Insulin Receptor Substrate-1 Degradation Research Articles

Abstract 5181: Early activity and biomarker evaluation of NT219 in combination with cetuximab in a Phase 1/2 study of recurrent/metastatic squamous cell carcinoma of the head and neck (R/M SCCHN)

Abstract Background. NT219 is a novel small molecule, dual inhibitor, that uniquely triggers the degradation of Insulin Receptor Substrates 1/2 (IRS) and the dephosphorylation and suppression of STAT3. These two major oncogenic targets in the tumor and tumor microenvironment (TME) play a key role in drug resistance, particularly to EGFR inhibitors. Previously, we demonstrated synergistic effect of NT219 and cetuximab in multiple SCCHN patient-derived xenograft (PDX) models. HPV-negative R/M SCCHN patients have poor survival and demonstrate IGF1R/IRS and STAT3 upregulation. We report biomarker results from dose escalation of NT219 in combination with cetuximab in patients (pts) with immunotherapy-refractory R/M SCCHN (NCT04474470). Method. Biopsies from 11 enrolled SCCHN patients treated with different dose levels of NT219 (6, 12, 24 and 50 mg/kg) were obtained and evaluated using Immunohistochemistry and pathological analysis. Evaluation of potential biomarkers included measurements of IRS1/2 levels, as well as IGF1R and STAT3 phosphorylation (pIGF1R, pSTAT3) in tissue biopsies, and H-score was defined for tumor cells, infiltrating immune cells, and fibroblasts in the TME. Membrane completeness (mc) parameter was included in pIGF1R scoring. Results. NT219 Cmax and AUC demonstrated a dose dependent increase, with 50mg/kg dose mean human AUC reaching Human Equivalent Dose efficacy of animal models. Indeed, at this dose, anti-tumor activity in humans was also demonstrated with 2 confirmed partial responses (PR) reported. At the 50 mg/kg NT219 dose, out of 4 SCCHN pts, for whom pre-treatment biopsies were available, 3 were HPV negative and 2 of them achieved PR. Pretreatment biopsies of these 2 pts demonstrated high activated IGF1R membranal staining (Avg. mcH-score: 285, p-value:) and high activated STAT3 nuclear staining (Avg. H-score: 285), in contrast to the non-responding SCCHN pts (30 and 33, with p-value 0.017, 0.004 respectively). Inhibition of intratumoral IGF1R/IRS and STAT3 was demonstrated in on-treatment biopsies. Analysis will be presented at the conference. Conclusion. NT219 in combination with cetuximab demonstrated safety and early activity with 2 confirmed PRs at 50mg/kg dose level in R/M SCCHN pts. This dose level was the first dose at which the observed NT219 exposure was within the range of the efficacious dose levels in pre-clinical models. Detection of activated IGF1R and STAT3 in responding pts, compared to the non-responding pts, suggests a potential use of these targets as biomarkers for future patient selection. This notion is further substantiated by the demonstration of NT219 on-target effects in on-treatment tumor biopsies. Upregulation of NT219 targets, IGF1R and STAT3, in HPV-negative SCCHN pts and their response to the NT219+cetuximab treatment, support further evaluation of this patient population. Citation Format: Ari J. Rosenberg, Shumei Kato, Daniel Johnson, Aron Popovtzer, Vi K. Chiu, Ravit Geva, Hava Ben-David, Tomer Meirson, Michael Schickler, Hadas Reuveni. Early activity and biomarker evaluation of NT219 in combination with cetuximab in a Phase 1/2 study of recurrent/metastatic squamous cell carcinoma of the head and neck (R/M SCCHN) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5181.

Abstract 1939: NT219, a dual inhibitor of IRS1/2 and STAT3, suppresses cancer stem cell mediated resistance to KRASG12C and KRASG12D inhibitors in solid tumors

