes. Herein, we retrospectively analyzed pancreatic lesions in autopsy tissues from 67 SARS-CoV-2 infected non-human primates (NHPs) models and 121 vaccinated and infected NHPs from 2020 to 2023 and COVID-19 patients. Multi-label immunofluorescence revealed direct infection of both exocrine and endocrine pancreatic cells by the virus in NHPs and humans. Minor and limited phenotypic and histopathological changes were observed in adult models. Systemic proteomics and metabolomics results indicated metabolic disorders, mainly enriched in insulin resistance pathways, in infected adult NHPs, along with elevated fasting C-peptide and C-peptide/glucose ratio levels. Furthermore, in elder COVID-19 NHPs, SARS-CoV-2 infection causes loss of beta (ß) cells and lower expressed-insulin in situ characterized by islet amyloidosis and necrosis, activation of α-SMA and aggravated fibrosis consisting of lower collagen in serum, an increase of pancreatic inflammation and stress markers, ICAM-1 and G3BP1, along with more severe glycometabolic dysfunction. In contrast, vaccination maintained glucose homeostasis by activating insulin receptor α and insulin receptor ß. Overall, the cumulative risk of diabetes post-COVID-19 is closely tied to age, suggesting more attention should be paid to blood sugar management in elderly COVID-19 patients.

The serine phosphorylations in the IRS-1 PIR domain abrogate IRS-1 and IR interaction.

Serine phosphorylations on insulin receptor substrate 1 (IRS-1) by diverse kinases aoccur widely during obesity-, stress-, and inflammation-induced conditions in models of insulin resistance and type 2 diabetes. In this study, we define a region within the human IRS-1, which is directly C-terminal to the PTB domain encompassing numerous serine phosphorylation sites including Ser307 (mouse Ser302) and Ser312 (mouse 307) creating a phosphorylation insulin resistance (PIR) domain. We demonstrate that the IRS-1 PTB-PIR with its unphosphorylated serine residues interacts with the insulin receptor (IR) but loses the IR-binding when they are phosphorylated. Surface plasmon resonance studies further confirm that the PTB-PIR binds stronger to IR than just the PTB domain, and that phosphorylations at Ser307, Ser312, Ser315, and Ser323 within the PIR domain result in abrogating the binding. Insulin-responsive cells containing the mutant IRS-1 with all these four serines changed into glutamates to mimic phosphorylations show decreased levels of phosphorylations in IR, IRS-1, and AKT compared to the wild-type IRS-1. Hydrogen-deuterium exchange mass spectrometry experiments indicating the PIR domain interacting with the N-terminal lobe and the hinge regions of the IR kinase domain further suggest the possibility that the IRS-1 PIR domain protects the IR from the PTP1B-mediated dephosphorylation.

Insulin receptor signaling engages bladder urothelial defenses that limit urinary tract infection.

Urinary tract infections (UTIs) commonly afflict people with diabetes. To better understand the mechanisms that predispose diabetics to UTIs, we employ diabetic mouse models and altered insulin signaling to show that insulin receptor (IR) shapes UTI defenses. Our findings are validated in human biosamples. We report that diabetic mice have suppressed IR expression and are more susceptible to UTIs caused by uropathogenic Escherichia coli (UPEC). Systemic IR inhibition increases UPEC susceptibility, while IR activation reduces UTIs. Localized IR deletion in bladder urothelium promotes UTI by increasing barrier permeability and suppressing antimicrobial peptides. Mechanistically, IR deletion reduces nuclear factor κB (NF-κB)-dependent programming that co-regulates urothelial tight junction integrity and antimicrobial peptides. Exfoliated urothelial cells or urine samples from diabetic youths show suppressed expression of IR, barrier genes, and antimicrobial peptides. These observations demonstrate that urothelial insulin signaling has a role in UTI prevention and link IR to urothelial barrier maintenance and antimicrobial peptide expression.

Activation of the insulin receptor by insulin-like growth factor 2.

