Insulin-receptor Complex Research Articles

Specific interaction of insulin receptor and GLP-1 receptor mediates crosstalk between their signaling

Both GLP-1 receptor (GLP-1R) and insulin receptor (IR) transmit signals for insulin release and/or cellular metabolism although using distinct sets of transducing molecules and separate pathways. We proposed a possible association of IR and GLP-1R, since they are coexpressed in diverse tissues including the pancreatic β-cells and crosstalk between their signaling was frequently reported. We showed a specific interaction between GLP-1R and IR which was independent of intracellular receptor domains and not responsive to ligand binding. In signaling, the IRS-1 was coupled more to GLP-1R and less to IR in the receptor complex at IR activation, with subsequent IRS-1 degradation suppressed rather than its activation inhibited. The Gsα recruitment to the activated GLP-1R was inhibited in the GLP-1R/IR complex, with the signaling in cAMP pathway suppressed at IR activation. Therefore, the identified GLP-1R/IR complex recruits their signaling molecules which are differentially modified, leading to a crosstalk between their signaling.

Codon-optimized FAM132b gene therapy prevents dietary obesity by blockading adrenergic response and insulin action

FAM132b (myonectin) has been identified as a muscle-derived myokine with exercise and has hormone activity in circulation to regulate iron homeostasis and lipid metabolism via unknown receptors. Here, we aim to explore the potential of adeno-associated virus to deliver FAM132b in vivo to develop a gene therapy against obesity. Adeno-associated virus AAV9 were engineered to induce overexpression of FAM132b with two mutations, A136T and P159A. Then, AAV9 was delivered into high-fat diet mice through tail vein, and glucose homeostasis and obesity development of mice were observed. Methods of structural biology were used to predict the action site or receptor of the FAM132b mutant. Treatment of high-fat diet-fed mice with AAV9 improved glucose intolerance and insulin resistance, and resulted in reductions in body weight, fat depot, and adipocyte size. Codon-optimized FAM132b (coFAM132b) reduced the glycemic response to epinephrine (EPI) in the whole body and increased the lipolytic response to EPI in adipose tissues. However, FAM132b knockdown by shRNA significantly increased the glycemic response to EPI in vivo and reduced adipocyte response to EPI and adipose tissue browning. Structural analysis predicted that the FAM132b mutant with A136T and P159A may form a weak bond with β2 adrenergic receptor (ADRB2) and may have more affinity for insulin and insulin-receptor complexes. Our study underscores the potential of FAM132b gene therapy with codon optimization to treat obesity by modulating the adrenergic response and insulin action. Both structural biological analysis and in vivo experiments suggest that the adrenergic response and insulin action are most likely blockaded by FAM132b mutants.

Splitting Hairs: Folliculin Parts the Good From the Bad in mTORC1 Control Over Lipid Metabolism