Abstract Background NT219 is a novel small molecule, a dual inhibitor which uniquely triggers the degradation of Insulin Receptor Substrates 1/2 (IRS) and the dephosphorylation and suppression of STAT3, two major oncogenic targets that play a key role in drug resistance. Mutated KRAS (mKRAS) has long been referred to as undruggable until recently when KRASG12C inhibitors were approved. While KRASG12C is the most common oncogenic driver in NCSLC, KRASG12D is found in 42% of pancreatic tumors. We present NT219 suppressive effect on mKRASi resistant and sensitive cells of both PDAC and NSCLC, as well as on cancer stem cells (CSC) known to contribute to disease recurrence. Method The inhibitory effects of NT219 as a monotherapy or in combination with either KRASG12C inhibitors (sotorasib/adagrasib) or KRASG12D inhibitor (MTRX1133) were tested in 2D cell growth and colony formation assays using mKRAS inhibitor sensitive and resistant cancer cell lines. The effect of NT219 and mKRASi on cancer stem cells was assessed using 3D spheroid assay evaluating spheroid size (microscopic measurements), cell viability (CellTiterGlo) and stemness marker expression (FACS analysis). Results NT219 in combination with either sotorasib or MTRX1133 suppressed established spheroids of NSCLC(KRASG12C) and PDAC(KRASG12D) respectively, in terms of size, disintegration and viability. In NSCLC H358, spheroid satellites appeared following treatment with sotorasib, while the combination with NT219 suppressed it. CSC marker ALDH increased 7-fold in NSCLC spheroids as compared to 2D grown cells, while NT219 and sotorasib suppressed it. The expression of SOX2 stemness marker was reduced by NT219 alone (5 fold), unexpectedly induced by sotorasib (4 fold) and suppressed by NT219+sotorasib (15 fold vs sotorasib alone). In addition, significant induction of STAT3, was observed following sotorasib treatment and completely reversed by the addition of NT219. We also demonstrate that NT219 induces growth inhibition in both KRASiG12C resistant PDAC and NSCLC, and KRASiG12D resistant PDAC cells. NT219 exhibited synergistic effects with adagrasib and MTRX1133, in suppressing the proliferation of KRASG12C PDAC and NSCLC cells and KRASG12D PDAC cells, respectively. This combination showed a synergistic effect in xenograft model. Conclusion We first showed the effect of NT219 in suppressing cancer stem cells, known to promote resistance and tumor recurrence. Synergistic effect of NT219 and mKRAS inhibitors, G12Cand G12D, in NSCLC and PDAC respectively, was demonstrated. Initial results suggest involvement of NT219 target proteins in resistance to mKRASG12C/D inhibitors. Treatment with sotorasib induced enhancement in stemness potential of mKRASG12C NSCLC, which NT219 efficiently suppressed, suggesting a novel therapy for this unmet need and a novel mechanism to combat resistance to mKRAS inhibitors Citation Format: Hadas Reuveni, Mohammed Najeeb Al-Hallak, Sarah Motorwala, Eliza Beal, Steve Kim, Rafic Beydoun, Gergory Dyson, Bassel El-Rayes, Herbert Chen, Philip A. Philip, Anthony F. Shields, Boris Pasche, Katy Sigalov, Hava Ben-David, Daphna Miron, Asfar S. Azmi. NT219, a dual inhibitor of IRS1/2 and STAT3, suppresses cancer stem cell mediated resistance to KRASG12C and KRASG12D inhibitors in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1939.

Human cytomegalovirus attenuates AKT activity by destabilizing insulin receptor substrate proteins.

Human cytomegalovirus (HCMV) requires inactivation of AKT to efficiently replicate, yet how AKT is shut off during HCMV infection has remained unclear. We show that UL38, an HCMV protein that activates mTORC1, is necessary and sufficient to destabilize insulin receptor substrate 1 (IRS1), a model insulin receptor substrate (IRS) protein. Degradation of IRS proteins in settings of excessive mTORC1 activity is an important mechanism for insulin resistance. When IRS proteins are destabilized, PI3K cannot be recruited to growth factor receptor complexes, and hence, AKT membrane recruitment, a rate limiting step in its activation, fails to occur. Despite its penchant for remodeling host cell signaling pathways, our results reveal that HCMV relies upon a cell-intrinsic negative regulatory feedback loop to inactivate AKT. Given that pharmacological inhibition of PI3K/AKT potently induces HCMV reactivation from latency, our findings also imply that the expression of UL38 activity must be tightly regulated within latently infected cells to avoid spontaneous reactivation.

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.

Cathepsin K activity controls cachexia-induced muscle atrophy via the modulation of IRS1 ubiquitination.