Insulin receptor (IR) controls growth and metabolism. Insulin-like growth factor 2 (IGF2) has different binding properties on two IR isoforms, mimicking insulin's function. However, the molecular mechanism underlying IGF2-induced IR activation remains unclear. Here, we present cryo-EM structures of full-length human long isoform IR (IR-B) in both the inactive and IGF2-bound active states, and short isoform IR (IR-A) in the IGF2-bound active state. Under saturated IGF2 concentrations, both the IR-A and IR-B adopt predominantly asymmetric conformations with two or three IGF2s bound at site-1 and site-2, which differs from that insulin saturated IR forms an exclusively T-shaped symmetric conformation. IGF2 exhibits a relatively weak binding to IR site-2 compared to insulin, making it less potent in promoting full IR activation. Cell-based experiments validated the functional importance of IGF2 binding to two distinct binding sites in optimal IR signaling and trafficking. In the inactive state, the C-terminus of α-CT of IR-B contacts FnIII-2 domain of the same protomer, hindering its threading into the C-loop of IGF2, thus reducing the association rate of IGF2 with IR-B. Collectively, our studies demonstrate the activation mechanism of IR by IGF2 and reveal the molecular basis underlying the different affinity of IGF2 to IR-A and IR-B.

Sensitive and label-free SPR biosensing platforms for high-throughput screening of plasma membrane receptors interactions with insulin-like targets of hypoglycaemic activity.

Progress in medical sciences aims for tailored therapy of civilization diseases like diabetes. Preclinical screening of new medicines superior to insulin should include the verification of their affinity to the membrane receptors naturally stimulated by this hormone: insulin receptor isoforms A and B and insulin-like growth factor receptor. Considering that the affinity constants obtained using different experimental conditions are incomparable, it is essential to develop a robust and reliable method to analyze these interactions. The versatile SPR platform developed in this study enables the evaluation of the bioactivity of hypoglycaemic molecules. Thanks to the comprehensive characterization of miscellaneous aspects of the analytical platform, including the design of the SPR biosensor receptor layer, ensuring interaction specificity, as well as the quality control of the standards used (human insulin, HI; long-acting insulin analog: glargine, Gla), the feasibility of the method of equilibrium and kinetic constants determination for insulin-like targets was confirmed. SPR assays constructed in the direct format using IR-A, IR-B, and IGF1-R receptor proteins show high sensitivities and low detection limits towards insulin and glargine detection in the range of 18.3-53.3 nM with no signs of mass transport limitations. The improved analytical performance and stability of SPR biosensors favor the acquisition of good-quality kinetic data, while preservation of receptors activity after binding to long-chain carboxymethyldextran, combined with spontaneous regeneration, results in stability and long shelf life of the biosensor, which makes it useful for label-free insulin analogs biosensing and thus extensive screening in diabetic drugs discovery.

A Novel Mutation in the INSR Gene Causes Severe Insulin Resistance and Rabson-Mendenhall Syndrome in a Paraguayan Patient.

Rabson-Mendenhall syndrome (RMS) is a rare autosomal recessive disorder characterized by severe insulin resistance, resulting in early-onset diabetes mellitus. We report the first case of RMS in a Paraguayan patient. The patient is a 6-year-old girl who presented with hypertrichosis, acanthosis nigricans, nephrocalcinosis, and elevated levels of glucose and insulin that served as diagnostic indicators for RMS. Genetic testing by next-generation sequencing (NGS) revealed two pathogenic variants in exons 2 and 19 of the INSR gene: c.332G>T (p.Gly111Val) and c.3485C>T (p.Ala1162Val), in combined heterozygosis. The novel INSR c. 332G>T variant leads to the substitution of glycine to valine at position 111 in the protein, and multiple in silico software programs predicted it as pathogenic. The c.3485C>T variant leads to the substitution of alanine to valine at position 1162 in the protein previously described for insulin resistance and RMS. The management of RMS is particularly challenging in children, and the use of metformin is often limited by its side effects. The patient was managed with nutritional measures due to the early age of onset. This report expands the knowledge of RMS to the Paraguayan population and adds a novel pathogenic variant to the existing literature.

Sialic acids cleavage induced by elastin-derived peptides impairs the interaction between insulin and its receptor in adipocytes 3T3-L1.

The insulin receptor (IR) plays an important role in insulin signal transduction, the defect of which is believed to be the root cause of type 2 diabetes. In 3T3-L1 adipocytes as in other cell types, the mature IR is a heterotetrameric cell surface glycoprotein composed of two α subunits and two ß subunits. Our objective in our study, is to understand how the desialylation of N-glycan chains, induced by elastin-derived peptides, plays a major role in the function of the IR. Using the 3T3-L1 adipocyte line, we show that removal of the sialic acid from N-glycan chains (N893 and N908), induced by the elastin receptor complex (ERC) and elastin derived-peptides (EDPs), leads to a decrease in the autophosphorylation activity of the insulin receptor. We demonstrate by molecular dynamics approaches that the absence of sialic acids on one of these two sites is sufficient to generate local and general modifications of the structure of the IR. Biochemical approaches highlight a decrease in the interaction between insulin and its receptor when ERC sialidase activity is induced by EDPs. Therefore, desialylation by EDPs is synonymous with a decrease of IR sensitivity in adipocytes and could thus be a potential source of insulin resistance associated with diabetic conditions.

