Multi-layered proteomics identifies insulin-induced upregulation of the EphA2 receptor via the ERK pathway which is dependent on low IGF1R level.

Insulin resistance impairs the cellular insulin response, and often precedes metabolic disorders, like type 2 diabetes, impacting an increasing number of people globally. Understanding the molecular mechanisms in hepatic insulin resistance is essential for early preventive treatments. To elucidate changes in insulin signal transduction associated with hepatocellular resistance, we employed a multi-layered mass spectrometry-based proteomics approach focused on insulin receptor (IR) signaling at the interactome, phosphoproteome, and proteome levels in a long-term hyperinsulinemia-induced insulin-resistant HepG2 cell line with a knockout of the insulin-like growth factor 1 receptor (IGF1R KO).

Glucose-sensitive insulin with attenuation of hypoglycaemia.

The risk of inducing hypoglycaemia (low blood glucose) constitutes the main challenge associated with insulin therapy for diabetes. Insulin doses must be adjusted to ensure that blood glucose values are within the normal range, but matching insulin doses to fluctuating glucose levels is difficult because even a slightly higher insulin dose than needed can lead to a hypoglycaemic incidence, which can be anything from uncomfortable to life-threatening. It has therefore been a long-standing goal to engineer a glucose-sensitive insulin that can auto-adjust its bioactivity in a reversible manner according to ambient glucose levels to ultimately achieve better glycaemic control while lowering the risk of hypoglycaemia.

Preclinical exploration of combined glucagon inhibition and liver-preferential insulin for treatment of diabetes using in vitro assays and rat and mouse models.

Aims/hypothesis: Normalisation of blood glucose in individuals with diabetes is recommended to reduce development of diabetic complications. However, risk of severe hypoglycaemia with intensive insulin therapy is a major obstacle that prevents many individuals with diabetes from obtaining the recommended reduction in HbA. Inhibition of glucagon receptor signalling and liver-preferential insulin action have been shown individually to have beneficial effects in preclinical models and individuals with diabetes (i.

Molecular Engineering of Efficacious Mono-Valent Ultra-Long Acting Two-Chain Insulin-Fc Conjugates.

Here, we describe molecular engineering of monovalent ultra-long acting two-chain insulin-Fc conjugates. Insulin-Fc conjugates were synthesized using trifunctional linkers with one amino reactive group for reaction with a lysine residue of insulin and two thiol reactive groups used for re-bridging of a disulfide bond within the Fc molecule. The ultra-long pharmacokinetic profile of the insulin-Fc conjugates was the result of concertedly slowing insulin receptor-mediated clearance by (1) introduction of amino acid substitutions that lowered the insulin receptor affinity and (2) conjugating insulin to the Fc element.

Insulin signalling in tanycytes gates hypothalamic insulin uptake and regulation of AgRP neuron activity.

Insulin acts on neurons and glial cells to regulate systemic glucose metabolism and feeding. However, the mechanisms of insulin access in discrete brain regions are incompletely defined. Here we show that insulin receptors in tanycytes, but not in brain endothelial cells, are required to regulate insulin access to the hypothalamic arcuate nucleus.

Impaired glucocorticoid receptor expression in liver disrupts feeding-induced gene expression, glucose uptake, and glycogen storage.

The transition from a fasted to a fed state is associated with extensive transcriptional remodeling in hepatocytes facilitated by hormonal- and nutritional-regulated transcription factors. Here, we use a liver-specific glucocorticoid receptor (GR) knockout (L-GRKO) model to investigate the temporal hepatic expression of GR target genes in response to feeding. Interestingly, in addition to the well-described fasting-regulated genes, we identify a subset of hepatic feeding-induced genes that requires GR for full expression.

Molecular and pharmacological characterization of insulin icodec: a new basal insulin analog designed for once-weekly dosing.

Introduction: Insulin icodec is a novel, long-acting insulin analog designed to cover basal insulin requirements with once-weekly subcutaneous administration. Here we describe the molecular engineering and the biological and pharmacological properties of insulin icodec.