Gosis BS, Wada S, Thorsheim C, et al. Inhibition of nonalcoholic fatty liver disease in mice by selective inhibition of mTORC1. Science 2022;376(6590):eabf8271. The mechanistic target of rapamycin complex 1 (mTORC1) is a signaling kinase complex that mediates several fundamental processes, including de novo lipogenesis. Therefore, mTORC1 is an attractive target to treat nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). However, a barrier to effectively target mTORC1 is an incomplete understanding of how mTORC1 regulates liver fat metabolism. To illustrate this point, both hyperactivating and inactivating mTORC1 signaling attenuates de novo lipogenesis and hepatic fat accumulation in mice. What is known is that mTORC1 activates de novo lipogenesis by phosphorylating 2 primary downstream effectors—p70 S6 kinase (P70S6K) and transcription factor E3 (TFE3)—which both promote de novo lipogenesis. At the same time, these de novo lipogenesis–promoting actions of mTORC1 are counterbalanced by an important negative feedback loop; mTORC1-P70S6K signaling inhibits the insulin receptor complex to attenuate further anabolic insulin signaling. Therefore, selective targeting of mTORC1 through TFE3, which prevents de novo lipogenesis but does not break the P70S6K anabolic insulin signaling feedback loop, could be an ideal target for NAFLD. Gosis and colleagues provided exciting new data to unify prior disparate observations and identify a potential therapeutic target for NAFLD. They identified the GTPase-activating protein folliculin (FLCN) as a factor that confers mTORC1 substrate specificity for TFE3, but not p70S6K. Using a variety of diet-induced models for NAFLD/NASH, they showed that liver-specific FLCN deletion blocked mTORC1-mediated TFE3 phosphorylation, thereby promoting lipid catabolic gene transcription. However, FLCN deletion preserved the insulin-mediated P70S6K-negative feedback loop, thereby reducing further anabolic insulin signaling. In these diet-induced NAFLD/NASH models, FLCN deletion promoted fat oxidation and prevented fat accumulation and fibrosis in the liver. Furthermore, in established diet-induced NASH, inhibiting FLCN reversed fat accumulation and fibrosis marker gene expression, but not fibrosis scores. The numerous strengths of this study include the detailed mechanistic in vivo genetic models used to demonstrate the cell biology, physiology, and translational significance of this pathway. Equally exciting, the clinical and translational impact is that these data nominate FLCN as a novel and tractable target to precisely inhibit mTORC1 signaling to treat NAFLD, NASH, and their complications, albeit a specific mechanism to target FLCN in patients will need to be developed. Although both of these studies (Science 2022;376(6590):eabf8271; Ann Intern Med 2022;175:74–83) offered insights into promising therapeutic options for NAFLD, the challenge of preventing the ensuing liver fibrosis remains, and additional studies are needed to determine whether either of these options will “crack that nut.” Further Insights Into the Impact of Bariatric Surgery on the Progression of Nonalcoholic Fatty Liver DiseaseGastroenterologyVol. 163Issue 2PreviewSeeberg KA, Borgeraas H, Hofsø H, et al. Gastric bypass versus sleeve gastrectomy in type 2 diabetes: effects on hepatic steatosis and fibrosis: a randomized controlled trial. Ann Intern Med 2022;175:74–83. Full-Text PDF

The insulin receptor discovery is 50 years old - A review of achieved progress

The isolation of insulin from the pancreas and its purification to a degree permitting its safe administration to type 1 diabetic patients were accomplished 100 years ago at the University of Toronto by Banting, Best, Collip and McLeod and constitute undeniably one of the major medical therapeutic revolutions, recognized by the attribution of the 1923 Nobel Prize in Physiology or Medicine to Banting and McLeod. The clinical spin off was immediate as well as the internationalization of insulin's commercial production. The outcomes regarding basic research were much slower, in particular regarding the molecular mechanisms of insulin action on its target cells. It took almost a half-century before the determination of the tri-dimensional structure of insulin in 1969 and the characterization of its cell receptor in 1970-1971. The demonstration that the insulin receptor is in fact an enzyme named tyrosine kinase came in the years 1982-1985, and the crystal structure of the intracellular kinase domain 10 years later. The crystal structure of the first intracellular kinase substrate (IRS-1) in 1991 paved the way for the elucidation of the intracellular signalling pathways but it took 15 more years to obtain the complete crystal structure of the extracellular receptor domain (without insulin) in 2006. Since then, the determination of the structure of the whole insulin-receptor complex in both the inactive and activated states has made considerable progress, not least due to recent improvement in the resolution power of cryo-electron microscopy. I will here review the steps in the development of the concept of hormone receptor, and of our knowledge of the structure and molecular mechanism of activation of the insulin receptor.

Role of surface-exposed charged basic amino acids (Lys, Arg) and guanidination in insulin on the interaction and stability of insulin–insulin receptor complex

Naturally occurring proteins are emerging as novel therapeutics in the protein-based biopharmaceutical industry for the treatment of diabetes and obesity. However, proteins are not suitable for oral delivery due to short half-life, reduced physical and chemical stability and low permeability across the membrane. Chemical modification has been identified as a formulation strategy to enhance the stability and bioavailability of protein drugs. The present study aims to study the effect of charge-specific modification of basic amino acids (Lys, Arg) and guanidination on the interaction of insulin with its receptor using molecular modelling. Our investigation revealed that the guanidination of insulin (Lys-NHC = NHNH2) enhanced and exerted stronger binding of the protein to its receptor through electrostatic interaction than native insulin (Lys-NH3+). Point mutations of Lys and Arg (R22, K29; R22K, K29; R22, K29R; R22K, K29R) were attempted and the effects on the interaction and stability between insulin/modified insulins and insulin receptor were also analyzed in this study. The findings from the study are expected to provide a better understanding of the possible mechanism of action of the modified protein at a molecular level before advancing to real experiments.

