Insulin's Discovery: New Insights on Its Hundredth Birthday: From Insulin Action and Clearance to Sweet Networks.

In 2021, the 100th anniversary of the isolation of insulin and the rescue of a child with type 1 diabetes from death will be marked. In this review, we highlight advances since the ingenious work of the four discoverers, Frederick Grant Banting, John James Rickard Macleod, James Bertram Collip and Charles Herbert Best. Macleoad closed his Nobel Lecture speech by raising the question of the mechanism of insulin action in the body. This challenge attracted many investigators, and the question remained unanswered until the third part of the 20th century. We summarize what has been learned, from the discovery of cell surface receptors, insulin action, and clearance, to network and precision medicine.

Network medicine-travelling with the insulin receptor: Encounter of the second type.

Important progress has been made in understanding many aspects of insulin action in the last 10 years. Attention will be focused here on the physical protein interaction network of the internalized insulin receptor (IR) and its relationships with the genetic architecture of type 2 diabetes mellitus (T2D). The IR recognizes signals from the outside (circulating insulin) and engages the insulin signaling response. Within seconds, the IR is also involved in insulin internalization and its subsequent degradation in endosomes (physiological clearance of insulin). A T2D disease module sharing functional similarities with insulin secretion in pancreatic islets was recently identified in the close neighborhood of the internalized IR in liver. This module brought a new light on the apparent functional heterogeneity of numerous genes at risk to T2D by linking them to a few noncanonical layers of signaling feedback loops. These findings should be translated into a better understanding of the primary mechanisms of the disease and consequently a more precise sub-classification of T2D, ultimately leading to precision medicine and the development of new therapeutical drugs.

A type 2 diabetes disease module with a high collective influence for Cdk2 and PTPLAD1 is localized in endosomes.

Despite the identification of many susceptibility genes our knowledge of the underlying mechanisms responsible for complex disease remains limited. Here, we identified a type 2 diabetes disease module in endosomes, and validate it for functional relevance on selected nodes. Using hepatic Golgi/endosomes fractions, we established a proteome of insulin receptor-containing endosomes that allowed the study of physical protein interaction networks on a type 2 diabetes background. The resulting collated network is formed by 313 nodes and 1147 edges with a topology organized around a few major hubs with Cdk2 displaying the highest collective influence. Overall, 88% of the nodes are associated with the type 2 diabetes genetic risk, including 101 new candidates. The Type 2 diabetes module is enriched with cytoskeleton and luminal acidification-dependent processes that are shared with secretion-related mechanisms. We identified new signaling pathways driven by Cdk2 and PTPLAD1 whose expression affects the association of the insulin receptor with TUBA, TUBB, the actin component ACTB and the endosomal sorting markers Rab5c and Rab11a. Therefore, the interactome of internalized insulin receptors reveals the presence of a type 2 diabetes disease module enriched in new layers of feedback loops required for insulin signaling, clearance and islet biology.

Annexin A2 is SUMOylated on its N-terminal domain: regulation by insulin.

Insulin receptor (IR) endocytosis requires a remodelling of the actin cytoskeleton. We show here that ANXA2 is SUMOylated at the K10 located in a non-consensus SUMOylation motif in the N-terminal domain. The Y24F mutation decreased the SUMOylation signal, whereas insulin stimulation increased ANXA2 SUMOylation. A survey of protein SUMOylation in hepatic Golgi/endosome (G/E) fractions after insulin injections revealed the presence of a SUMOylation pattern and confirmed the SUMOylation of ANXA2. The construction of an IR/ANXA2/SUMO network (IRASGEN) in the G/E context reveals the presence of interacting nodes whereby SUMO1 connects ANXA2 to actin and microtubule-mediated changes in membrane topology. Heritable variants associated with type 2 diabetes represent 41% of the IRASGEN thus pointing out the physio-pathological importance of this subnetwork.

The last enzyme of the de novo purine synthesis pathway 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC) plays a central role in insulin signaling and the Golgi/endosomes protein network.

