Role In Insulin Signaling Research Articles

Abstract Tu082: Novel Structural and Biochemical Effects of Nexilin Associated Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy is primarily a genetic disease that results in abnormal cardiac muscle phenotypes characterized by ventricular hypertrophy, fibrosis, and contractile dysfunction. Both sarcomere structural and signaling genes are associated with HCM. Among various genes, one of the implicated genes for HCM is Nexilin (NEXN). Nexilin is an F-actin binding protein widely expressed in muscle cells and localized at sarcomere and T-tubules. It is known to have a surprising role in insulin signaling. The role of Nexilin mutations is poorly studied. We hypothesized that Nexilin missense variants in genetically engineered human pluripotent stem cells (hPSCs) perturb essential cardiac metabolic signaling pathways leading to HCM. In our Indian HCM cohort, we identified seven Nexilin mutations through exome sequencing and categorized them as pathogenic per ACMG/AMP classification. A representative novel mutant in Nexilin p.Q131T was introduced in hPSC with the CRISPR-Cas9 knock-in method and was differentiated into cardiomyocytes, recapitulating patient-specific heterozygous mutation. Our data shows that Nexilin p.Q131T causes an increase in cell size and activation of fetal gene transcriptional programs in hPSC-derived cardiomyocytes. Global transcriptomics was performed to understand transcriptional changes and investigate further. We observe the significant activation of insulin-related signaling pathways, including AKT/MTOR and MAPK, confirmed by immunoblotting. Akt-mTOR and MAPK cascades are well-known to be regulated by IRS-1 signaling. Our studies show hyperactivation of these cascades, suggesting IRS-1 involvement in the pathogenesis. Nexilin and IRS-1 interaction were probed and found to be perturbed, hence causing higher activation of IRS-1 and its downstream targets. In addition, Nexilin wild type and mutant protein structure-functional analysis were performed with purified proteins. Using cryo-EM structural analysis, we identified unique F-actin binding sites for Nexilin for the first time, and the mutant p.Q131T showed decreased binding affinity to F-actin. We report Nexilin's high-resolution cryo-EM structure and a novel metabolic signaling mechanism where mutations in Nexilin cause dysregulation of IRS-1 signaling in the gene-edited cardiomyocytes, leading to HCM pathogenesis.

The role of insulin signaling with FOXO and FOXK transcription factors.

Insulin is an essential hormone for animal activity and survival, and it controls the metabolic functions of the entire body. Throughout the evolution of metazoan animals and the development of their brains, a sustainable energy supply has been essential to overcoming the competition for survival under various environmental stresses. Managing energy for metabolism, preservation, and consumption inevitably involves high oxidative stress, causing tissue damage in various organs. In both mice and humans, excessive dietary intake can lead to insulin resistance in various organs, ultimately displaying metabolic syndrome and type 2 diabetes. Insulin signals require thorough regulation to maintain metabolism across diverse environments. Recent studies demonstrated that two types of forkhead-box family transcription factors, FOXOs and FOXKs, are related to the switching of insulin signals during fasting and feeding states. Insulin signaling plays a role in supporting higher activity during periods of sufficient food supply and in promoting survival during times of insufficient food supply. The insulin receptor depends on the tyrosine phosphatase feedback of insulin signaling to maintain adipocyte insulin responsiveness. α4, a regulatory subunit of protein phosphatase 2A (PP2A), has been shown to play a crucial role in modulating insulin signaling pathways by regulating the phosphorylation status of key proteins involved in these pathways. This short review summarizes the current understanding of the molecular mechanism related to the regulation of insulin signals.

How Does CBG Administration Affect Sphingolipid Deposition in the Liver of Insulin-Resistant Rats?

