Aberrant Insulin Signaling Research Articles

Role of miR‐802 in brain insulin signaling and its impact on Down syndrome

AbstractBackgroundDown syndrome (DS) individuals are characterized by a variety of pathological phenotypes that manifest with wide variability in the different tissues. At the level of the central nervous system, the accelerated aging phenotype is associated with the risk to develop Alzheimer‐like dementia. A central aspect of neurodegeneration is the close association between metabolic disorders and cognitive decline. Multiple studies have suggested a link between metabolic disorder and microRNAs (miR), small non‐coding RNAs acting as post‐transcriptional regulators of a plethora of genes. Among triplicated miRNAs on chromosome 21, we focus on miR‐802 because recent studies demonstrated its association with development on insulin resistance in obesity and diabetes. Considering these findings and based on the “gene dosage hypothesis” of DS, the goal of the study is to decipher how miR‐802 may contribute to aberrant insulin signaling (IS) and, in parallel to the risk to develop dementia early in life in DS. MethodThe miR‐802 expression, protein levels and activation state of main components of the IS were evaluated (i) in the brain of autoptic cases from DS, DSAD and age‐matched controls and (ii) in the brain of euploid and Ts65Dn mice (a model of DS). Further, using bioinformatic tools we identified miR‐802 predicted target genes that are involved in the IS (PTEN and GSK‐3β).ResultThe IS alterations worsen in the transition from DS to DS/AD and similar findings were collected inTs65Dn mice where IS dysregulation persists with aging where neurodegeneration becomes significant. Intriguingly, these latter changes were driven by the over‐expression of miR‐802, which negatively regulates PTEN and GSK‐3β mRNA in the brain.ConclusionIn this picture, the identification of specific targets modulated by miR‐802 and involved in IS pathway, will provide molecular basis to develop novel therapeutic strategies to prevent/delay the onset of brain insulin resistance in DS.

Endometrial protein expression and phosphorylation landscape decipher aberrant insulin and mTOR signalling in patients with recurrent pregnancy loss

What are the proteomic and phosphoproteomic differences between the endometrium of women with recurrent pregnancy loss (RPL) and the endometrium of healthy control women during the proliferative and secretory phases of the menstrual cycle? In total, 54 endometrial samples were collected during the proliferative and secretory phases from women with RPL (n = 28) and healthy controls (n = 26). Comprehensive proteomic and phosphoproteomic analyses were conducted using label-free liquid chromatography-tandem mass spectrometry (n = 44), and verified through Western blotting (n = 10). Three comparison groups were established: total RPL endometrium versus total control endometrium; RPL proliferative endometrium versus control proliferative endometrium; and RPL secretory endometrium versus control secretory endometrium. Differentially expressed proteins and differentially phosphorylated proteins were identified in the three comparison groups. Combining pathway enrichment, network analysis and soft clustering analysis, the insulin/cyclic nucleotide signalling pathway and AMPK/mTOR signalling pathway were identified as the major contributors to the aberration of RPL endometrium. Western blotting verified altered expression of four proteins: cAMP-dependent protein kinase type I-β regulatory subunit, adenylate cyclase type 3, 5'-AMP-activated protein kinase catalytic subunit α-2 and phosphatidate phosphatase LPIN2. This exploratory study provides insights into the differentiated protein expression and phosphorylation profiles of the endometrium of women with RPL in both the proliferative and sectretory phases of the menstrual cycle. The results highlight potential proteins associated with the pathogenesis of RPL that may serve as potential indicators for RPL. The findings contribute to the identification of potential targets for RPL treatment as well as its pathogenesis.

Reinstated Activity of Human Tau-induced Enhanced Insulin Signaling Restricts Disease Pathogenesis by Regulating the Functioning of Kinases/Phosphatases and Tau Hyperphosphorylation in Drosophila.