BackgroundCachexia is a complicated metabolic disorder that is characterize by progressive atrophy of skeletal muscle. Cathepsin K (CTSK) is a widely expressed cysteine protease that has garnered attention because of its enzymatic and non‐enzymatic functions in signalling in various pathological conditions. Here, we examined whether CTSK participates in cancer‐induced skeletal muscle loss and dysfunction, focusing on protein metabolic imbalance.MethodsMale 9‐week‐old wild‐type (CTSK+/+, n = 10) and CTSK‐knockout (CTSK−/−, n = 10) mice were injected subcutaneously with Lewis lung carcinoma cells (LLC; 5 × 105) or saline, respectively. The mice were then subjected to muscle mass and muscle function measurements. HE staining, immunostaining, quantitative polymerase chain reaction, enzyme‐linked immunosorbent assay, and western blotting were used to explore the CTSK expression and IRS1/Akt pathway in the gastrocnemius muscle at various time points. In vitro measurements included CTSK expression, IRS1/Akt pathway‐related target molecule expressions, and the diameter of C2C12 myotubes with or without LLC‐conditioned medium (LCM). An IRS1 ubiquitin assay, and truncation, co‐immunoprecipitation, and co‐localization experiments were also performed.ResultsCTSK+/+ cachectic animals exhibited loss of skeletal muscle mass (muscle weight loss of 15%, n = 10, P < 0.01), muscle dysfunction (grip strength loss > 15%, n = 10, P < 0.01), and fibre area (average area reduction > 30%, n = 5, P < 0.01). Compared with that of non‐cachectic CTSK+/+ mice, the skeletal muscle of cachectic CTSK+/+ mice exhibited greater degradation of insulin receptor substrate 1 (IRS1, P < 0.01). In this setting, cachectic muscles exhibited decreases in the phosphorylation levels of protein kinase B (Akt308, P < 0.01; Akt473, P < 0.05) and anabolic‐related proteins (the mammalian target of rapamycin, P < 0.01) and increased levels of catabolism‐related proteins (muscle RING‐finger protein‐1, P < 0.01; MAFbx1, P < 0.01) in CTSK+/+ mice (n = 3). Although there was no difference in LLC tumour growth (n = 10, P = 0.44), CTSK deletion mitigated the IRS1 degradation, loss of the skeletal muscle mass (n = 10, P < 0.01), and dysfunction (n = 10, P < 0.01). In vitro, CTSK silencing prevented the IRS1 ubiquitination and loss of the myotube myosin heavy chain content (P < 0.01) induced by LCM, and these changes were accelerated by CTSK overexpression even without LCM. Immunoprecipitation showed that CTSK selectively acted on IRS1 in the region of amino acids 268 to 574. The results of co‐transfection of IRS1‐N‐FLAG or IRS1‐C‐FLAG with CTSK suggested that CTSK selectively cleaves IRS1 and causes ubiquitination‐related degradation of IRS1.ConclusionsThese results demonstrate that CTSK plays a novel role in IRS1 ubiquitination in LLC‐induced muscle wasting, and suggest that CTSK could be an effective therapeutic target for cancer‐related cachexia.

Serum‐ and glucocorticoid‐inducible kinase 1 promotes insulin resistance in adipocytes via degradation of insulin receptor substrate 1

Accumulating evidence indicates that serum- and glucocorticoid-inducible kinase 1 (SGK1) plays a role in the development of metabolic syndrome via a poorly understood mechanism. This study aimed to investigate the direct effect of SGK1 on insulin sensitivity in adipose tissue. We ectopically expressed or silenced SGK1 in adipocytes via lentiviral transfection, measured glucose uptake and evaluated insulin signalling using western blotting. In vivo insulin resistance was measured at the whole-body and adipose tissue levels in db/db mice treated with an inhibitor of SGK1. After 8 weeks of SGK1 inhibitor treatment, the serum insulin level andhomeostasis model assessment of insulin resistance index were significantly decreased, and AKT phosphorylation in adipose tissue was enhanced in db/db mice. Overexpression of constitutively active SGK1 in adipocytes in vitro decreased AKT phosphorylation and insulin-stimulated glucose uptake. Dexamethasone and oleic acid increased SGK1 expression and decreased AKT phosphorylation and insulin receptor substrate expression in adipocytes. Administration of an inhibitor of SGK1 or Lv-shSGK1 reversed the suppression of insulin signalling induced by dexamethasone and oleic acid. SGK1 overexpression increased FoxO1 phosphorylation, and administration of Lv-shSGK1 reversed an increase in FoxO1 phosphorylation induced by dexamethasone and oleic acid. Thus, SGK1 mediates the effect of glucocorticoids and high-fat feeding and induces insulin resistance in adipocytes. Our data suggest that SGK1 is a possible therapeutic target for metabolic syndrome and related complications.