Cannabigerol (CBG), a non-psychotropic phytocannabinoid found in Cannabis sativa plants, has been the focus of recent studies due to its potential therapeutic properties. We proposed that by focusing on sphingolipid metabolism, which plays a critical role in insulin signaling and the development of insulin resistance, CBG may provide a novel therapeutic approach for metabolic disorders, particularly insulin resistance. In a rat model of insulin resistance induced by a high-fat, high-sucrose diet (HFHS), we aimed to elucidate the effect of intragastrically administered CBG on hepatic sphingolipid deposition and metabolism. Moreover, we also elucidated the expression of sphingolipid transporters and changes in the sphingolipid concentration in the plasma. The results, surprisingly, showed a lack of changes in de novo ceramide synthesis pathway enzymes and significant enhancement in the expression of enzymes involved in ceramide catabolism, which was confirmed by changes in hepatic sphingomyelin, sphinganine, sphingosine-1-phosphate, and sphinganine-1-phosphate concentrations. The results suggest that CBG treatment may modulate sphingolipid metabolism in the liver and plasma, potentially protecting the liver against the development of metabolic disorders such as insulin resistance.

SAT047 Regulation Of The Insulin Receptor And The Insulin-like Growth Factor 1 Receptor By miR-322-5p, A miR-16 Family Member, In Hypothalamic Neuronal Models

Abstract Disclosure: W. He: None. N. Loganathan: None. K.W. Mak: None. E. McIlwraith: None. D.D. Belsham: None. Insulin signals through the insulin receptor (INSR) and the insulin-like growth factor 1 receptor (IGF1R) in hypothalamic neurons to control food intake and peripheral metabolism. A contributor to obesity, and subsequent comorbidities such type 2 diabetes and heart disease, is the development of cellular insulin resistance in hypothalamic neurons. However, the molecular changes to neuronal insulin signaling remain to be fully elucidated. MicroRNAs (miRNAs) inhibit translation of specific mRNAs; thus, this study aimed to understand the involvement of miRNAs in the regulation of insulin signaling and resistance in hypothalamic neurons. To profile miRNAs expressed in hypothalamic neurons, RNA from the whole hypothalami of 14-week-old CD1 male mice (n = 4), as well as the immortalized hypothalamic neuronal cell lines mHypoE-46 (n = 3) and mHypoA-59 (n = 3), each co-expressing neuropeptide Y (NPY) and agouti-related peptide (AgRP), were assessed using the Affymetrix GeneChip miRNA 4.0 Array. Notably, miR-16 family members, including miR-16-5p, miR-15b-5p, and miR-322-5p, were among the most highly expressed miRNAs in each case. In the mHypoA-59 neurons, overexpression of miR-322-5p for 24 hours decreased the mRNA levels of Igf1r (-26.1%; p = 0.0007; n = 4) and the protein level of INSR-beta (-31.8%; p = 0.0023). These results suggest that the miR-16 family plays an inhibitory role in insulin signaling in hypothalamic neurons. To study the involvement of miRNAs in hyperinsulinemia-induced neuronal insulin resistance, the mHypoE-46 neurons were treated with 100 nM insulin for 24 hours to induce cellular insulin resistance, as characterized by decreased INSR-beta protein (-96.3%; p < 0.0001; n = 4) and a resulting decrease in insulin-induced phosphorylation of protein kinase B (AKT) (-67.6%; p = 0.01; n = 4). GeneChip miRNA array analysis showed that insulin overexposure disrupted the expression of 48 miRNAs (p < 0.05, n = 3), including the upregulation of miR-18a-3p, mir-322, miR-494-3p, and miR-671-3p. Upon RT-qPCR validation (n = 3), we established that prolonged (24 hours), but not acute (1-6 hours), insulin exposure upregulated miR-18a-3p (+62%; p = 0.0104), miR-671-3p (+111%; p = 0.0079), and miR-1983 (+97%; p = 0.0181). miR-671-3p, miR-494-3p, and miR-18a-3p have been shown to decrease phosphatase and tensin homolog (PTEN) levels, which can promote neuronal insulin resistance based on bioinformatic analysis. Overall, these results suggest hyperinsulinemia disrupts the expression of specific miRNAs in hypothalamic neurons to promote cellular insulin resistance. Knowledge derived from these studies will provide insight into hypothalamus-derived miRNAs that could be targeted for miRNA-based diagnostics and therapeutics for early central insulin resistance in humans.(Supported by the CRC, CIHR, NSERC, EndoSoc, and BBDC) Presentation: Saturday, June 17, 2023

Non-coding RNA genes modulate PI3K/AKT signaling pathway in polycystic ovary syndrome.