Tauopathies such as Alzheimer's disease (AD), Frontotemporal dementia, and parkinsonism linked to chromosome 17 (FTDP-17), etc. are characterized by tau hyperphosphorylation and distinguished accumulation of paired helical filaments (PHFs)/or neurofibrillary tangles (NFTs) in a specific-neuronal subset of the brain. Among different reported risk factors, type 2 diabetes (T2D) has gained attention due to its correlation with tau pathogenesis. However, mechanistic details and the precise contribution of insulin pathway in tau etiology is still debatable. We demonstrate that expression of human tau causes overactivation of insulin pathway in Drosophila disease models. We subsequently noted that tissue-specific downregulation of insulin signaling or even exclusive reduction of its growth-promoting sub-branch effectively reinstates the overactivated insulin signaling pathway in human tau expressing cells, which in turn restricts pathogenic tau hyperphosphorylation and aggregate formation. It was further noted that restored tau phosphorylation was achieved due to a reestablished balance between the levels of different kinase(s) (GSK3β and ERK/P38 MAP kinase) and phosphatase (PP2A). Taken together, our study demonstrates a precise involvement of the insulin pathway and associated molecular events in the pathogenesis of human tauopathies in Drosophila, which will be immensely helpful in developing novel therapeutic options against these devastating human brain disorders. Moreover, our study reveals an interesting link between tau etiology and aberrant insulin signaling, which is a characteristic feature of Type 2 Diabetes.

Mitochondrial Cholesterol Metabolites in a Bile Acid Synthetic Pathway Drive Nonalcoholic Fatty Liver Disease: A Revised "Two-Hit" Hypothesis.

The rising prevalence of nonalcoholic fatty liver disease (NAFLD)-related cirrhosis highlights the need for a better understanding of the molecular mechanisms responsible for driving the transition of hepatic steatosis (fatty liver; NAFL) to steatohepatitis (NASH) and fibrosis/cirrhosis. Obesity-related insulin resistance (IR) is a well-known hallmark of early NAFLD progression, yet the mechanism linking aberrant insulin signaling to hepatocyte inflammation has remained unclear. Recently, as a function of more distinctly defining the regulation of mechanistic pathways, hepatocyte toxicity as mediated by hepatic free cholesterol and its metabolites has emerged as fundamental to the subsequent necroinflammation/fibrosis characteristics of NASH. More specifically, aberrant hepatocyte insulin signaling, as found with IR, leads to dysregulation in bile acid biosynthetic pathways with the subsequent intracellular accumulation of mitochondrial CYP27A1-derived cholesterol metabolites, (25R)26-hydroxycholesterol and 3β-Hydroxy-5-cholesten-(25R)26-oic acid, which appear to be responsible for driving hepatocyte toxicity. These findings bring forth a "two-hit" interpretation as to how NAFL progresses to NAFLD: abnormal hepatocyte insulin signaling, as occurs with IR, develops as a "first hit" that sequentially drives the accumulation of toxic CYP27A1-driven cholesterol metabolites as the "second hit". In the following review, we examine the mechanistic pathway by which mitochondria-derived cholesterol metabolites drive the development of NASH. Insights into mechanistic approaches for effective NASH intervention are provided.

Hyperinsulinemia-induced microglial mitochondrial dynamic and metabolic alterations lead to neuroinflammation in vivo and in vitro.

Numerous studies have demonstrated that type 2 diabetes (T2D) is closely linked to the occurrence of Alzheimer's disease (AD). Nevertheless, the underlying mechanisms for this association are still unknown. Insulin resistance (IR) hallmarked by hyperinsulinemia, as the earliest and longest-lasting pathological change in T2D, might play an important role in AD. Since hyperinsulinemia has an independent contribution to related disease progressions by promoting inflammation in the peripheral system, we hypothesized that hyperinsulinemia might have an effect on microglia which plays a crucial role in neuroinflammation of AD. In the present study, we fed 4-week-old male C57BL/6 mice with a high-fat diet (HFD) for 12 weeks to establish IR model, and the mice treated with standard diet (SD) were used as control. HFD led to obesity in mice with obvious glucose and lipid metabolism disorder, the higher insulin levels in both plasma and cerebrospinal fluid, and aberrant insulin signaling pathway in the whole brain. Meanwhile, IR mice appeared impairments of spatial learning and memory accompanied by neuroinflammation which was characterized by activated microglia and upregulated expression of pro-inflammatory factors in different brain regions. To clarify whether insulin contributes to microglial activation, we treated primary cultured microglia and BV2 cell lines with insulin in vitro to mimic hyperinsulinemia. We found that hyperinsulinemia not only increased microglial proliferation and promoted M1 polarization by enhancing the production of pro-inflammatory factors, but also impaired membrane translocation of glucose transporter 4 (GLUT4) serving as the insulin-responding glucose transporter in the processes of glucose up-taking, reduced ATP production and increased mitochondrial fission. Our study provides new perspectives and evidence for the mechanism underlying the association between T2D and AD.