Insulin Resistance the Hinge Between Hypertension and Type 2 Diabetes.

Epidemiological studies have documented a high incidence of diabetes in hypertensive patients.Insulin resistance is defined as a less than expected biologic response to a given concentration of the hormone and plays a pivotal role in the pathogenesis of diabetes. However, over the last decades, it became evident that insulin resistance is not merely a metabolic abnormality, but is a complex and multifaceted syndrome that can also affect blood pressure homeostasis. The dysregulation of neuro-humoral and neuro-immune systems is involved in the pathophysiology of both insulin resistance and hypertension. These mechanisms induce a chronic low grade of inflammation that interferes with insulin signalling transduction. Molecular abnormalities associated with insulin resistance include the defects of insulin receptor structure, number, binding affinity, and/or signalling capacity. For instance, hyperglycaemia impairs insulin signalling through the generation of reactive oxygen species, which abrogate insulin-induced tyrosine autophosphorylation of the insulin receptor. Additional mechanisms have been described as responsible for the inhibition of insulin signalling, including proteasome-mediated degradation of insulin receptor substrate 1/2, phosphatase-mediated dephosphorylation and kinase-mediated serine/threonine phosphorylation of both insulin receptor and insulin receptor substrates. Insulin resistance plays a key role also in the pathogenesis and progression of hypertension-induced target organ damage, like left ventricular hypertrophy, atherosclerosis and chronic kidney disease. Altogether these abnormalities significantly contribute to the increase the risk of developing type 2 diabetes.

Stat3 activation induces insulin resistance via a muscle-specific E3 ubiquitin ligase Fbxo40.

Cellular mechanisms causing insulin resistance (IR) in chronic kidney disease (CKD) are poorly understood. One potential mechanism is that CKD-induced inflammation activates the signal transducer and activator of transcription 3 (Stat3) in muscle. We uncovered increased p-Stat3 in muscles of mice with CKD or mice fed high-fat diet (HFD). Activated Stat3 stimulates the expression of Fbxo40, a muscle-specific E3 ubiquitin ligase that stimulates ubiquitin conjugation leading to degradation of insulin receptor substrate 1 (IRS1). Evidence that Stat3 activates Fbxo40 includes 1) potential Stat3 binding sites in Fbxo40 promoters; 2) Stat3 binding to the Fbxo40 promoter; and 3) constitutively active Stat3 stimulating both Fbxo40 expression and its promoter activity. We found that IL-6 activates Stat3 in myotubes, increasing Fbxo40 expression with reduced IRS1 and p-Akt. Knockdown Fbxo40 using siRNA from myotubes results in higher levels of IRS1 and p-Akt despite the presence of IL-6. We treated mice with a small-molecule inhibitor of Stat3 (TTI-101) and found improved glucose tolerance and insulin signaling in skeletal muscles of mice with CKD or fed an HFD. Finally, we uncovered improved glucose tolerance in mice with muscle-specific Stat3 KO versus results in Stat3f/f mice in response to the HFD. Thus Stat3 activation in muscle increases IR in mice. Inhibition of Stat3 by TTI-101 could be developed into clinical strategies to improve muscle insulin signaling in inflammation and other catabolic diseases.

Triptolide induces atrophy of myotubes by triggering IRS-1 degradation and activating the FoxO3 pathway

Triptolide is an active ingredient isolated from an ancient Chinese herb (Tripterygium wilfordii Hook. f) for inflammatory and immune disorders. It has been shown to inhibit the proliferation of skeletal muscle; however, mechanisms of this effect remain unclear. We used mouse C2C12 myotubes as an in vitro model to investigate the effects of triptolide on skeletal muscle. Triptolide markedly inhibited the expression of myosin heavy chain and upregulated the expression of muscle atrophy-related proteins, leading to atrophy of the myotubes. Triptolide dose-dependently decreased the phosphorylation of Forkhead box O3 (FoxO3) and activated FoxO3 transcription activity, which regulates the expression of muscle atrophy-related proteins. Furthermore, triptolide inhibited the phosphorylation of Akt on the site of S473 and T308, and decreased the phosphorylation of insulin receptor substrate-1 (IRS-1) on the site of S302. In addition, triptolide reduced the protein level, but not mRNA level of IRS-1, whereas other upstream regulators of the Akt signaling pathway were not affected. Finally, a time-course experiment showed that the triptolide-induced degradation of IRS-1 in myotubes occurred 12 h prior to both inhibition of Akt activity and the activation of FoxO3. These data indicate that triptolide triggers IRS-1 degradation to promote FoxO3 activation, which subsequently led to atrophy of myotubes, providing us a potential target to prevent triptolide-induced skeletal muscle atrophy.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A ameliorates insulin resistance in neurons by up-regulating IRS-1 expression