The PI3K protein kinase B (PI3K/AKT) signaling pathway has crucial roles in insulin signaling and other endocrine disorders. The purpose of this study is to validate the association of PCOS with PI3K/AKT pathway target genes, miRNA486-5p, and miRNA483-5p as well as to evaluate the outcome of metformin on the pathogenesis of PCOS. METHODS: This case-controlled study included 3 subject groups: twenty healthy females (control group), twenty PCOS females before treatment, and twenty PCOS females treated with metformin at a dose (500mg 3 times per day for 3 months). The following gene expressions were assessed by real-time PCR: PI3K, AKT, ERK, GLUT4, miRNA486-5p, and miRNA483-5p in the whole blood. There was a significant decrease in miRNA486-5p and miRNA483-5p in the PCOS group with a significant negative correlation between miRNA486-5p and PI3K and a significant negative correlation between miRNA483-5p and ERK. Metformin treatment resulted in significant elevation of the studied miRNA, significant downregulation of PI3K/AKT target genes, and significant amelioration of the gonadotrophic hormonal imbalance and insulin resistance markers: fasting blood glucose, HBA1C, fasting insulin, and GLUT4 gene expression. miRNA486 and miRNA483 downregulation may contribute to the etiology of PCOS, influence glucose metabolism, and result in IR in PCOS. Metformin's upregulation of those miRNAs affects glucose metabolism by controlling the expression of GLUT4, ameliorates PCOS-related insulin resistance, and improves PCOS-related hormonal imbalance by controlling the PI3K/AKT signaling pathway.

Exploring Metabolites Of Green Algae Caulerpa Spp. To Discover Putative Inhibitors Of Dpp-4, A New Antidiabetic Target Protein

Diabetes mellitus (DM) remains a serious global health threat, claiming a million lives every year and affecting nearly 9% of the adult population who suffer from this impaired insulin sensitivity disease. The Dipeptidyl peptidase-4 (DPP-4) enzyme has recently attracted attention because of its crucial role in insulin signaling, making this enzyme an interesting and emerging target for antidiabetic drug discovery. This study aimed to explore reported metabolites from marine algae of the genus Caulerpa as DPP-4 inhibitors through computational studies using CB-dock 2, Protein-Ligand Interaction Profiler, SwissAdme, and pkCMS. Molecular docking allowed the identification of 7 hit compounds with strong binding affinities (8.4 kcal to 9.3 kcal/mol) against DPP-4 target enzyme PDB ID: 3G4I. Four hits showed stronger binding affinity than two DPP-4 specific inhibitors and FDA-approved antidiabetic drugs, sitagliptin (8.4 kcal/mol) and linagliptin (9.0 kcal/mol). Following a molecular modification of the hit compounds using a bioisosterism-like approach and ADMET evaluation with pkCMS, three putative DPP-4 inhibitors were identified. They showed either stronger binding affinities or better ADMET profiles than sitagliptin and linagliptin, suggesting their promising potential as DPP-4 inhibitors. However, further optimized bioisoterism, in silico and in vivo studies, and clinical-based trials are required to confirm their antidiabetic activity. Keywords: Antidiabetic, Caulerpa racemosa, caulerpin, DPP-4

LRP6 Bidirectionally Regulates Insulin Sensitivity through Insulin Receptor and S6K Signaling in Rats with CG-IUGR