Blocking MG53S255 Phosphorylation Protects Diabetic Heart From Ischemic Injury.

As an integral component of cell membrane repair machinery, MG53 (mitsugumin 53) is important for cardioprotection induced by ischemia preconditioning and postconditioning. However, it also impairs insulin signaling via its E3 ligase activity-mediated ubiquitination-dependent degradation of IR (insulin receptor) and IRS1 (insulin receptor substrate 1) and its myokine function-induced allosteric blockage of IR. Here, we sought to develop MG53 into a cardioprotection therapy by separating its detrimental metabolic effects from beneficial actions. Using immunoprecipitation-mass spectrometry, site-specific mutation, in vitro kinase assay, and in vivo animal studies, we investigated the role of MG53 phosphorylation at serine 255 (S255). In particular, utilizing recombinant proteins and gene knock-in approaches, we evaluated the potential therapeutic effect of MG53-S255A mutant in treating cardiac ischemia/reperfusion injury in diabetic mice. We identified S255 phosphorylation as a prerequisite for MG53 E3 ligase activity. Furthermore, MG53S255 phosphorylation was mediated by GSK3β (glycogen synthase kinase 3 beta) and markedly elevated in the animal models with metabolic disorders. Thus, IR-IRS1-GSK3β-MG53 formed a vicious cycle in the pathogenesis of metabolic disorders where aberrant insulin signaling led to hyper-activation of GSK3β, which in turn, phosphorylated MG53 and enhanced its E3 ligase activity, and further impaired insulin sensitivity. Importantly, S255A mutant eliminated the E3 ligase activity while retained cell protective function of MG53. Consequently, the S255A mutant, but not the wild type MG53, protected the heart against ischemia/reperfusion injury in db/db mice with advanced diabetes, although both elicited cardioprotection in normal mice. Moreover, in S255A knock-in mice, S255A mutant also mitigated ischemia/reperfusion-induced myocardial damage in the diabetic setting. S255 phosphorylation is a biased regulation of MG53 E3 ligase activity. The MG53-S255A mutant provides a promising approach for the treatment of acute myocardial injury, especially in patients with metabolic disorders.

Design, Synthesis, and Preliminary Evaluation of [68Ga]Ga-NOTA-Insulin as a PET Probe in an Alzheimer's Disease Mouse Model.

Aberrant insulin signaling has been considered one of the risk factors for the development of Alzheimer’s disease (AD) and has drawn considerable attention from the research community to further study its role in AD pathophysiology. Herein, we describe the development of an insulin-based novel positron emission tomography (PET) probe, [68Ga]Ga-NOTA-insulin, to noninvasively study the role of insulin in AD. The developed PET probe [68Ga]Ga-NOTA-insulin showed a significantly higher uptake (0.396 ± 0.055 SUV) in the AD mouse brain compared to the normal (0.140 ± 0.027 SUV) mouse brain at 5 min post injection and also showed a similar trend at 10, 15, and 20 min post injection. In addition, [68Ga]Ga-NOTA-insulin was found to have a differential uptake in various brain regions at 30 min post injection. Among the brain regions, the cortex, thalamus, brain stem, and cerebellum showed a significantly higher standard uptake value (SUV) of [68Ga]Ga-NOTA-insulin in AD mice as compared to normal mice. The inhibition of the insulin receptor (IR) with an insulin receptor antagonist peptide (S961) in normal mice showed a similar brain uptake profile of [68Ga]Ga-NOTA-insulin as it was observed in the AD case, suggesting nonfunctional IR in AD and the presence of an alternative insulin uptake route in the absence of a functional IR. The Gjedde–Patlak graphical analysis was also performed to predict the input rate of [68Ga]Ga-NOTA-insulin into the brain using MicroPET imaging data and supported the in vivo results. The [68Ga]Ga-NOTA-insulin PET probe was successfully synthesized and evaluated in a mouse model of AD in comparison with [18F]AV1451 and [11C]PIB to noninvasively study the role of insulin in AD pathophysiology.

Trilobatin, a Natural Food Additive, Exerts Anti-Type 2 Diabetes Effect Mediated by Nrf2/ARE and IRS-1/GLUT2 Signaling Pathways.