Insulin resistance in the brain is a pathological mechanism that is shared between Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM). Although aberrant expression and phosphorylation of insulin receptor substrate 1 (IRS-1) contribute to insulin resistance, the underlying mechanism remains elusive. In this study, we used several approaches, including adeno-associated virus-based protein overexpression, immunoblotting, immunoprecipitation, immunohistochemistry, and in situ proximal ligation assays, to investigate the function of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) in IRS-1 regulation and the downstream insulin signaling in neurons. We found that DYRK1A overexpression up-regulated IRS-1 expression by slowing turnover of the IRS-1 protein. We further observed that DYRK1A directly interacted with IRS-1 and phosphorylated IRS-1's multiple serine residues. Of note, DYRK1A and IRS-1 were coordinately up-regulated in the prefrontal cortex of db/db mice brain. Furthermore, DYRK1A overexpression ameliorated chronic high insulin-induced insulin resistance in SH-SY5Y cells as well as in primary rat neurons. These findings suggest that DYRK1A protects against insulin resistance in the brain by elevating IRS-1 expression.

Deficiency of the autophagy gene ATG16L1 induces insulin resistance through KLHL9/KLHL13/CUL3-mediated IRS1 degradation

Connections between deficient autophagy and insulin resistance have emerged, however, the mechanism through which reduced autophagy impairs insulin-signaling remains unknown. We examined mouse embryonic fibroblasts lacking Atg16l1 (ATG16L1 KO mouse embryonic fibroblasts (MEFs)), an essential autophagy gene, and observed deficient insulin and insulin-like growth factor-1 signaling. ATG16L1 KO MEFs displayed reduced protein content of insulin receptor substrate-1 (IRS1), pivotal to insulin signaling, whereas IRS1myc overexpression recovered downstream insulin signaling. Endogenous IRS1 protein content and insulin signaling were restored in ATG16L1 KO mouse embryonic fibroblasts (MEF) upon proteasome inhibition. Through proximity-dependent biotin identification (BioID) and co-immunoprecipitation, we found that Kelch-like proteins KLHL9 and KLHL13, which together form an E3 ubiquitin (Ub) ligase complex with cullin 3 (CUL3), are novel IRS1 interactors. Expression of Klhl9 and Klhl13 was elevated in ATG16L1 KO MEFs and siRNA-mediated knockdown of Klhl9, Klhl13, or Cul3 recovered IRS1 expression. Moreover, Klhl13 and Cul3 knockdown increased insulin signaling. Notably, adipose tissue of high-fat fed mice displayed lower Atg16l1 mRNA expression and IRS1 protein content, and adipose tissue KLHL13 and CUL3 expression positively correlated to body mass index in humans. We propose that ATG16L1 deficiency evokes insulin resistance through induction of Klhl9 and Klhl13, which, in complex with Cul3, promote proteasomal IRS1 degradation.

Impact of the Anticancer Drug NT157 on Tyrosine Kinase Signaling Networks.

The small-molecule drug NT157 has demonstrated promising efficacy in preclinical models of a number of different cancer types, reflecting activity against both cancer cells and the tumor microenvironment. Two known mechanisms of action are degradation of insulin receptor substrates (IRS)-1/2 and reduced Stat3 activation, although it is possible that others exist. To interrogate the effects of this drug on cell signaling pathways in an unbiased manner, we have undertaken mass spectrometry-based global tyrosine phosphorylation profiling of NT157-treated A375 melanoma cells. Bioinformatic analysis of the resulting dataset resolved 5 different clusters of tyrosine-phosphorylated peptides that differed in the directionality and timing of response to drug treatment over time. The receptor tyrosine kinase AXL exhibited a rapid decrease in phosphorylation in response to drug treatment, followed by proteasome-dependent degradation, identifying an additional potential target for NT157 action. However, NT157 treatment also resulted in increased activation of p38 MAPK α and γ, as well as the JNKs and specific Src family kinases. Importantly, cotreatment with the p38 MAPK inhibitor SB203580 attenuated the antiproliferative effect of NT157, while synergistic inhibition of cell proliferation was observed when NT157 was combined with a Src inhibitor. These findings provide novel insights into NT157 action on cancer cells and highlight how globally profiling the impact of a specific drug on cellular signaling networks can identify effective combination treatments. Mol Cancer Ther; 17(5); 931-42. ©2018 AACR.