Insulin is internalized with its cognate receptor into the endosomal apparatus rapidly after binding to hepatocytes. We performed a bioinformatic screen of Golgi/endosome hepatic protein fractions and found that ATIC, which is a rate-limiting enzyme in the de novo purine biosynthesis pathway, and PTPLAD1 are associated with insulin receptor (IR) internalization. The IR interactome (IRGEN) connects ATIC to AMPK within the Golgi/endosome protein network (GEN). Forty-five percent of the IR Golgi/endosome protein network have common heritable variants associated with type 2 diabetes, including ATIC and AMPK. We show that PTPLAD1 and AMPK are rapidly compartmentalized within the plasma membrane (PM) and Golgi/endosome fractions after insulin stimulation and that ATIC later accumulates in the Golgi/endosome fraction. Using an in vitro reconstitution system and siRNA-mediated partial knockdown of ATIC and PTPLAD1 in HEK293 cells, we show that both ATIC and PTPLAD1 affect IR tyrosine phosphorylation and endocytosis. We further show that insulin stimulation and ATIC knockdown readily increase the level of AMPK-Thr172 phosphorylation in IR complexes. We observed that IR internalization was markedly decreased after AMPKα2 knockdown, and treatment with the ATIC substrate AICAR, which is an allosteric activator of AMPK, increased IR endocytosis in cultured cells and in the liver. These results suggest the presence of a signaling mechanism that senses adenylate synthesis, ATP levels, and IR activation states and that acts in regulating IR autophosphorylation and endocytosis.

Preclinical evaluation of dipotassium bisperoxo (picolinato) oxovanadate V for the treatment of pediatric low-grade gliomas.

AIM: The treatment of pediatric low-grade gliomas with current treatment modalities still remains ineffective among a subset of patients; hence, justifying the need to further investigate more effective therapies. Dipotassium bisperoxo (picolinato) oxovanadate V (Bpv[pic]), is a derivative of the trace metal vanadium and a potent inhibitor of protein tyrosine phosphatases, which are important mediators of oncogenic and tumor suppressive activities in cancers. In this study, we undertook a preclinical evaluation of the antineoplastic functions of Bpv(pic) in the treatment of pediatric low-grade gliomas. MATERIALS & METHODS: We utilized pediatric low-grade glioma cell lines (Res186, Res259 and R286) in a wide variety of cancer assays to determine whether Bpv(pic) can abrogate the neoplastic properties of these cells. RESULTS: Our preclinical evaluation of the antineoplastic properties of Bpv(pic) in pediatric low-grade gliomas reveals a significant dose-dependent decrease in cell viability as a consequence of decreased proliferation and sustained induction of growth arrest and apoptosis. Bpv(pic) significantly decreases cell migration/invasion and anchorage-independent growth in soft agarose. Within cells, Bpv(pic) functions by attenuating CDC25A activity, and by decreasing the expression of multiple protein tyrosine phosphatases, DNA repair genes, microtubule-associated genes, such as PLK1, AURKA and HDAC6, and conversely augmenting the expression of proapoptotic mediators such as BAK, AIFM and CTSL1. CONCLUSION: Collectively, our data strongly suggest novel evidence of Bpv(pic) being a potent antineoplastic drug and a suitable alternative for the treatment of pediatric low-grade gliomas.

Annexin A1 is regulated by domains cross-talk through post-translational phosphorylation and SUMOYlation.

Mouse prostate membrane-associated proteins of the annexin family showed changes in SUMOylation during androgen treatment. Among these the calcium-binding annexin A1 protein (ANXA1) was chosen for further characterization given its role in protein secretion and cancer. SUMOylation of ANXA1 was confirmed by overexpressing SUMO-1 in LNCaP cells. Site-directed mutagenesis indicated that K257 located in a SUMOylation consensus motif in the C-terminal calcium-binding DA3 repeat domain is SUMOylated. Mutation of the N-terminal Y21 decreased markedly the SUMOylation signal while EGF stimulation increased ANXA1 SUMOylation. A structural analysis of ANXA1 revealed that K257 is located in a hot spot where Ca(2+) and SUMO-1 bind and where a nuclear export signal and a polyubiquitination site are also present. Also, Y21 is buried inside an α-helix structure in the Ca(2+)-free conformation implying that Ca(2+) binding, and the subsequent expelling of the N-terminal α-helix in a disordered conformation, is permissive for its phosphorylation. These results show for the first time that SUMOylation can be regulated by an external signal (EGF) and indicate the presence of a cross-talk between the N-terminal and C-terminal domains of ANXA1 through post-translational modifications.

Oleodaphnoic acid and coriaceol, two new natural products from the stem bark of Wikstroemia coriacea.