Oxidative stress and aberrant insulin signaling transduction play vital roles in type 2 diabetes mellitus (T2DM). Our previous research has demonstrated that trilobatin (TLB), derived from the leaves of Lithocarpus Polystachyus (Wall.), exhibits a potent antioxidative profile. In the current study, we investigated the anti-T2DM effect of TLB on KK-Ay diabetic mice and further explored the potential mechanisms. Our results showed that TLB significantly reduced the high fasting blood glucose level and insulin resistance and promoted the tolerances to exogenous glucose and insulin in KK-Ay mice. Moreover, TLB reduced the content of reactive oxygen species; enhanced antioxidant enzymes activities, including serum catalase, glutathione peroxidase, and superoxide dismutase; and regulated the abnormal parameters of lipid metabolism, including triglyceride, high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol, and free fatty acid, as evidenced by enzyme-linked immunosorbent assay. Additionally, TLB markedly ameliorated the pancreatic islet morphology near normal and increased the insulin expression of the islet. Whereafter, TLB promoted Nrf2 that was translocated from cytoplasm to nucleus. Moreover, it increased the protein expressions of HO-1, NQO-1, and GLUT-2, and phosphorylation levels of Akt and GSK-3β Ser 9 and decreased the protein expressions of keap1 and phosphorylation levels of IRS-1Ser 307 and GSK-3β Tyr 216. Taken together, our findings reveal that TLB exhibits an anti-T2DM effect in KK-Ay mice by activating the Nrf2/ARE signaling pathway and regulating insulin signaling transduction pathway, and TLB is promising to be developed into a novel candidate for the treatment of T2DM in clinic due to its favorable druggability.

Integrative Metabolic and Proteomic Profiling of the Brainstem in Spontaneously Hypertensive Rats.

The brainstem, the core of the central nervous system, plays a vital role in controlling arterial blood pressure and its elevation of hypertension subtypes, especially essential hypertension. Integrative metabolic and proteomic profiling was performed on the brainstem samples of 11 week old spontaneously hypertensive rats (SHRs) and age-matched normotensive Wistar rats, using hydrophilic interaction liquid chromatography quadrupole/time-of-flight mass spectrometry (HILIC-Q/TOFMS) (PeptideAtlas: PASS01621) and nano-liquid chromatography-high-resolution-MS (nano-LC-high-resolution) combined with quantitative tandem mass tags (ProteomeXchange: PXD021210). The results showed a potentially significant measure of metabolic disorders in the brainstem of SHRs, including purine and pyrimidine metabolism and carnitine and acylcarnitine deficiency. By integrating the differential metabolites (VIP > 1 and p < 0.1) with the differentially expressed proteins (>1.2-fold and p < 0.05), the results revealed aberrant insulin signaling in the brainstem of SHRs, including reduced carnitine and acetylcarnitine; increased arginine; and increased flotillin-1 (FLOT1), hemoglobin subunit alpha-1/2, and hemoglobin subunit beta-2 proteins verified by the parallel reaction monitoring analysis (PeptideAtlas: PASS01622). The aberrant insulin signaling pathway in the brainstem of SHRs might help explain the correlation between essential hypertension and insulin resistance. These findings on the brainstem of SHRs could provide new insights into the dysregulation of the central nervous system in hypertension, especially as it relates to metabolite and protein levels.

Insulin and adipokine signaling and their cross-regulation in postmortem human brain

Aberrant insulin and adipokine signaling has been implicated in cognitive decline associated with both type 2 diabetes mellitus and neurodegenerative diseases. We established methods that reliably measure insulin, adiponectin and leptin signaling, and their crosstalk, in thawed postmortem mid-frontal cortical tissue from cognitively normal older subjects with a short postmortem interval. Insulin-evoked insulin receptor (IR) activation increases activated, tyrosine-phosphorylated IRβ on tyrosine residues 960, 1150, and 1151, insulin receptor substrate-1 recruitment to IRβ and phosphorylated RAC-α-serine/threonine-protein kinase. Adiponectin augments, but leptin inhibits, insulin signaling. Adiponectin activates adiponectin receptors to induce APPL1 binding to adiponectin receptor 1 and 2 and T-cadherin and downstream adenosine monophosphate–dependent protein kinase phosphorylation. Insulin inhibited adiponectin-induced signaling. In addition, leptin-induced leptin receptor (OB-R) signaling promotes Janus kinase 2 recruitment to OB-R and Janus kinase 2 and downstream signal transducer and activator of transcription 3 phosphorylation. Insulin enhanced leptin signaling. These data demonstrate insulin and adipokine signaling interactions in human brain. Future studies can use these methods to examine insulin, adiponectin, and leptin metabolic dysregulation in aging and disease states, such as type 2 diabetes and Alzheimer's disease–related dementias.