Ubiquitinated CD36 sustains insulin-stimulated Akt activation by stabilizing insulin receptor substrate 1 in myotubes

Both the magnitude and duration of insulin signaling are important in executing its cellular functions. Insulin-induced degradation of insulin receptor substrate 1 (IRS1) represents a key negative feedback loop that restricts insulin signaling. Moreover, high concentrations of fatty acids (FAs) and glucose involved in the etiology of obesity-associated insulin resistance also contribute to the regulation of IRS1 degradation. The scavenger receptor CD36 binds many lipid ligands, and its contribution to insulin resistance has been extensively studied, but the exact regulation of insulin sensitivity by CD36 is highly controversial. Herein, we found that CD36 knockdown in C2C12 myotubes accelerated insulin-stimulated Akt activation, but the activated signaling was sustained for a much shorter period of time as compared with WT cells, leading to exacerbated insulin-induced insulin resistance. This was likely due to enhanced insulin-induced IRS1 degradation after CD36 knockdown. Overexpression of WT CD36, but not a ubiquitination-defective CD36 mutant, delayed IRS1 degradation. We also found that CD36 functioned through ubiquitination-dependent binding to IRS1 and inhibiting its interaction with cullin 7, a key component of the multisubunit cullin-RING E3 ubiquitin ligase complex. Moreover, dissociation of the Src family kinase Fyn from CD36 by free FAs or Fyn knockdown/inhibition accelerated insulin-induced IRS1 degradation, likely due to disrupted IRS1 interaction with CD36 and thus enhanced binding to cullin 7. In summary, we identified a CD36-dependent FA-sensing pathway that plays an important role in negative feedback regulation of insulin activation and may open up strategies for preventing or managing type 2 diabetes mellitus.

IGF-1R signalling contributes to IL-6 production and T cell dependent inflammation in rheumatoid arthritis

BackgroundSignalling through insulin-like growth factor 1 receptor (IGF-1R) is essential for cell survival, but may turn pathogenic in uncontrolled tissue growth in tumours. In rheumatoid arthritis (RA), the IGF-1R signalling is activated and supports expansion of the inflamed synovia. AimIn the present study, we assess if disruption of IGF-1R signalling resolves arthritis. Material and methodsClinical associations of IGF-1R expression in leukocytes of the peripheral blood were studied in 84 RA patients. Consequences of the IGF-1R signalling inhibition for arthritis were studied in mBSA immunised Balb/c mice treated with NT157 compound promoting degradation of insulin receptor substrates. ResultsIn RA patients, high expression of IGF-1R in leukocytes was associated with systemic inflammation as verified by higher expression of NF-kB, serum levels of IL6 and erythrocyte sedimentation rate, and higher pain perception. Additionally, phosphorylated IGF-1R and STAT3 enriched T cells infiltrate in RA synovia. Treatment with NT157 inhibited the phosphorylation of IGF-1R and STAT3 in synovia, and alleviated arthritis and joint damage in mice. It also reduced expression of IGF-1R and despaired ERK and Akt signalling in spleen T cells. This limited IL-6 production, changed RoRgt/FoxP3 balance and IL17 levels. ConclusionIGF-1R signalling contributes to T cell dependent inflammation in arthritis. Inhibition of IGF-1R on the level of insulin receptor substrates alleviates arthritis by restricting IL6-dependent formation of Th17 cells and may open for new treatment strategies in RA.

MG53-IRS-1 (Mitsugumin 53-Insulin Receptor Substrate-1) Interaction Disruptor Sensitizes Insulin Signaling in Skeletal Muscle

Mitsugumin 53 (MG53) is an E3 ligase that interacts with and ubiquitinates insulin receptor substrate-1 (IRS-1) in skeletal muscle; thus, an MG53-IRS-1 interaction disruptor (MID), which potentially sensitizes insulin signaling with an elevated level of IRS-1 in skeletal muscle, is an excellent candidate for treating insulin resistance. To screen for an MID, we developed a bimolecular luminescence complementation system using an N-terminal luciferase fragment fused with IRS-1 and a C-terminal luciferase fragment fused with an MG53 C14A mutant that binds to IRS-1 but does not have E3 ligase activity. An MID, which was discovered using the bimolecular luminescence complementation system, disrupted the molecular association of MG53 with IRS-1, thus abolishing MG53-mediated IRS-1 ubiquitination and degradation. Thus, the MID sensitized insulin signaling and increased insulin-elicited glucose uptake with an elevated level of IRS-1 in C2C12 myotubes. These data indicate that this MID holds promise as a drug candidate for treating insulin resistance.