Fractionation of the chloroform extract of Wikstroemia coriacea led to the isolation of two new compounds, oleodaphnoic acid (1), a guaiane-type sesquiterpenoid, and coriaceol (2), an 1,5-diphenyl-1-pentanone analogue, together with nine known compounds. The structures of 1 and 2 were elucidated by extensive spectroscopic data analysis. The known compounds were oleodaphnal (3), indicanone (4), (5R,8R,8aR)-3,8-dimethyl-4,5,6,7,8,8a-hexahydro-5-(1-methylethenyl)-2(1H)-azulenone, (5), 1,5 diphenyl-1-pentanone (6), (+)-3-hydroxy-1,5-diphenyl-1-pentanone (7), umbelliferone (8), daphnoretin (9), ß-sitostenone (10) and (-)-hinokinin (11).

Keratin 8/18 regulation of glucose metabolism in normal versus cancerous hepatic cells through differential modulation of hexokinase status and insulin signaling.

As differentiated cells, hepatocytes primarily metabolize glucose for ATP production through oxidative phosphorylation of glycolytic pyruvate, whereas proliferative hepatocellular carcinoma (HCC) cells undergo a metabolic shift to aerobic glycolysis despite oxygen availability. Keratins, the intermediate filament (IF) proteins of epithelial cells, are expressed as pairs in a lineage/differentiation manner. Hepatocyte and HCC (hepatoma) cell IFs are made solely of keratins 8/18 (K8/K18), thus providing models of choice to address K8/K18 IF functions in normal and cancerous epithelial cells. Here, we demonstrate distinctive increases in glucose uptake, glucose-6-phosphate formation, lactate release, and glycogen formation in K8/K18 IF-lacking hepatocytes and/or hepatoma cells versus their respective IF-containing counterparts. We also show that the K8/K18-dependent glucose uptake/G6P formation is linked to alterations in hexokinase I/II/IV content and localization at mitochondria, with little effect on GLUT1 status. In addition, we find that the insulin-stimulated glycogen formation in normal hepatocytes involves the main PI-3 kinase-dependent signaling pathway and that the K8/K18 IF loss makes them more efficient glycogen producers. In comparison, the higher insulin-dependent glycogen formation in K8/K18 IF-lacking hepatoma cells is associated with a signaling occurring through a mTOR-dependent pathway, along with an augmentation in cell proliferative activity. Together, the results uncover a key K8/K18 regulation of glucose metabolism in normal and cancerous hepatic cells through differential modulations of mitochondrial HK status and insulin-mediated signaling.

Proteomic analysis identifies new biomarkers for postmenopausal and dry skin.

A proteomic analysis of stratum corneum (SC) samples of normal healthy skin revealed the presence of more than 70 proteins by 2D electrophoresis. The majority of these proteins to our knowledge have not yet been described in normal SC. We analysed by Western blot the levels of 25 proteins in the SC taken from postmenopausal and dry skin compared with young and normal skin, respectively. In postmenopausal skin, there was a significantly increased amount of heat shock protein 27, plakoglobin and desmoglein 1, whereas transglutaminase 3, apolipoprotein D and acid ceramidase levels were significantly reduced compared with the SC of young skin. We confirmed corneodesmosin as a marker of dry skin. In addition, we showed for the first time that the levels of both phosphatidylethanolamine-binding protein 1 and annexin A2 were significantly increased in the SC of dry skin compared with the SC of normal skin. These results suggest that a proteomic analysis of the SC obtained using a non-invasive varnish stripping method is an attractive alternative to invasive methods to better characterize changes in the physiology of ageing and dry skin.

Compartmentalized CDK2 is connected with SHP-1 and ß-catenin and regulates insulin internalization.

The cyclin-dependant kinase Cdk2 is compartmentalized in endosomes but its role is poorly understood. Here we show that Cdk2 present in hepatic endosome fractions is strictly located in a Triton X-100-resistant environment. The endosomal Cdk2 was found to be associated with the protein tyrosine phosphatase SHP-1, a regulator of insulin clearance, and the actin anchor ß-catenin, a known substrate for both Cdk2 and SHP-1. In the plasma membranes and endosome fractions, ß-catenin is associated with CEACAM1, also known as regulator of insulin clearance. We show that ß-catenin, not CEACAM1, is a substrate for Cdk2. Partial down-modulation of Cdk2 in HEK293 cells increased the rate of insulin internalization. These findings reveal that Cdk2 functions, at least in part, via a Cdk2/SHP-1/ß-catenin/CEACAM1 axis, and show for the first time that Cdk2 has the capacity to regulate insulin internalization.