Pericyte Control of Gene Expression in the Blood-Brain Barrier Endothelium: Implications for Alzheimer's Disease.

Background: A strong body of evidence suggests that cerebrovascular pathologies augment the onset and progression of Alzheimer's disease (AD). One distinctive aspect of this cerebrovascular dysfunction is the degeneration of brain pericytes-often overlooked supporting cells of blood-brain barrier endothelium. Objective: The current study investigates the influence of pericytes on gene and protein expressions in the blood-brain barrier endothelium, which is expected to facilitate the identification of pathophysiological pathways that are triggered by pericyte loss and lead to blood-brain barrier dysfunction in AD. Methods: Bioinformatics analysis was conducted on the RNA-Seq expression counts matrix (GSE144474), which compared solo-cultured human blood-brain barrier endothelial cells against endothelial cells co-cultured with human brain pericytes in a non-contact model. We constructed a similar cell culture model to verify protein expression using western blots. Results: The insulin resistance and ferroptosis pathways were found to be enriched. Western blots of the insulin receptor and heme oxygenase expressions were consistent with those observed in RNA-Seq data. Additionally, we observed more than 5-fold upregulation of several genes associated with neuroprotection, including insulin-like growth factor 2 and brain-derived neurotrophic factor. Conclusions: Results suggest that pericyte influence on blood-brain barrier endothelial gene expression confers protection from insulin resistance, iron accumulation, oxidative stress, and amyloid deposition. Since these are conditions associated with AD pathophysiology, they imply mechanisms by which pericyte degeneration could contribute to disease progression.

Down-Regulation of INSR Restores Th17/Treg Immune Balance and Alleviates Airway Hyperviscosity in Asthmatic Mice via Inactivation of STAT3 Pathway.

BACKGROUND: Allergic asthma (AA) is a prevalent chronic airway inflammation disease. In this study, this study aims to investigate the biological functions and potential regulatory mechanisms of the insulin receptor (INSR) in the progression of AA. METHODS: BALB/c mice (n = 48) were randomly divided into the following groups: control group, AA group, AA+Lentivirus (Lv)-vector short hairpin RNA (shRNA) group, AA+Lv-vector group, AA+Lv-INSR shRNA group, and AA+Lv-INSR group. The pulmonary index was calculated. mRNA and protein expression levels of INSR, signal transducer and activator of transcription 3 (STAT3), Janus kinase 2 (JAK2), phosphorylated-STAT3 (p-STAT3), phosphorylated-JAK2 (p-JAK2), alpha-smooth muscle actin (α-SMA), febrile neutropenia (FN), mucin 5AC (MUC5AC), and mucin 5B (MUC5B) were examined using reverse-transcription quantitative PCR (RT-qPCR) and western blot assays. Positive expressions of INSR, retinoic acid-related orphan receptor gamma-t (RORγt), and forkhead box protein P3 (Foxp3) were quantified by immunohistochemistry. Fluorescence intensities of α-SMA and FN were detected by immunofluorescence. Pathological morphology was observed through hematoxylin-eosin (H&E) staining, Masson staining, and Periodic Acid-Schiff (PAS) staining. Contents of immunoglobulin E (IgE), interleukin-6 (IL-6), eotaxin, interleukin-4 (IL-4), interleukin-13 (IL-13), interferon-γ (IFN-γ), interleukin-17 (IL-17), and interleukin-10 (IL-10) were quantified using enzyme-linked immunosorbent assay (ELISA). The percentage of T helper 17 (Th17) and regulatory T (Treg) cells was determined through flow cytometry. RESULTS: Compared to the control group, expression levels of INSR, p-STAT3, p-JAK2, α-SMA, FN, MUC5AC, MUC5B, RORγt, and Foxp3, as well as IgE, IL-6, eotaxin, IL-4, IL-13, and IL-17 contents, pulmonary index, glycogen-positive area (%), and Th17 cell percentage significantly increased (p < 0.05). Additionally, pulmonary histopathological deterioration and collagen deposition were aggravated, while Treg cell percentage and IFN-γ and IL-10 contents remarkably decreased (p < 0.05). The overexpression of INSR further exacerbated the progression of allergic asthma, but the down-regulation of INSR reversed the trends of the above indicators. CONCLUSIONS: The down-regulation of INSR alleviates airway hyperviscosity, inflammatory infiltration, and airway remodeling, restoring Th17/Treg immune balance in AA mice by inactivating the STAT3 pathway.