Intrauterine growth restriction followed by postnatal catch-up growth (CG-IUGR) increases the risk of insulin resistance-related diseases. Low-density lipoprotein receptor-related protein 6 (LRP6) plays a substantial role in glucose metabolism. However, whether LRP6 is involved in the insulin resistance of CG-IUGR is unclear. This study aimed to explore the role of LRP6 in insulin signaling in response to CG-IUGR. The CG-IUGR rat model was established via a maternal gestational nutritional restriction followed by postnatal litter size reduction. The mRNA and protein expression of the components in the insulin pathway, LRP6/β-catenin and mammalian target of rapamycin (mTOR)/S6 kinase (S6K) signaling, was determined. Liver tissues were immunostained for the expression of LRP6 and β-catenin. LRP6 was overexpressed or silenced in primary hepatocytes to explore its role in insulin signaling. Compared with the control rats, CG-IUGR rats showed higher homeostasis model assessment for insulin resistance (HOMA-IR) index and fasting insulin level, decreased insulin signaling, reduced mTOR/S6K/ insulin receptor substrate-1 (IRS-1) serine307 activity, and decreased LRP6/β-catenin in the liver tissue. The knockdown of LRP6 in hepatocytes from appropriate-for-gestational-age (AGA) rats led to reductions in insulin receptor (IR) signaling and mTOR/S6K/IRS-1 serine307 activity. In contrast, LRP6 overexpression in hepatocytes of CG-IUGR rats resulted in elevated IR signaling and mTOR/S6K/IRS-1 serine307 activity. LRP6 regulated the insulin signaling in the CG-IUGR rats via two distinct pathways, IR and mTOR-S6K signaling. LRP6 may be a potential therapeutic target for insulin resistance in CG-IUGR individuals.

Arsenic induces the global hypophosphorylation of insulin receptor substrate proteins in differentiated human neuroblastoma SH-SY5Y cells

We recently reported that arsenic disrupted neuronal insulin signaling. Here, we further investigated the effect of arsenic on insulin receptor substrate (IRS) proteins, which are crucial downstream signaling molecules of insulin in differentiated human neuroblastoma SH-SY5Y cells. We also found that prolonged arsenic treatment accelerated the migration of IRS1 and IRS2 on SDS-PAGE. Treatment with phosphatases abolished the arsenic-induced increased mobility of IRS, suggesting that the electrophoretic mobility shift of IRS on SDS-PAGE by arsenic was phosphorylation-dependent. By using label-free mass spectrometry, the phosphorylation sites of IRS1 were found to be S24, S345, S636, T774, S1057, S1058, and S1070, while those of IRS2 were at S645, Y653, T657, S665, S667, S669, S672, S915, and S1203, which were at least 2-fold lower than found in the control. These findings indicated a global hypophosphorylation of IRS proteins after prolonged arsenic treatment. In addition, four novel phosphorylation sites were identified on IRS1 (T774, S1057, S1058, and S1070), with another two on IRS2 (S665 and S667). As basal IRS phosphorylation plays an important role in insulin signaling, the reduction of IRS phosphorylation on multiple residues may underlie arsenic-impaired insulin signaling in neurons.

The Role of Insulin Signaling in Hippocampal-Related Diseases: A Focus on Alzheimer's Disease.

Alzheimer's disease (AD) is a global concern and has become a major public health event affecting human health. Insulin is a metabolic hormone secreted mainly by the peripheral tissue pancreas. In recent years, more and more evidence has proved that insulin regulates various functions of the brain. The hippocampus, one of the earliest brain regions affected by AD, is widely distributed with insulin receptors. Studies have shown that type 2 diabetes mellitus, characterized by insulin resistance, is closely related to AD, which has drawn extensive attention to the relationship between hippocampal insulin signaling and AD. Therefore, we provide an overview of intranasal insulin administration on memory and its underlying mechanism. We also highlight the molecular link between hippocampal insulin resistance and AD and provide a theoretical basis for finding new therapeutic targets for AD in clinical practice.