Amyloid-beta impairs insulin signaling by accelerating autophagy-lysosomal degradation of LRP-1 and IR-β in blood-brain barrier endothelial cells in vitro and in 3XTg-AD mice

Aberrant insulin signaling constitutes an early change in Alzheimer's disease (AD). Insulin receptors (IR) and low-density lipoprotein receptor-related protein-1 (LRP-1) are expressed in brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB). There, insulin may regulate the function of LRP-1 in Aβ clearance from the brain. Changes in IR-β and LRP-1 and insulin signaling at the BBB in AD are not well understood. Herein, we identified a reduction in cerebral and cerebrovascular IR-β levels in 9-month-old male and female 3XTg-AD (PS1M146V, APPSwe, and tauP301L) as compared to NTg mice, which is important in insulin mediated signaling responses. Reduced cerebral IR-β levels corresponded to impaired insulin signaling and LRP-1 levels in brain. Reduced cerebral and cerebrovascular IR-β and LRP-1 levels in 3XTg-AD mice correlated with elevated levels of autophagy marker LC3B. In both genotypes, high-fat diet (HFD) feeding decreased cerebral and hepatic LRP-1 expression and elevated cerebral Aβ burden without affecting cerebrovascular LRP-1 and IR-β levels. In vitro studies using primary porcine (p)BCEC revealed that Aβ peptides 1–40 or 1–42 (240 nM) reduced cellular levels and interaction of LRP-1 and IR-β thereby perturbing insulin-mediated signaling. Further mechanistic investigation revealed that Aβ treatment accelerated the autophagy-lysosomal degradation of IR-β and LRP-1 in pBCEC. LRP-1 silencing in pBCEC decreased IR-β levels through post-translational pathways further deteriorating insulin-mediated responses at the BBB. Our findings indicate that LRP-1 proves important for insulin signaling at the BBB. Cerebral Aβ burden in AD may accelerate LRP-1 and IR-β degradation in BCEC thereby contributing to impaired cerebral and cerebromicrovascular insulin effects.

ROLE OF ACUTE EXERCISE-INDUCED BRAIN BDNF ON APP PROCESSING AND BACE1

Perturbations in metabolism results in the accumulation of beta-amyloid, which is a pathological feature of Alzheimer's Disease (AD) (1). Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the rate limiting enzyme in the pathway responsible for beta-amyloid production (2). Exercise has been shown to reduce BACE1 activity, although the exact neurobiological mechanisms responsible are unknown (3). Exercise has also been shown to increase brain-derived neurotropic factor (BDNF) content and signaling (4), however whether this neurotrophic factor mediates the effects of exercise on BACE1 regulation requires further investigation. C57BL6 male mice were placed on a low (LFD; n=24) or high fat diet (HFD; n=24) for 10-weeks. Following the intervention, the mice either remained sedentary (n=12) or underwent an acute bout of treadmill running (n=12), proceeded by glucose/insulin tolerance tests and a Barnes maze test (spatial memory). Mice were euthanized and the prefrontal cortex was collected for protein quantification. Markers of the amyloidogenic pathway, along with targets of the BDNF signaling pathway, were examined. The 10-week HFD intervention led to increased body mass, inguinal and epididymal white adipose tissue mass, and liver mass (p < 0.05). HFD groups had impairments in glucose and insulin tolerance, as well as spatial memory (time in incorrect zones) (p < 0.05). A HFD led to increases in Akt phosphorylation (insulin signalling) and decreases in ERK phosphorylation (downstream of BDNF) (p < 0.05). An acute bout of exercise was able to recover ERK phosphorylation in the HFD group (p < 0.05) and significantly increase BDNF protein content in both LFD and HFD groups (p < 0.05). However, there were no changes in BACE1 protein content with diet or exercise (p > 0.05). Results from this study suggest that exercise can increase BDNF protein content in an obesogenic rodent model. Furthermore, a HFD potentially contributes to memory impairments by aberrant insulin signalling and impairments in BDNF signalling, which can be recovered through exercise. Although BACE1 protein content did not change, future analysis will examine BACE1 activity. Through these mechanisms, we gain further insight into using BDNF, mediated through exercise, as a therapeutic intervention.