Insulin and IGF-1 have both overlapping and distinct effects on CD4+ T cell mitochondria, metabolism, and function.

Insulin and insulin-like growth factor 1 (IGF-1) are metabolic hormones with known effects on CD4+ T cells through insulin receptor (IR) and IGF-1 receptor (IGF-1R) signaling. Here, we describe specific and distinct roles for these hormones and receptors. We have found that IGF-1R, but not IR, expression is increased following CD4+ T cell activation or following differentiation toward Th17 cells. Although both insulin and IGF-1 increase the metabolism of CD4+ T cells, insulin has a more potent effect. However, IGF-1 has a unique role and acts specifically on Th17 cells to increase IL-17 production and Th17 cell metabolism. Furthermore, IGF-1 decreases mitochondrial membrane potential and mitochondrial reactive oxygen species (mROS) in Th17 cells, providing a cytoprotective effect. Interestingly, both IR and IGF-1R are required for this effect of IGF-1 on mitochondria, which suggests that the hybrid IR/IGF-1R may be required for mediating the effect of IGF-1 on mitochondrial membrane potential and mROS production.

Integrated network pharmacological analysis revealed that Smilax glabra Roxb. alleviates IMQ-induced psoriatic skin inflammation through regulating T cell immune response.

ETHNOPHARMACOLOGICAL RELEVANCE: Psoriasis is an autoimmune disease characterized by dysfunctional T cells and dysregulated immune responses. Smilax glabra Roxb. (SGR) is a formulation used in Traditional Chinese Medicine for the treatment of inflammatory skin disorders, including psoriasis. This study explores the scientific basis for its use by examining the effects of SGR on T cell differentiation and insulin receptor signaling, relevant pathways implicated in the pathophysiology of psoriasis. AIM OF THE STUDY: This study investigates the therapeutic potential of SGR (a Chinese medicine) in psoriasis and its impact on T cell differentiation. MATERIALS AND METHODS: An integrated network pharmacology and bioinformatics approach was employed to elucidate the mechanisms of SGR in regulating T cell differentiation. A psoriasis mouse model was utilized to evaluate the effects of SGR on T cell subsets. Immunohistochemistry and gene expression analyses were conducted to investigate the modulation of insulin receptor signaling pathways by SGR. RESULTS: SGR treatment effectively reset the expression of various T cell subsets in the psoriasis mouse model, suggesting its ability to regulate T cell differentiation and immune function. Furthermore, SGR treatment inhibited insulin receptor signaling and downstream pathways, including PI3K/AKT and ERK, in psoriatic skin lesions. This indicates that SGR may exert its therapeutic effects through modulation of the insulin receptor signaling pathway. CONCLUSIONS: This study provides novel insights into the therapeutic potential of SGR in psoriasis. By modulating T cell differentiation and targeting the insulin receptor signaling pathway, SGR holds promise as a potential treatment option for psoriasis.

Pancreatic ß cell derived extracellular vesicles containing surface preproinsulin are involved in glucose stimulated insulin secretion.

AIMS: Extracellular vesicles (EVs) are involved in intercellular communication and are a topic of increasing interest due to their therapeutic potential. The aim of this study was to determine whether human islet-derived EVs contain insulin, and if so, what role do they play in glucose stimulated insulin secretion. MAIN METHODS: We isolated EVs from human islets culture and plasma to probe for insulin. Plasma from hyperglycemic glucose clamp experiments were also used to isolate and measure EV insulin content in response to a secretory stimulus. We performed immunogold electron microscopy for insulin presence in EVs. Co-culture experiments of isolated EVs with fresh islets were performed to examine the effect of EV cargo on insulin receptor signaling. KEY FINDINGS: EVs isolated from culture medium contained insulin. Glucose treatment of islets increased the level of EV insulin. Hyperglycemic glucose clamp experiments in humans also lead to increased levels of insulin in plasma-derived EVs. Immunogold electron microscopy and proteinase K-digestion experiments demonstrated that insulin in EVs predominantly associated with the exterior surface of EVs while western blot analyses uncovered the presence of only preproinsulin in EVs. Membrane-bound preproinsulin in EVs was capable of activating insulin signaling pathway in an insulin receptor-dependent manner. The physiological relevance of this finding was observed in priming of human naïve islets by EVs during glucose stimulated insulin secretion. SIGNIFICANCE: Our data suggest that (1) human islets secret insulin via an alternate pathway (EV-mediated) other than conventional granule-mediated insulin secretion, and (2) EV membrane bound preproinsulin is biologically active.