Role of insulin signaling and its associated signaling in glomerulus for diabetic kidney disease.

The number of patients with diabetic kidney disease (DKD) has been rising significantly over the last several decades and is one of the most frequent causes of chronic kidney disease (CKD) in the United States. Hyperglycemia accelerates development of DKD, a direct result of increased intracellular glucose availability. Two large clinical studies, the Diabetes Control and Complications Trial in type 1 diabetes and the United Kingdom Prospective Diabetes Study in type 2 diabetes showed that intensive glycemic control delayed the onset and the progression of DKD. On the other hand, it is reported that glycemic control alone is not sufficient to control DKD progression. Recent data support that insulin signaling and its associated signaling contribute significantly to preserve glomerular function. However, little is known about the key regulators of insulin signaling in glomerular component cells. In this review, we summarize the novel knowledge regarding the reno-protective effects of insulin signaling or its associated signaling in glomerular constituent cells on DKD.

Type 2 diabetes mellitus augments Parkinson's disease risk or the other way around: Facts, challenges and future possibilities.

About 10% of the adult population is living with type 2 diabetes mellitus (T2DM) and 1% of the population over 60 years of age is suffering from Parkinson's disease (PD). A school of thought firmly believes that T2DM, an age-related disease, augments PD risk. Such relationship is reflected from the severity of PD symptoms in drug naive subjects possessing T2DM. Onset of Parkinsonian feature in case controls possessing T2DM corroborates the role of hyperglycemia in PD. A few cohort, meta-analysis and animal studies have shown an increased PD risk owing to insulin resistance. High fat diet and role of insulin signaling in the regulation of sugar metabolism, oxidative stress, α-synuclein aggregation and accumulation, inflammatory response and mitochondrial function in PD models and sporadic PD further connect the two. Although little is reported about the implication of PD in hyperglycemia and T2DM, a few studies have also contradicted. Ameliorative effect of anti-diabetic drugs on Parkinsonian symptoms and vague outcome of anti-PD medications in T2DM patients also suggest a link. The article reviews the literature supporting augmented risk of one by the other, analysis of proof of the concept, facts, challenges, future possibilities and standpoint on the subject.

Role of mitochondria-associated endoplasmic reticulum membranes in insulin sensitivity, energy metabolism, and contraction of skeletal muscle.

Skeletal muscle has a critical role in the regulation of the energy balance of the organism, particularly as the principal tissue responsible for insulin-stimulated glucose disposal and as the major site of peripheral insulin resistance (IR), which has been related to accumulation of lipid intermediates, reduced oxidative capacity of mitochondria and endoplasmic reticulum (ER) stress. These organelles form contact sites, known as mitochondria-associated ER membranes (MAMs). This interconnection seems to be involved in various cellular processes, including Ca2+ transport and energy metabolism; therefore, MAMs could play an important role in maintaining cellular homeostasis. Evidence suggests that alterations in MAMs may contribute to IR. However, the evidence does not refer to a specific subcellular location, which is of interest due to the fact that skeletal muscle is constituted by oxidative and glycolytic fibers as well as different mitochondrial populations that appear to respond differently to stimuli and pathological conditions. In this review, we show the available evidence of possible differential responses in the formation of MAMs in skeletal muscle as well as its role in insulin signaling and the beneficial effect it could have in the regulation of energetic metabolism and muscular contraction.