Selenium overexposure induces insulin resistance: In silico study

Background and aimsSeveral studies raise concerns about the possible association of high selenium exposure with insulin resistance and type 2 diabetes. This in silico study proposes a possible mechanism of insulin resistance in the case of overexposure to selenium. MethodA study was carried out using molecular modeling, where cysteines of the insulin-receptor are replaced by selenocysteines. Calculation of the interaction energy of the receptor was performed in both cases with Auto Dock Tools and Vina 4.2 software to predict whether the substitution of amino acid could lead to destabilization of the protein-ligand complex and therefore possibly insulin resistance. Finally, the docked complex was analyzed by using BIOVIA Discovery Studio Visualizer to show the type of interactions between the ligands and insulin-receptor, and to determine the distance of the ligands from the binding site on insulin-receptor. ResultsThe results show that the substitution of cysteine by selenocysteine in the insulin receptor does not lead to stabilization of the complex receptor/insulin, but to its disruption.In addition, the types and the number of bonds between insulin and its receptor in the two cases are different, where 7 strong bonds between insulin and its receptor were found in the case of the cysteine complex compared to 6 weak bonds in the second case. ConclusionFindings of this study suggest that misincorporation of selenocysteines in insulin receptor could lead to destabilization of the insulin-receptor complex and therefore may possibly cause an insulin resistance.

Sequential and Asynchronous Strengthening of the Influence of Temperature on the Endo- and Exocytosis of Insulin in the Isolated Vertebrata Hepatocytes: Summing up Previous Studies.

Insulin internalization and processing of the Insulin Receptor Complex (IRC) inside the cell are important components of the intracellular Mechanism of Insulin Action (MIA). They define the continuation of intracellular signaling of IRC and allow utilization of the parts of the complex after ligand dissociation. Traditionally, changes in the insulin regulatory system associated with the vertebrate phylogenesis have been evaluated by changes of its two elements: the hormone and its receptor. A hormone-competent cell was considered as an evolutionarily completed element of insulin regulatory system. However, previous studies of the isolated hepatocytes of four classes of vertebrates (lamprey, frog, chicken, and rat) revealed significant differences in the state of internalization of 125I-insulin and intracellular IRC processing. Radical differences were noted in the regulation of 125I-insulin internalization and the intracellular fate of the IRC. Here, cytosolic efficient insulin degradation and a complete lack of 125I-insulin exocytosis were observed in the cyclostome cells, whereas in amphibians the hormone underwent lysosomal degradation and showed low levels of exocytosis, while birds and mammals were characterized by high volumes of the excreted 125Iinsulin containing proteolytic 125I-insulin fragments. Despite the established recognition of the importance of the temperature factor, a complete understanding of the molecular mechanisms underlying the temperature effects on MIA is still missing. This poorly studied problem of the MIA temperature dependence can be behind the differences in the effect of temperature on the intracellular action of insulin and IGF-I. In fact, at different phylogenetic stages, successive changes were reported for the temperature dependence of the 125Iinsulin internalization and exocytosis. The following regularities were reported for the effect of temperature on the 125I-insulin internalization in isolated hepatocytes of different origin: complete lack of receptibility of the process to temperature in lampreys, receptibility of the process in a narrow range of low temperatures (0-5°C) in amphibians, and flexible regulation of 125I-insulin internalization in a wide temperature range (6- 37°C) in the cells from endothermic organisms. Reported data make it possible to observe three stages in the alteration of temperature regulation of 125I-insulin internalization (in cells of cyclostomes, amphibians, and endothermic organisms) and two stages of temperature regulation of 125I-insulin exocytosis in cells of amphibians, birds, and mammals. The data presented in this study reflect the specificity of the developmental reorganization of the intracellular MIA regulation and hormone utilization, and emphasize the central role of temperature in selective MIA formation during vertebrate phylogenesis.