Aberrant insulin receptor expression is associated with insulin resistance and skeletal muscle atrophy in myotonic dystrophies.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant multisystemic disorders linked to two different genetic loci and characterized by several features including myotonia, muscle atrophy and insulin resistance. The aberrant alternative splicing of insulin receptor (IR) gene and post-receptor signalling abnormalities have been associated with insulin resistance, however the precise molecular defects that cause metabolic dysfunctions are still unknown. Thus, the aims of this study were to investigate in DM skeletal muscle biopsies if beyond INSR missplicing, altered IR protein expression could play a role in insulin resistance and to verify if the lack of insulin pathway activation could contribute to skeletal muscle wasting. Our analysis showed that DM skeletal muscle exhibits a lower expression of the insulin receptor in type 1 fibers which can contribute to the defective activation of the insulin pathway. Moreover, the aberrant insulin signalling activation leads to a lower activation of mTOR and to an increase in MuRF1 and Atrogin-1/MAFbx expression, possible explaining DM skeletal muscle fiber atrophy. Taken together our data indicate that the defective insulin signalling activation can contribute to skeletal muscle features in DM patients and are probably linked to an aberrant specific-fiber type expression of the insulin receptor.

Heart Failure in Type 2 Diabetes Mellitus.

Patients with diabetes mellitus have >2× the risk for developing heart failure (HF; HF with reduced ejection fraction and HF with preserved ejection fraction). Cardiovascular outcomes, hospitalization, and prognosis are worse for patients with diabetes mellitus relative to those without. Beyond the structural and functional changes that characterize diabetic cardiomyopathy, a complex underlying, and interrelated pathophysiology exists. Despite the success of many commonly used antihyperglycemic therapies to lower hyperglycemia in type 2 diabetes mellitus the high prevalence of HF persists. This, therefore, raises the possibility that additional factors beyond glycemia might contribute to the increased HF risk in diabetes mellitus. This review summarizes the state of knowledge about the impact of existing antihyperglycemic therapies on HF and discusses potential mechanisms for beneficial or deleterious effects. Second, we review currently approved pharmacological therapies for HF and review evidence that addresses their efficacy in the context of diabetes mellitus. Dysregulation of many cellular mechanisms in multiple models of diabetic cardiomyopathy and in human hearts have been described. These include oxidative stress, inflammation, endoplasmic reticulum stress, aberrant insulin signaling, accumulation of advanced glycated end-products, altered autophagy, changes in myocardial substrate metabolism and mitochondrial bioenergetics, lipotoxicity, and altered signal transduction such as GRK (g-protein receptor kinase) signaling, renin angiotensin aldosterone signaling and β-2 adrenergic receptor signaling. These pathophysiological pathways might be amenable to pharmacological therapy to reduce the risk of HF in the context of type 2 diabetes mellitus. Successful targeting of these pathways could alter the prognosis and risk of HF beyond what is currently achieved using existing antihyperglycemic and HF therapeutics.

Identification of novel mutations of Insulin Receptor Substrate 1 (IRS1) in tumor samples of non-small cell lung cancer (NSCLC): Implications for aberrant insulin signaling in development of cancer.

Lung cancer is the leading cause of cancer-related death, and NSCLC constitutes nearly 85%–90% of all cases. The IRS proteins function as adaptors and transmit signals from multiple receptors. Upon binding of insulin to the insulin receptor (IR), IRS1 is phosphorylated at several YXXM motifs creating docking sites for the binding of PI3Kp85, which activates AKT kinase. Therefore, we thought that gain of function mutantions of IRS1 could be related to development of lung cancer. In line with this, we wanted determine whether the IRS1 gene was mutated in the coding regions surrounding YXXM motifs. We sequenced the coding regions surrounding YXXM motifs of IRS1 using tumor samples of 42 NSCLC patients and 40 matching controls and found heterozygote p.S668T mutation in nine of 42 samples and four of nine also had the p.D674H mutation. We generated IRS1 expression vectors harboring p.S668T, p.D674H and double mutants. Expression of the mutants differentially affected insulin-induced phosphorylation of IRS1, AKT, ERK, and STAT3. Also, our mutants induced proliferation, glucose uptake, inhibited the migration of 293T cells and affected the responsiveness of the cells to cisplatin and radiation. Our results suggest that these novel mutations play a role in the phenotype of lung cancer.