Research Design And Methods: A number of in vitro assays measuring receptor binding, intracellular signaling as well as cellular metabolic and mitogenic responses were used to characterize the biological properties of insulin icodec.

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots.

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFRβ+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice.

Cross-species reactive monoclonal antibodies against the extracellular domains of the insulin receptor and IGF1 receptor.

Translation across species of immunoassay results is often challenging due to the lack of cross-species reactivity of antibodies. In order to investigate the biology of insulin and IGF1 receptors, we generated new versatile monoclonal assay antibodies using the extracellular domain of the insulin/IGF1 hybrid receptor as the bait protein in the Adimab yeast antibody discovery platform and as the antigen in a rabbit monoclonal antibody platform. The resulting antibody clones were screened for receptor specificity as well as cross-species reactivity to both tissue and cell line derived samples.

The insulin receptor is expressed and functional in cultured blood-brain barrier endothelial cells but does not mediate insulin entry from blood to brain.

Insulin and its receptor are known to be present and functional in the brain. Insulin cerebrospinal fluid concentrations have been shown to correlate with plasma levels of insulin in a nonlinear fashion, indicative of a saturable transport pathway from the blood to the brain interstitial fluid. The aim of the present study was to investigate whether insulin was transported across brain endothelial cells in vitro via an insulin receptor-dependent pathway.

Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin.

A specific covalently linked dimeric species of insulin high molecular weight products (HMWPs), formed during prolonged incubation of a neutral pharmaceutical formulation of human insulin, were characterized in terms of tertiary structure, self-association, biological activity, and fibrillation properties. The dimer was formed by a covalent link between A21Asn and B29Lys. It was analyzed using static and dynamic light scattering and small-angle X-ray scattering to evaluate its self-association behavior.

Liver X receptor regulates hepatic nuclear O-GlcNAc signaling and carbohydrate responsive element-binding protein activity.

Liver X receptor (LXR)α and LXRβ play key roles in hepatic de novo lipogenesis through their regulation of lipogenic genes, including sterol regulatory element-binding protein (SREBP)-1c and carbohydrate responsive element-binding protein (ChREBP). LXRs activate lipogenic gene transcription in response to feeding, which is believed to be mediated by insulin. We have previously shown that LXRs are targets for glucose-hexosamine-derived O-linked β-N-acetylglucosamine (O-GlcNAc) modification enhancing their ability to regulate SREBP-1c promoter activity in vitro.

Treatment of diabetic rats with insulin or a synthetic insulin receptor agonist peptide leads to divergent metabolic responses.

In addition to lowering of blood glucose, treatment with insulin also induces lipid synthesis and storage. Patients with type 2 diabetes often suffer from lipid-related comorbidities including dyslipidemia, obesity, and fatty liver disease. We examined here in two separate studies changes in lipid dynamics in Zucker diabetic fatty (ZDF) rats, in response to 7 days of treatment with either insulin or the insulin receptor agonist peptide S597.

Adipose weight gain during chronic insulin treatment of mice results from changes in lipid storage without affecting de novo synthesis of palmitate.

Insulin treatment is associated with increased adipose mass in both humans and mice. However, the underlying dynamic basis of insulin induced lipid accumulation in adipose tissue remains elusive. To assess this, young female C57BL6/J mice were fed a low fat diet for 3 weeks, treated subsequently with 7 days of constant subcutaneous insulin infusion by osmotic minipumps and compared to mice with only buffer infused.

Agonism and antagonism at the insulin receptor.

Insulin can trigger metabolic as well as mitogenic effects, the latter being pharmaceutically undesirable. An understanding of the structure/function relationships between insulin receptor (IR) binding and mitogenic/metabolic signalling would greatly facilitate the preclinical development of new insulin analogues. The occurrence of ligand agonism and antagonism is well described for G protein-coupled receptors (GPCRs) and other receptors but in general, with the exception of antibodies, not for receptor tyrosine kinases (RTKs).