LPIN1 promotes epithelial cell transformation and mammary tumourigenesis via enhancing insulin receptor substrate 1 stability.

LPIN1 is a protein that exhibits dual functions as a phosphatidic acid phosphatase enzyme in regulation of triglyceride and glycerophospholipid metabolism and a transcriptional coregulator. Through unknown tumour-promoting mechanism, LPIN1 frequently observed in various human cancer cell lines controls main cellular processes involved in cancer progression. Here, we demonstrate that LPIN1 enhances the tumour-promoting function of insulin receptor substrate 1 (IRS1) by controlling IRS1 stability. LPIN1 interacts with IRS1 in an insulin growth factor-1-dependent signalling pathway and inhibits its serine phosphorylation, and thereby eliminating ubiquitin-dependent degradation of IRS1 via proteasomal and lysosomal pathways. Consequently, LPIN1 overexpression increases IRS1 abundance and enhances IRS1's ability to induce epithelial cell proliferation and mammary tumourigenesis. By contrast, depletion or inhibition of LPIN1 in breast cancer cells leads to a decreased IRS1 level, which subsequently inhibits the RAF1-mediated signalling pathway and AP-1 activity. In the syngeneic 4T1 breast cancer model, LPIN1 overexpression increased tumour development, whereas inhibition of LPIN1 and IRS1 suppressed it. Consistent with these observations, LPIN1 levels were positively correlated with IRS1 expression in human breast cancer. Thus, our results indicate a mechanism by which IRS1 expression is increased in breast cancer, and LPIN1 may be a promising drug target for anticancer therapy.

Inhibition of Protein Tyrosine Phosphatase 1B Improves IGF-I Receptor Signaling and Protects Against Inflammation-Induced Gliosis in the Retina.

Insulin-like growth factor-I receptor (IGF-IR) signaling mediates retinal growth and survival and its failure may contribute to aggravate diabetic retinopathy (DR). Protein tyrosine phosphatase 1B (PTP1B) negatively modulates IGF-IR signaling, but its involvement in inflammation during DR remains unknown. We investigated whether PTP1B participates in the cross-talk between proinflammatory signaling pathways and IGF-IR-mediated signaling in the retina. 661W photoreceptors or mouse retinal explants were treated with TNFα, IL6, and IL1β. Insulin-like growth factor-I receptor signaling cascade was evaluated in the absence or presence of PTP1B. db/db mice were used to test a PTP1B inhibitor in retinal gliosis. 661W retinal cells and retinal explants responded to IGF-I by inducing IGF-IR tyrosine (13-fold) and Akt phosphorylations (7- and 3-fold for serine 473 and threonine 308, respectively). Cytokines triggered early activation of stress kinases (c-jun [NH2] terminal kinase [JNK] and p38 MAPK), resulting in insulin receptor substrate 1 (IRS1) serine 307 phosphorylation that precedes its degradation. Pretreatment of 661W cells or retinal explants with cytokines upregulated PTP1B protein levels (1.45- and 4.5-fold, respectively), induced IRS1 degradation and decreased IGF-I-mediated IGF-IR/Akt phosphorylation. Silencing or deficiency in PTP1B ameliorated the negative effects of cytokines on IGF-IR signaling. Cytokines increased glial fibrillary acidic protein (GFAP) expression in retinal explants by 4.5-fold, this response being reduced by 2-fold with a PTP1B inhibitor. Protein tyrosine phosphatase 1B protein levels increased by 3-fold in retinas from db/db mice and its inhibition reduced gliosis. Targeting PTP1B might be useful for modulating IGF-I effects in retinal cells during DR.