Activation mechanism of the insulin receptor revealed by cryo-EM structure of the fully liganded receptor-ligand complex.

Insulin signaling controls metabolic homeostasis. Here, we report the cryo-EM structure of full-length insulin receptor (IR) and insulin complex in the active state. This structure unexpectedly reveals that maximally four insulins can bind the 'T'-shaped IR dimer at four distinct sites related by 2-fold symmetry. Insulins 1 and 1' bind to sites 1 and 1', formed by L1 of one IR protomer and α-CT and FnIII-1 of the other. Insulins 2 and 2' bind to sites 2 and 2' on FnIII-1 of each protomer. Mutagenesis and cellular assays show that both sites 1 and 2 are required for optimal insulin binding and IR activation. We further identify a homotypic FnIII-2-FnIII-2 interaction in mediating the dimerization of membrane proximal domains in the active IR dimer. Our results indicate that binding of multiple insulins at two distinct types of sites disrupts the autoinhibited apo-IR dimer and stabilizes the active dimer.

Dephosphorylation of clustered phosphoserine residues in human Grb14 by protein phosphatase 1 and its effect on insulin receptor complex formation.

The physical interaction of the human growth factor receptor-bound protein 14 (hGrb14) and the insulin receptor (IR) represses insulin signaling. With respect to the recruiting mechanism of hGrb14 to IR respond to insulin stimulus, our previous reports have suggested that phosphorylation of Ser358 , Ser362 , and Ser366 in hGrb14 by glycogen synthase kinase-3 repressed hGrb14-IR complex formation. In this study, we investigated phosphatase-mediated dephosphorylation of the hGrb14 phosphoserine residues. An in vitro phosphatase assay with hGrb14-derived synthetic phosphopeptides suggested that protein phosphatase 1 (PP1) is involved in the dephosphorylation of Ser358 and Ser362 . Furthermore, coimmunoprecipitation experiments suggested that insulin-induced hGrb14-IR complex formation was repressed by the substitution of Ser358 or Ser362 with glutamic acid. These findings suggested that phosphate groups on Ser358 and Ser362 in hGrb14 are dephosphorylated by PP1, and the dephosphorylation facilitates hGrb14-IR complex formation.

Functional characterization of β2-adrenergic and insulin receptor heteromers

This study aimed to functionally characterize β2-adrenergic (β2AR) and insulin receptor (IR) heteromers in regard to β-arrestin 2 (βarr2) recruitment and cAMP signaling and to examine the involvement of the cytoplasmic portion of the IR β chain in heteromerization with β2AR. Evidence for β2AR:IR:βarr2 complex formation and the specificity of the IR:βarr2 interaction was first provided by bioinfomatics analysis. Receptor-heteromer investigation technology (HIT) then provided functional evidence of β2AR:IR heterodimerization by showing isoproterenol-induced but not insulin-induced GFP2-βarr2 recruitment to the β2AR:IR complex; the IR:βarr2 interaction was found to only be constitutive. The constitutive IR:βarr2 BRET signal (BRETconst) was significantly smaller in cells coexpressing IR-RLuc8 and a GFP2-βarr2 1–185 mutant lacking the proposed IR binding domain. β2AR:IR heteromerization also influenced the pharmacological phenotype of β2AR, i.e., its efficacy in recruiting βarr2 and activating cAMP signaling. Evidence suggesting involvement of the cytoplasmic portion of the IR β chain in the interaction with β2AR was provided by BRET2 saturation and HIT assays using an IR 1–1271 stop mutant lacking the IR C-terminal tail region. For the complex consisting of IR 1–1271–RLuc8:β2AR-GFP2, saturation was not reached, most likely reflecting random collisions between IR 1–1271 and β2AR. Furthermore, in the HIT assay, no substantial agonist-induced increase in the BRET2 signal was detected that would be indicative of βarr2 recruitment to the IR 1–1271:β2AR heteromer. Complementary 3D visualization of β2AR:IR provided supporting evidence for stability of the heterotetramer complex and identified amino acid residues involved in β2AR:IR heteromerization.This article is part of the Special Issue entitled ‘Receptor heteromers and their allosteric receptor-receptor interactions’.