Role of microRNAs in regulation of insulin secretion and insulin signaling involved in type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder due to insulin resistance that can be caused by both genetic and environmental factors. In 2018, the American Diabetes Association (ADA) estimated more than 500 million T2DM cases globally. In recent years, studies conducted on humans and animals have suggested that non-coding RNAs, namely, microRNAs (miRNAs), post-transcriptionally regulate gene expression that can bring changes in normal physiology, resulting in the development of metabolic diseases. miRNAs also regulate different cellular processes including insulin synthesis and its secretion from pancreatic β-islet cells, its development and function, insulin signaling and glucose homeostasis. Dysregulation of miRNA can affect the functioning of different tissues during the progression of T2DM. This review focuses on various miRNAs that influence the development of β-cells and insulin secretion, various protein cascades that play an important role in insulin signaling and glucose uptake, and their role in insulin resistance. Similarly, the long noncoding RNAs also known as lncRNAs and their β-cell characteristics involved in T2DM have been discussed. Finally, the significance of miRNAs and their mRNA targets as effective biomarkers and therapeutics in studying the early onset and progression of T2DM have been highlighted.

Retraction of: Insulin-Like Growth Factor 1 Alleviates High-Fat Diet-Induced Myocardial Contractile Dysfunction: Role of Insulin Signaling and Mitochondrial Function.

Retraction of: Insulin-Like Growth Factor 1 Alleviates High-Fat Diet-Induced Myocardial Contractile Dysfunction: Role of Insulin Signaling and Mitochondrial Function.

229-OR: Role of Insulin Signaling in the Regulation of Proteostasis in Astrocytes

Insulin is a principal metabolic hormone. It regulates a plethora of metabolic pathways in sensitive peripheral tissues. Recent studies show insulin signaling plays fundamental roles in the brain. Loss of insulin action in different neuronal and glial cells contributes to a variety of metabolic and behavioral abnormalities in mice. In particular, loss of astrocytic insulin signaling leads to impaired brain glucose sensing and increased depressive-like behavior in mice. How insulin signaling modulates astrocytic cellular functions and neural circuits is not completely known. Using next-generation RNA sequencing, we show that insulin triggers a robust transcriptional regulation on multiple metabolic pathways in primary astrocytes. As expected, insulin increased expression of genes involved in cholesterol biosynthesis, important for supporting synaptic integrity and remodeling. Further, autophagy is a quintessential pathway subjected to insulin regulation, indicating the role of insulin action on proteostasis in astrocytes. Thus, insulin suppresses the expression of most critical genes involved in autophagy, including p62, Ulk1/2, several Atg genes. The inhibitory effect is potent, as 1 nM is sufficient to suppress gene expression. To determine whether insulin receptors (IR) are required for such regulation, we examined the insulin response in astrocytes without endogenous IR. IR loss only partially reverses insulin-dependent suppression of autophagic genes, indicating that insulin may act through closely related IGF-1 receptors in astrocytes. In summary, insulin potently suppresses autophagy in astrocytes through transcriptional regulation, indicating a potential important role of astrocytic insulin signaling on regulating proteostasis in accordance with systemic nutritional status. Impairment of this regulation in insulin resistance and diabetes may affect the modulation of neural circuits by astrocytes and contribute to neurological complications linked to diabetes. Disclosure S.Geffken: None. C.Smith: None. H.Lee: None. W.Cai: None. Funding KDK120740

1367-P: A Submembrane Cytoskeleton-Dependent Mechanism Modulates Mitochondrial Homeostasis and Skeletal Muscle Bioenergetics