Insulin analog with additional disulfide bond has increased stability and preserved activity.

Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use.

Phosphorylation of serine 248 of C/EBPα is dispensable for myelopoiesis but its disruption leads to a low penetrant myeloid disorder with long latency.

Background: Transcription factors play a key role in lineage commitment and differentiation of stem cells into distinct mature cells. In hematopoiesis, they regulate lineage-specific gene expression in a stage-specific manner through various physical and functional interactions with regulatory proteins that are simultanously recruited and activated to ensure timely gene expression. The transcription factor CCAAT/enhancer binding protein α (C/EBPα) is such a factor and is essential for the development of granulocytic/monocytic cells.

Novel covalently linked insulin dimer engineered to investigate the function of insulin dimerization.

An ingenious system evolved to facilitate insulin binding to the insulin receptor as a monomer and at the same time ensure sufficient stability of insulin during storage. Insulin dimer is the cornerstone of this system. Insulin dimer is relatively weak, which ensures dissociation into monomers in the circulation, and it is stabilized by hexamer formation in the presence of zinc ions during storage in the pancreatic β-cell.

Identification of anchor points for chemical modification of a small cysteine-rich protein by using a cysteine scan.

Chemical modifications of proteins are increasingly important in the development of protein drugs with fine-tuned properties. Regioselective modification, such as the chemoselective alkylation of an unpaired cysteine residue, is a prerequisite for obtaining homogenous protein products. The introduction of an unpaired Cys into the Cys-rich protein, insulin, was investigated by using a Cys scan.

Characterization of murine melanocortin receptors mediating adipocyte lipolysis and examination of signalling pathways involved.

The melanocortin receptors (MCRs) belong to the G-protein coupled receptors (family A). So far, 5 different subtypes have been described (MC1R-MC5R) and of these MC2R and MC5R have been proposed to act directly in adipocytes and regulate lipolysis in rodents. Using ACTH and α-melanocyte stimulating hormone (α-MSH) generated from proopiomelanocortin (POMC), as well as synthetic MSH analogues to stimulate lipolysis in murine 3T3-L1 adipocytes it is shown that MC2R and MC5R are lipolytic mediators in differentiated 3T3-L1 adipocytes.

Rexinoid bexarotene modulates triglyceride but not cholesterol metabolism via gene-specific permissivity of the RXR/LXR heterodimer in the liver.

Objective: Bexarotene (Targretin) is a clinically used antitumoral agent which exerts its action through binding to and activation of the retinoid-X-receptor (RXR). The most frequent side-effect of bexarotene administration is an increase in plasma triglycerides, an independent risk factor of cardiovascular disease. The molecular mechanism behind this hypertriglyceridemia remains poorly understood.

Genome-wide profiling of PPARgamma:RXR and RNA polymerase II occupancy reveals temporal activation of distinct metabolic pathways and changes in RXR dimer composition during adipogenesis.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is a key regulator of adipocyte differentiation in vivo and ex vivo and has been shown to control the expression of several adipocyte-specific genes. In this study, we used chromatin immunoprecipitation combined with deep sequencing to generate genome-wide maps of PPARgamma and retinoid X receptor (RXR)-binding sites, and RNA polymerase II (RNAPII) occupancy at very high resolution throughout adipocyte differentiation of 3T3-L1 cells. We identify >5000 high-confidence shared PPARgamma:RXR-binding sites in adipocytes and show that during early stages of differentiation, many of these are preoccupied by non-PPARgamma RXR-heterodimers.

Modeling of C/EBPalpha mutant acute myeloid leukemia reveals a common expression signature of committed myeloid leukemia-initiating cells.

Mutations in the CEBPA gene are present in 7%-10% of human patients with acute myeloid leukemia (AML). However, no genetic models exist that demonstrate their etiological relevance. To mimic the most common mutations affecting CEBPA-that is, those leading to loss of the 42 kDa C/EBPalpha isoform (p42) while retaining the 30kDa isoform (p30)-we modified the mouse Cebpa locus to express only p30.