Molecular adaptations in human subcutaneous adipose tissue after ten weeks of endurance exercise training in healthy males.

Endurance exercise training induces adaptations in metabolically active organs, but adaptations in human subcutaneous adipose tissue (scAT) remains incompletely understood. On the basis of animal studies, we hypothesized that endurance exercise training would increase the expression of proteins involved in lipolysis and glucose uptake in scAT. To test these hypotheses, 19 young and healthy males were randomized to either endurance exercise training (TR; age 18-24 yr; BMI 19.0-25.4 kg/m2) or a nonexercising control group (CON; age 21-35 yr; BMI 20.5-28.8 kg/m2). Abdominal subcutaneous fat biopsies and blood were obtained at rest before and after intervention. By using Western blotting and PCR, we determined expression of lipid droplet-associated proteins, various proteins involved in substrate metabolism, and mRNA abundance of cell surface G protein-coupled receptors (GPCRs). Adipose tissue insulin sensitivity was determined from fasting plasma insulin and nonesterified fatty acids (adipose tissue insulin resistance index; Adipo-IR). Adipo-IR improved in TR compared with CON ( P = 0.03). This was accompanied by increased insulin receptor (IR) protein expression in scAT with a 1.54-fold (SD 0.79) change from baseline in TR vs. 0.85 (SD 0.30) in CON ( P = 0.007). Additionally, hexokinase II (HKII) and succinate dehydrogenase complex subunit A (SDHA) protein increased in TR compared with CON ( P = 0.006 and P = 0.04, respectively). We did not observe changes in lipid droplet-associated proteins or mRNA abundance of GPCRs. Collectively, 10 weeks of endurance exercise training improved adipose tissue insulin sensitivity, which was accompanied by increased IR, HKII, and SDHA protein expression in scAT. We suggest that these adaptations contribute to an improved metabolic flexibility. NEW & NOTEWORTHY This study is the first to investigate the molecular adaptations in human subcutaneous adipose tissue (scAT) after endurance exercise training compared with a nonexercising control group. We show that endurance exercise training improves insulin sensitivity in human scAT, and this is accompanied by increased expression of insulin receptor, hexokinase II, and succinate dehydrogenase complex subunit A. Collectively, our data suggest that endurance exercise training induces molecular adaptations in human scAT, which may contribute to an improved metabolic flexibility.

Loss of Hepatic CEACAM1: A Unifying Mechanism Linking Insulin Resistance to Obesity and Non-Alcoholic Fatty Liver Disease.

The pathogenesis of human non-alcoholic fatty liver disease (NAFLD) remains unclear, in particular in the context of its relationship to insulin resistance and visceral obesity. Work on the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in mice has resolved some of the related questions. CEACAM1 promotes insulin clearance by enhancing the rate of uptake of the insulin-receptor complex. It also mediates a negative acute effect of insulin on fatty acid synthase activity. This positions CEACAM1 to coordinate the regulation of insulin and lipid metabolism. Fed a regular chow diet, global null mutation of Ceacam1 manifest hyperinsulinemia, insulin resistance, obesity, and steatohepatitis. They also develop spontaneous chicken-wire fibrosis, characteristic of non-alcoholic steatohepatitis. Reduction of hepatic CEACAM1 expression plays a significant role in the pathogenesis of diet-induced metabolic abnormalities, as bolstered by the protective effect of hepatic CEACAM1 gain-of-function against the metabolic response to dietary fat. Together, this emphasizes that loss of hepatic CEACAM1 links NAFLD to insulin resistance and obesity.

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*[S

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.

Insight into the structural and biological relevance of the T/R transition of the N-terminus of the B-chain in human insulin.