Human variants in the ubiquitously expressed cytoskeletal scaffolding protein Ankyrin B (AnkB) , which is enriched in skeletal muscle (SKM) , have been identified as risk factors for diabetes and obesity. Mice harboring pathogenic variants exhibit reductions in levels of the predominant 220-kDa AnkB isoform in several tissues, including SKM and impaired systemic glucose regulation. To understand the specific metabolic role of AnkB in SKM, we developed a conditional knock-out mouse in which 220-kDa AnkB is knocked out only in SKM (SKM-AnkB-KO) . SKM-AnkB-KO mice display glucose mishandling that persists with age, as well as decreases in activity and exercise capacity, measured by indirect calorimetry and voluntary running wheel analysis. Histological evaluations of aged SKM-AnkB-KO mice showed general disorganization of muscle fibers and the presence of centralized nuclei. This is accompanied by increases in lean muscle mass and in the cross-sectional area of mitochondria-rich soleus muscle, suggesting the development of muscle hypertrophy over time. Electron microscopy analysis revealed the presence of hyperfused mitochondria in SKM-AnkB-KO mice. This morphological change was concomitant with up-regulation of transcripts in pathways associated with energetic stress, including AMPK signaling, redox signaling and mTOR signaling, assessed by RNA-seq, and with alterations in mitochondrial respiration in isolated muscle fibers. Interestingly, SKM-AnkB-KO mice mi SKM-AnkB-KO mice also exhibit increases in protein levels of electron transport chain proteins. Through proteomics and biochemical analysis, we uncovered that AnkB directly associates with several mitochondrial proteins and co-fractionates with mitochondrial in SKM. Together, these data suggest a direct role of AnkB in SKM mitochondrial metabolism that is distinct from its previously characterized role in insulin signaling in other tissues and cytoskeletal organization in SKM. Disclosure K.Voos: n/a. J.Tzeng: None. H.Choi: None. T.Pharr: None. D.Lorenzo: None. Funding American Diabetes Association; National Institutes of Health (1T32GM119999-01A1)

1285-P: Differentiating between Diabetic and Idiopathic Gastroparesis: A Genetic Perspective

Gastroparesis is a serious medical condition characterized by delayed gastric emptying and symptoms of nausea, vomiting, bloating, fullness after meals, and abdominal pain. An innate immune dysregulation and injury to the interstitial cells of Cajal and other components of the enteric nervous system is likely central to the pathogenesis of gastroparesis. Most common etiologies include diabetes, post-surgical but most cases are idiopathic. Thus far, little is known about the underlying genetic risk factors for gastroparesis. We have done a genome-wide association study comparing idiopathic and diabetic cases with controls as well as contrasting idiopathic versus diabetic cases. We report a novel genetic association of SLC15A4 locus with idiopathic gastroparesis. This signal is driven by multiple variants with top missense variant SLC15A4:NM145648:exon2:c.T716C:p.V239A, rs33990080. Altogether, among EUR cohort we report 69 carriers out of 214 versus controls 165 out of 896 (MAF: cases 0.18; MAF: controls 0.09) and OR: 1.9 (p-value<10^-6) . We furthermore differentiate idiopathic from diabetic gastroparesis with an established diabetes genetic risk score, - result that is statistically significant (p-value<0.0004) . We also delineate a number of significant loci implicated in diabetes risk including (HLA-DQB1,HLA-DQA2, EXOC6B) in the idiopathic versus diabetic gastroparesis analysis. Of particular interest are the Exocyst complex component, 6 (EXOC6/6B) with lead variant rs41420. previously identified to play a role in insulin signaling through its interaction with the glucose transporter GLUT4. Current genetic findings suggest that a mechanism directly connected to macrophage polarization may be implicated in the etiology gastroparesis. The GWAS picture that is emerging implicates novels loci implicated in the pathophysiology of gastroparesis differentiating those of diabetic versus idiopathic etiology. Disclosure S.P.Smieszek: None.

Hydrogen sulfide disrupts insulin-induced glucose uptake in L6 skeletal muscle cells