Synthesis and complete assignment of the 1H and 13C NMR signals of new acetamido and aminoflavonoid derivatives.

The complete (1)H and (13)C NMR assignment of 9 acetamidochalcones, 18 acetamidoflavones, 18 aminoflavones, 9 acetamidoflavonols and 9 aminoflavonols has been performed using one- and two-dimensional NMR techniques including COSY, HMQC and HMBC experiments.

Antimalarial compounds from the aerial parts of Flacourtia indica (Flacourtiaceae).

AIM OF THE STUDY: In the Comoros Islands, the aerial parts of Flacourtia indica are used in traditional medicine to treat malaria. Because of the important use of this plant, the phytochemistry of the aerial parts was investigated. MATERIALS AND METHODS: Three compounds were isolated from the decoction of this plant material, pyrocatechol, homaloside D and poliothrysoside. The in vitro antiplasmodial activity on the chloroquine-resistant strain (W2) of Plasmodium falciparum and the cytotoxicity on two complementary human cell lines (THP1, HepG2), of AcOEt extract obtained after liquid/liquid extraction of the decoction and pure compounds, were evaluated. RESULTS: The poliothrysoside isolated from the AcOEt extract presented a strong antiplasmodial activity (IC(50)=7.4 microM) and a good selectivity index (>28) similar to chloroquine. CONCLUSION: This study reports for the first time antiplasmodial activity for Flacourtia indica, for its AcOEt extract and the three major constituents and confirms its traditional use.

Src-family kinase signaling, actin-mediated membrane trafficking and organellar dynamics in the control of cell fate: lessons to be learned from the adenovirus E4orf4 death factor.

Evidence has accumulated that there are different modes of regulated cell death, which share overlapping signaling pathways. Cytoskeletal-dependent inter-organellar communication as a result of protein and lipid trafficking in and out of organelles has emerged as a common, key issue in the regulation of cell death modalities. The movement of proteins and lipids between cell compartments is believed to relay death signals in part through modifications of organelles dynamics. Little is known, however, regarding how trafficking is integrated within stress signaling pathways directing organelle-specific remodeling events. In this review, we discuss emerging evidence supporting a role for regulated changes in actin dynamics and intracellular membrane flow. Based on recent findings using the adenovirus E4orf4 death factor as a probing tool to tackle the mechanistic underpinnings that control alternative modes of cell death, we propose the existence of multifunctional platforms at the endosome-Golgi interface regulated by SFK-signaling. These endosomal platforms could be mobilized during cell activation processes to reorganize cellular membranes and promote inter-organelle signaling.

Proteomic analysis of Src family kinases signaling complexes in Golgi/endosomal fractions using a site-selective anti-phosphotyrosine antibody: identification of LRP1-insulin receptor complexes.

A role for Src Family Kinases (SFKs) in the dynamics of endocytic and secretory pathways has previously been reported. Identification of low-abundance compartmentalized complexes still remains challenging, highlighting the need for novel tools. Here we describe analysis of SFK-signaling complexes of hepatic Golgi/endosomes (G/E) fractions by sequential affinity enrichment of proteins. Mouse G/E permeabilized membranes were first validated in terms of electron microscopy, 1-D electrophoresis (1-DE), insulin-mediated endocytosis and protein content. With the use of quantitative N-terminal labeling of tryptic peptides (iTRAQ), 1-DE and IEF tryptic peptides separation methods, a total of 666 proteins were identified, including the SFK Lyn. Following insulin injection, a series of proteins were recognized by an anti-phosphotyrosine antibody (alpha P42-2) raised against the residue most frequently phosphorylated by SFK on the adenoviral protein E4orf4 and that cross-reacts with endosomal SFK targets. By using affinity chromatography coupled with mass spectrometry, we identified 16 proteins classified as (1) recycling receptors, (2) vesicular trafficking proteins, (3) actin network proteins, (4) metabolism proteins, or (5) signaling proteins. One of these proteins, low density lipoprotein-related protein 1 (LRP1), which is a known SFK substrate, was found to associate with the internalized insulin receptor (IR), suggesting the presence of a co-internalization process. The identification of these proteomes should, thus, contribute to a better understanding of the molecular mechanisms that regulate trafficking events and insulin clearance.