Insulin resistance disrupts cell integrity, mitochondrial function, and inflammatory signaling in lymphatic endothelium

Lymphatic vessel dysfunction and increased lymph leakage have been directly associated with several metabolic diseases. However, the underlying cellular mechanisms causing lymphatic dysfunction have not been determined. Aberrant insulin signaling affects the metabolic function of cells and consequently impairs tissue function. We hypothesized that insulin resistance in LECs decreases eNOS activity, disrupts barrier integrity increases permeability, and activates mitochondrial dysfunction and pro-inflammatory signaling pathways. LECs were treated with insulin and/or glucose to determine the mechanisms leading to insulin resistance. Acute insulin treatment increased eNOS phosphorylation and NO production in LECs via activation of the PI3K/Akt signaling pathway. Prolonged hyperglycemia and hyperinsulinemia induced insulin resistance in LECs. Insulin-resistant LECs produced less NO due to a decrease in eNOS phosphorylation and showed a significant decrease in impedance across an LEC monolayer that was associated with disruption in the adherence junctional proteins. Additionally, insulin resistance in LECs impaired mitochondrial function by decreasing basal-, maximal-, and ATP-linked OCRs and activated NF-κB nuclear translocation coupled with increased pro-inflammatory signaling. Our data provide the first evidence that insulin resistance disrupts endothelial barrier integrity, decreases eNOS phosphorylation and mitochondrial function, and activates inflammation in LECs.

Acute Exercise and Brain BACE1 Protein Content: a Time Course Study

Obesity and type 2 diabetes are significant risk factors in the development of neurodegenerative disorders, such as Alzheimer's disease (AD). A variety of cellular mechanisms, such as altered insulin and stress‐signaling contribute to neurodegeneration, and have been linked to increases in beta‐site amyloid precursor protein cleaving enzyme 1 (BACE1). BACE1 is the rate limiting enzyme in the production of amyloid‐beta peptides which are implicated in the pathology of AD. Previous work suggests that a single, acute bout of exercise may have beneficial neuro‐protective effects in the prefrontal cortex of obese mice, independent of weight loss. The purpose of this study was to examine‐ how long the exercise induced alterations in the prefrontal cortex persist following a single exercise bout. Male C57BL/6 mice were fed either a low (LFD, 10% kcals from lard) or a high fat diet (HFD, 60% kcals from lard) for 7 weeks. HFD mice then underwent an acute bout of exercise (treadmill running: 15 m/min, 5% incline, 120 min) followed by a recovery period of 2, 8, or 24 hours. The HFD resulted in increased body mass (LFD 27.8±1.05 vs HFD 41.7±0.60 g; p&lt;0.05) and glucose intolerance (AUC LFD 63.27±4.5 vs HFD 128.9±4.6; p&lt;0.05). In the prefrontal cortex, BACE1 protein content was reduced 2‐ and 8‐hours post‐exercise compared to the sedentary HFD group, however BACE1 protein content 24‐hours post‐exercise was not different (p&lt;0.05). Compared to the LFD, the HFD had higher prefrontal cortex phosphorylation of p38, JNK, and Akt (Ser473), with no changes in ERK phosphorylation, indicative of increased neuronal stress and aberrant insulin signaling. Post‐exercise p38 and JNK phosphorylation were no different from either the HFD or LFD groups, while ERK phosphorylation was significantly reduced by 24‐hours of recovery. These changes were accompanied by an increase in circulating BDNF (pg/ml) at 24‐hour recovery (p&lt;0.01). Our results suggest that a single, acute bout of exercise may have beneficial neuro‐protective effects in obesity. We provide evidence that an acute bout of exercise can reduce markers of stress‐signaling pathways as well as BACE1 protein content in the prefrontal cortex. However, the reduction in BACE1 content is short lived and highlights the importance of daily exercise interventions for long‐term improvements in early AD‐associated pathology.Support or Funding InformationSupported by Alzheimer Society of Brant, Haldimand, Norfolk, Hamilton, and Halton PI:REKMThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impaired insulin signaling upon loss of ovarian function is associated with a reduction of tristetraprolin and an increased stabilization of chemokine in adipose tissue