Tryptophanylation of insulin receptor by WARS attenuates insulin signaling.

Increased circulating amino acid levels have been linked to insulin resistance and development of type 2 diabetes (T2D), but the underlying mechanism remains largely unknown. Herein, we show that tryptophan modifies insulin receptor (IR) to attenuate insulin signaling and impair glucose uptake. Mice fed with tryptophan-rich chow developed insulin resistance. Excessive tryptophan promoted tryptophanyl-tRNA synthetase (WARS) to tryptophanylate lysine 1209 of IR (W-K1209), which induced insulin resistance by inhibiting the insulin-stimulated phosphorylation of IR, AKT, and AS160. SIRT1, but not other sirtuins, detryptophanylated IRW-K1209 to increase the insulin sensitivity. Collectively, we unveiled the mechanisms of how tryptophan impaired insulin signaling, and our data suggested that WARS might be a target to attenuate insulin resistance in T2D patients.

Neddylation of insulin receptor substrate acts as a bona fide regulator of insulin signaling and its implications for cancer cell migration.

Irregularities in insulin signaling have significantly increased the risk of various cancers, yet the precise underlying mechanisms remain unclear. Within our study, we observed that inhibiting neddylation enhances cancer cell migration across different cancer types by activating both insulin receptor substrates 1 and 2 (IRS1 and IRS2), along with the PI3K/AKT signaling pathway. Notably, in the context of high-grade serous carcinoma (HGSC) patients, whether they had type 2 diabetes mellitus or not, IRS1 and IRS2 displayed a parallel relationship with each other while exhibiting an inverse relationship with NEDD8. We also identified C-CBL as an E3 ligase responsible for neddylating IRS1 and IRS2, with clinical evidence further confirming a reciprocal relationship between C-CBL and pAKT, thereby reinforcing the tumor suppressive role of C-CBL. Altogether, these findings suggest that neddylation genuinely participates in IRS1 and IRS2-dependent insulin signaling, effectively suppressing cancer cell migration. Thus, caution is advised when considering neddylation inhibitors as a treatment option for cancer patients, particularly those presenting with insulin signaling dysregulations linked to conditions like obesity-related type 2 diabetes or hyperinsulinemia.

IRS1 promotes thyroid cancer metastasis through EMT and PI3K/AKT pathways.

OBJECTIVE: Insulin receptor substract 1 (IRS1) protein is an important signal transduction adapter for extracellular signal transduction from insulin-like growth factor-1 receptor and its family members to IRS1 downstream proteins. IRS1 has been reported to be involved in tumourigenesis and metastasis in some of solid tumors. Investigating the role of IRS1 in thyroid cancer can help to screen high risk patients at the initial diagnosis. DESIGN, PATIENTS AND MEASUREMENTS: Immunohistochemical assay was used to detect the expression levels of IRS1 in 131 metastatic thyroid cancer tissues. Wound healing, cell invasion and colony formation assays were used to study the functions of IRS1 in vitro. RNA sequencing (RNA-seq) and Western blot analysis analyses were performed to examine the underlying regulation mechanisms of IRS1 in thyroid cancer cells. RESULTS: IRS1 was highly expressed in thyroid cancers and its expression was positively associated with distant metastasis and advanced clinical stages. In vitro studies demonstrated that IRS1 is an important mediator of migration, invasion and colony formation of thyroid cancer cells. RNA-seq showed that IRS1 promoted the metastasis of thyroid cancer by regulating epithelial-mesenchymal transition and phosphoinositide 3-kinase (PI3K)/AKT pathway. CONCLUSIONS: IRS1 overexpression contributes to the aggressiveness of thyroid cancer and is expected to be a stratified marker and a potential therapeutic target for thyroid cancer.