Hydrogen sulfide (H2S) has been known for its toxicity. However, recent studies have focused on the mechanisms involved in endogenous production and function. To date, the H2S role in insulin signaling and glucose homeostasis is unclear. This uncertainty is even more evident in skeletal muscle, a physiological niche highly relevant for regulating glycemia in response to insulin. This study aimed to investigate the role of H2S on insulin signaling and glucose uptake in the L6 skeletal muscle cell line. We evaluated the endogenous synthesis with the fluorescent dye, 7-azido-4-methyl coumarin (7-AzMC). Glucose restriction-induced an increase in the endogenous levels of H2S, likely through stimulation of cystathionine γ-lyase activity, as its specific inhibitor, PAG (5 mM) prevented this increase, and mRNA levels of CSE decreased with glucose and amino acid restriction. Exogenous H2S reduced insulin-induced glucose uptake at 0.5 up to 24 h, an effect dissociated from the level of Akt phosphorylation. Our results show that glucose restriction induces endogenous production of H2S via CSE. In addition, H2S disrupts insulin-induced glucose uptake independent of the Akt pathway. These results suggest that H2S antagonism over insulin-induced glucose uptake could help maintain the plasmatic glucose levels in conditions that provoke hypoglycemia, which could serve as an H2S-regulated mechanism for maintaining glucose plasmatic levels through the inhibition of the skeletal muscle insulin-depended glucose uptake.

PTEN-induced kinase 1 deficiency alters albumin permeability and insulin signaling in podocytes.

Alterations of insulin signaling in diabetes are associated with podocyte injury, proteinuria, and renal failure. Insulin stimulates glucose transport to cells and regulates other intracellular processes that are linked to cellular bioenergetics, such as autophagy, gluconeogenesis, fatty acid metabolism, and mitochondrial homeostasis. The dysfunction of mitochondrial dynamics, including mitochondrial fusion, fission, and mitophagy, has been observed in high glucose-treated podocytes and renal cells from patients with diabetes. Previous studies showed that prolonged hyperglycemia is associated with the development of insulin resistance in podocytes, and high glucose-treated podocytes exhibit an increase in mitochondrial fission and decrease in markers of mitophagy. In the present study, we found that deficiency of the main mitophagy protein PTEN-induced kinase 1 (PINK1) significantly increased albumin permeability and hampered glucose uptake to podocytes. We suggest that PINK1 inhibition impairs the insulin signaling pathway, in which lower levels of phosphorylated Akt and membrane fractions of the insulin receptor and glucose transporter-4 were observed. Moreover, PINK1-depleted podocytes exhibited lower podocin and nephrin expression, thus identifying a potential mechanism whereby albumin leakage increases under hyperglycemic conditions when mitophagy is inhibited. In conclusion, we found that PINK1 plays an essential role in insulin signaling and the maintenance of proper permeability in podocytes. Therefore, PINK1 may be a potential therapeutic target for the treatment or prevention of diabetic nephropathy.

Incorporation of Oxidized Phenylalanine Derivatives into Insulin Signaling Relevant Proteins May Link Oxidative Stress to Signaling Conditions Underlying Chronic Insulin Resistance.

A link between oxidative stress and insulin resistance has been suggested. Hydroxyl free radicals are known to be able to convert phenylalanine (Phe) into the non-physiological tyrosine isoforms ortho- and meta-tyrosine (o-Tyr, m-Tyr). The aim of our study was to examine the role of o-Tyr and m-Tyr in the development of insulin resistance. We found that insulin-induced uptake of glucose was blunted in cultures of 3T3-L1 grown on media containing o- or m-Tyr. We show that these modified amino acids are incorporated into cellular proteins. We focused on insulin receptor substrate 1 (IRS-1), which plays a role in insulin signaling. The activating phosphorylation of IRS-1 was increased by insulin, the effect of which was abolished in cells grown in m-Tyr or o-Tyr media. We found that phosphorylation of m- or o-Tyr containing IRS-1 segments by insulin receptor (IR) kinase was greatly reduced, PTP-1B phosphatase was incapable of dephosphorylating phosphorylated m- or o-Tyr IRS-1 peptides, and the SH2 domains of phosphoinositide 3-kinase (PI3K) bound the o-Tyr IRS-1 peptides with greatly reduced affinity. According to our data, m- or o-Tyr incorporation into IRS-1 modifies its protein–protein interactions with regulating enzymes and effectors, thus IRS-1 eventually loses its capacity to play its role in insulin signaling, leading to insulin resistance.