Molecular mechanisms of insulin resistance in chronic kidney disease

Insulin resistance refers to reduced sensitivity of organs to insulin-initiated biologic processes that result in metabolic defects. Insulin resistance is common in patients with end-stage renal disease but also occurs in patients with chronic kidney disease (CKD), even when the serum creatinine is minimally increased. Following insulin binding to its receptor, auto-phosphorylation of the insulin receptor is followed by kinase reactions that phosphorylate insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3-kinase (PI3K) and Akt. In fact, low levels of Akt phosphorylation (p-Akt) identifies the presence of the insulin resistance that leads to metabolic defects in insulin-initiated metabolism of glucose, lipids and muscle proteins. Besides CKD, other complex conditions (e.g., inflammation, oxidative stress, metabolic acidosis, aging and excess angiotensin II) reduce p-Akt resulting in insulin resistance. Insulin resistance in each of these conditions is due to activation of different, E3 ubiquitin ligases which specifically conjugate ubiquitin to IRS-1 marking it for degradation in the ubiquitin-proteasome system (UPS). Consequently, IRS-1 degradation suppresses insulin-induced intracellular signaling, causing insulin resistance. Understanding mechanisms of insulin resistance could lead to therapeutic strategies that improve the metabolism of patients with CKD.

N-myristoylated ubiquitin ligase Cbl-b inhibitor prevents on glucocorticoid-induced atrophy in mouse skeletal muscle

A DGpYMP peptide mimetic of tyrosine608-phosphorylated insulin receptor substrate-1 (IRS-1), named Cblin, was previously shown to significantly inhibit Cbl-b-mediated IRS-1 ubiquitination. In the present study, we developed N-myristoylated Cblin and investigated whether it was effective in preventing glucocorticoid-induced muscle atrophy. Using HEK293 cells overexpressing Cbl-b, IRS-1 and ubiquitin, we showed that the 50% inhibitory concentrations of Cbl-b-mediated IRS-1 ubiquitination by N-myristoylated Cblin and Cblin were 30 and 120μM, respectively. Regarding the DEX-induced atrophy of C2C12 myotubes, N-myristoylated Cblin was more effective than Cblin for inhibiting the DEX-induced decreases in C2C12 myotube diameter and IRS-1 degradation. The inhibitory efficacy of N-myristoylated Cblin on IRS-1 ubiquitination in C2C12 myotubes was approximately fourfold larger than that of Cblin. Furthermore, N-myristoylation increased the incorporation of Cblin into HEK293 cells approximately 10-folds. Finally, we demonstrated that N-myristoylated Cblin prevented the wet weight loss, IRS-1 degradation, and MAFbx/atrogin-1 and MuRF-1 expression in gastrocnemius muscle of DEX-treated mice approximately fourfold more effectively than Cblin. Taken together, these results suggest that N-myristoylated Cblin prevents DEX-induced skeletal muscle atrophy in vitro and in vivo, and that N-myristoylated Cblin more effectively prevents muscle atrophy than unmodified Cblin.

Myostatin Induces Insulin Resistance via Casitas B-Lineage Lymphoma b (Cblb)-mediated Degradation of Insulin Receptor Substrate 1 (IRS1) Protein in Response to High Calorie Diet Intake

To date a plethora of evidence has clearly demonstrated that continued high calorie intake leads to insulin resistance and type-2 diabetes with or without obesity. However, the necessary signals that initiate insulin resistance during high calorie intake remain largely unknown. Our results here show that in response to a regimen of high fat or high glucose diets, Mstn levels were induced in muscle and liver of mice. High glucose- or fat-mediated induction of Mstn was controlled at the level of transcription, as highly conserved carbohydrate response and sterol-responsive (E-box) elements were present in the Mstn promoter and were revealed to be critical for ChREBP (carbohydrate-responsive element-binding protein) or SREBP1c (sterol regulatory element-binding protein 1c) regulation of Mstn expression. Further molecular analysis suggested that the increased Mstn levels (due to high glucose or fatty acid loading) resulted in increased expression of Cblb in a Smad3-dependent manner. Casitas B-lineage lymphoma b (Cblb) is an ubiquitin E3 ligase that has been shown to specifically degrade insulin receptor substrate 1 (IRS1) protein. Consistent with this, our results revealed that elevated Mstn levels specifically up-regulated Cblb, resulting in enhanced ubiquitin proteasome-mediated degradation of IRS1. In addition, over expression or knock down of Cblb had a major impact on IRS1 and pAkt levels in the presence or absence of insulin. Collectively, these observations strongly suggest that increased glucose levels and high fat diet, both, result in increased circulatory Mstn levels. The increased Mstn in turn is a potent inducer of insulin resistance by degrading IRS1 protein via the E3 ligase, Cblb, in a Smad3-dependent manner.