Loss of ovarian function can activate inflammation and lead to insulin resistance (IR). IR is also a core feature of obesity and obesity-associated metabolic dysfunction. Tristetraprolin/zinc finger protein 36 (TTP) interferes with TNF-α production by destabilizing TNF-α mRNA, and mice deficient in TTP develop a complex syndrome of inflammatory disease (Carballo et al., 1998; Taylor et al., 1999). We hypothesized that ovariectomy (OVX) might also prime inflammation by reducing tristetraprolin/zinc finger protein 36 (TTP) levels. We used a mouse OVX model to study impaired insulin signaling due to loss of ovarian function by evaluating Akt activity upon insulin stimulus. Impaired insulin signaling was initially detected in adipose tissue (AT) at 4 weeks after OVX, and then spread to liver and muscle, finally resulting in systemic IR at 12 weeks after OVX. OVX decreased TTP protein levels and increased adipocyte size, oxidative stress, chemokine expression and fat mass in AT by 4 weeks after surgery. TTP deficiency due to TTP gene deletion induced aberrant insulin signaling and increased chemokine expression and macrophage numbers in AT but did not increase adipocyte size, oxidative stress, or fat mass, suggesting that it promotes insulin signaling by decreasing AT inflammation independent of oxidative stress and adiposity. OVX, like TTP deficiency, increased the stability of chemokine transcripts as assessed from their half-lives. Our data indicate that the impaired insulin signaling resulting from OVX is due to an OVX-induced reduction of TTP and the resulting stabilization of inflammatory chemokines.

P 116 - Crosstalk between insulin resistance and oxidative stress in the development of Alzheimer-like neurodegeneration

Down Syndrome (DS) individuals by the age of 40ys develop a type of dementia that has the same characteristics as Alzheimer disease (AD). Previous studies in DS and AD brain suggest common neurodegenerative pathways including mitochondrial dysfunction, oxidative stress (OS) and reduced glucose metabolism. In addition, several studies suggest a link between insulin resistance and cognitive dysfunction in AD. The present study aims to analyze the crosstalk between the onset of brain insulin resistance (BIR) and OS as possible contributing factors to the neurodegenerative process in tg mouse model of DS (Ts65Dn). We longitudinally analyze (at 1–3-9–18 months) changes of i) IR/IRS1/ERK1/2/Akt levels and activation state iii) oxidative stress markers and iii) biliverdin reductase-A (BVR-A), SIRT1 and PTEN protein levels and activation, in the cortex of Ts65Dn mice. In parallel, changes of APP/Abeta levels have been analyzed. Our results show the mutual interaction between increased OS and BIR in Ts65dn, which does not correlate with Abeta levels. We found that OS negatively impacts the activation of insulin cascade since postnatal age that also persists with age. These findings highlight the role of BIR in the onset of AD-like neurodegeneration and suggest that aberrant insulin signaling strongly contributes to cognitive decline also in DS.

Hyperglycaemia and aberrated insulin signalling stimulate tumour progression via induction of the extracellular matrix component hyaluronan.

Epidemiological studies have detected a higher incidence of various tumour entities in diabetic patients. However, the underlying mechanisms remain insufficiently understood. Glucose-derived pericellular and extracellular hyaluronan (HA) promotes tumour progression and development. In our study, we tested the hypothesis that a diabetic metabolic state, characterised by hyperglycaemia and concomitant aberrant insulin signalling, stimulates tumour progression via the induction of HA synthesis. In a streptozotocin-induced diabetic nude mouse tumour xenograft model, hyperglycaemia and lack of insulin caused an increased formation of tumour-associated HA-matrix, which in turn accelerated tumour progression and neoangiogenesis. This process was effectively attenuated by treatment with 4-methylumbelliferone, a pharmacological inhibitor of HA-synthesis. To define the mechanisms behind these in vivo observations, we investigated the impact of hyperglycaemia and insulin on the glucose metabolism in oesophageal squamous cell cancer cells (ESCC). Hyperglycaemia induced HA synthesis while insulin diminished HA production by directing glucose metabolites to glycolysis. Vice versa, inhibition of glycolysis, either by knockdown of the glycolytic key enzyme phosphofructokinase or by an experimental abrogation of insulin signalling (knockdown of the insulin receptor and long-term treatment with insulin) augmented HA synthesis. Consequently, these processes induced invasion, anchorage-independent growth and adhesion of ESCC to endothelial cells in vitro. Thus, the cellular shift in glucose usage from catabolism of glucose to anabolism of HA driven by hyperglycaemia and insulin resistance may represent an important link between diabetes and cancer progression. Hence, therapeutical inhibition of HA synthesis may represent a promising approach for tumour treatment in diabetic patients.