Michael addition initiated carbocyclization sequences with nitroolefins for the stereoselective synthesis of functionalized heterocyclic and carbocyclic systems.

The synthesis of various heterocycles and carbocycles (tetrahydrofurans, pyrrolidines, cyclopentanes) has been achieved by using new and efficient ionic addition/cyclization sequences. Nitroolefins play an important role in the Michael addition induced ring-closing reactions (MIRC) reported in the present article, with various substituted alcohols, amines, Grignard reactants, or malonate derivatives acting as the nucleophile partner. The optimized cascade reactions were high yielding in most cases and highly stereoselective, creating up to three stereogenic centers starting from achiral substrates.

Fagraldehyde, a secoiridoid isolated from Fagraea fragrans.

A secoiridoid aglycone with an atypical skeleton, named fagraldehyde (1), together with several known secoiridoids (gentiopicroside (2), sweroside (3), and swertiamarin (4)) were isolated from the bark and leaves of Fagraea fragrans collected in Cambodia. The conformations of 1 were evaluated on the basis of molecular modeling and NOESY correlations. A hypothetical biogenesis of fagraldehyde was proposed to explain the unusual skeleton. Compound 1 was weakly active in vitro against Plasmodium falciparum.

Proteomic characterization of mouse cytosolic and membrane prostate fractions: high levels of free SUMO peptides are androgen-regulated.

The prostate is a relatively homogeneous tissue that is highly specialized in synthetic and secretory functions. The frequency of malignant growth explains its great clinical significance. We used here a combination of subcellular fractionation, 1-DE (one-dimensional gel electrophoresis) protein separation and mass spectrometry, to establish a prostate protein expression profile in mice. Analysis of proteins present in cytosolic (C) and membrane (P) prostate fractions led to the identification of 619 distinct proteins. A majority of abundant proteins were found to compose the metabolism and protein synthesis machinery. Those identified also correspond to known endoplasmic reticulum and Golgi residents, chaperones and anterograde cargos. They included a series of proteins involved in exocytic/endocytic trafficking. Among the signaling proteins, we identified the ubiquitin-like peptides smt3. We showed that both free small ubiquitin-related modifier SUMO-2/3 and SUMO-1 levels are subject to tight control by the androgen 5alpha-dihydrotestosterone (DHT). By contrast with SUMO-2/3, free SUMO-1 peptides are particularly abundant in the prostate when compared with other tissues. Therefore, we report prostate protein expression profiles of cytosolic and membrane fractions in mice. Our data suggest that the identified free SUMO peptides play an important role in this secretory tissue.

Marine bifunctional sphingolipids from the sponge Oceanapia ramsayi.

During the course of our continuing studies on marine natural lipid products,two known sphingolipids have been isolated for the first time from a specimen of the marine sponge Oceanapia ramsayi collected at Itampolo on the west coast of Madagascar in the Indian Ocean. The structures were elucidated using NMR data and by comparison with literature data. The occurrence of these sphingolipids within other Oceanapia spp. is discussed.

Protein tyrosine phosphatase inhibition induces anti-tumor activity: evidence of Cdk2/p27 kip1 and Cdk2/SHP-1 complex formation in human ovarian cancer cells.

The protein tyrosine phosphatase (PTP) superfamily of enzymes functions with protein tyrosine kinases to regulate a broad spectrum of fundamental physiological processes. Addition of the PTP inhibitor potassium bisperoxo(1,10-phenanthroline)oxo-vanadate(V) [bpV(phen)] to the culture medium of human ovarian cancer cells (OVCAR-3) resulted in a dose-dependent decrease in the formation of tumors in a 3-D culture system. An evaluation of the potency of bpV(phen) in vivo confirmed the anti-tumor activity. Further study of the mechanism of action revealed a 40% decrease in Cdk2 kinase activity, an elevated level of Cdk2/p27(kip1), and the appearance of Cdk2/SHP-1 complexes. Therefore, a cytostatic dose of a PTP inhibitor increases the intracellular levels of Cdk2/p27(kip) and Cdk2/SHP-1 complexes, which indicate the presence of additional mechanisms underlying the anti-tumor activity.