The N-terminus of the B-chain of insulin may adopt two alternative conformations designated as the T- and R-states. Despite the recent structural insight into insulin–insulin receptor (IR) complexes, the physiological relevance of the T/R transition is still unclear. Hence, this study focused on the rational design, synthesis, and characterization of human insulin analogues structurally locked in expected R- or T-states. Sites B3, B5, and B8, capable of affecting the conformation of the N-terminus of the B-chain, were subjects of rational substitutions with amino acids with specific allowed and disallowed dihedral φ and ψ main-chain angles. α-Aminoisobutyric acid was systematically incorporated into positions B3, B5, and B8 for stabilization of the R-state, and N-methylalanine and d-proline amino acids were introduced at position B8 for stabilization of the T-state. IR affinities of the analogues were compared and correlated with their T/R transition ability and analyzed against their crystal and nuclear magnetic resonance structures. Our data revealed that (i) the T-like state is indeed important for the folding efficiency of (pro)insulin, (ii) the R-state is most probably incompatible with an active form of insulin, (iii) the R-state cannot be induced or stabilized by a single substitution at a specific site, and (iv) the B1–B8 segment is capable of folding into a variety of low-affinity T-like states. Therefore, we conclude that the active conformation of the N-terminus of the B-chain must be different from the “classical” T-state and that a substantial flexibility of the B1–B8 segment, where GlyB8 plays a key role, is a crucial prerequisite for an efficient insulin–IR interaction.

Involvement of the endosomal compartment in cellular insulin signaling

The insulin receptor and insulin signaling proteins downstream the receptor reside in different subcellular compartments and undergo redistribution within the cell upon insulin activation. Endocytosis of the insulin-receptor complex, by mediating ligand degradation and receptor dephosphorylation, is generally viewed as a mechanism which attenuates or arrests insulin signal transduction. However, several observations suggest that insulin receptor endocytosis and/or recruitement of insulin signaling proteins to endosomes are also involved in a positive regulation of insulin signaling: (1) upon internalization, the insulin receptor remains transiently phosphorylated and activated; (2) in insulin-stimulated cells or tissues, signaling proteins of the PI3K/Akt and Ras/Raf/Mek/Erk pathways are recruited to endosomes or other intracellular compartments, in which they undergo phosphorylation and/or activation; and (3) depletion or overexpression of proteins involved in the regulation of membrane trafficking and endocytosis interfere with insulin signaling. These observations support a spatial and temporal regulation of insulin signal transduction and reinforce the concept that, as for other membrane signaling receptors, endocytosis and signaling are functionally linked.

Differences in time course of internalization of receptors of insulin and insulin-like growth factor (IGF-1) in isolated rat hepatocytes

Insulin and IGF-I are two related peptides performing in the mammalian body functionally different roles of the metabolic and growth hormones, respectively. Internalization of the insulin-receptor complex (IRC) is a most important chain of mechanism of the action of hormone. To elucidate differences in the main stages of internalization of the two related hormones at isolated rat hepatocytes, the internalization time course of 125I-insulin and 125I-IGF-I are traced at 37 and 12°C. There are established marked differences in the process of internalization of labeled hormones, which is stimulated by insulin and IGF-I. At 37°C the insulin-stimulated internalization, unlike the process initiated by IGF-I, did not reach the maximal level for 1 h of incubation. But essential differences in the internalization course of these two related peptides were obvious at the temperature of 12°C. The internalization level of insulin receptors at 12°C decreased by one third in spite of a significant increase of the insulin receptor binding on the hepatocyte plasma membrane. At 12°C a slight decrease of the proportion of intracellular 125I-IGF-I correlated with a decrease in the 125I-IGF-I binding to receptors on the cell membrane. Internalization of IGF-I receptors was not affected by low temperature, as neither its level, nor the rate changed at 12°C. The paradoxical decrease of the insulin-stimulated internalization at low temperature seems to represent a peculiar “inhibition mechanism” of immersion of IRC into the cell, which leads to accumulation of the complexes on the cell surface and possibly to a readjustment of the insulin biological activity. The resistance of internalization of the IGF-I receptor to action of cold seems to be related to the more ancient origin of this mechanism in the poikilothermal vertebrates.

First structural insights into insulin–insulin receptor complex

First structural insights into insulin–insulin receptor complex

Structural Integrity of the B24 Site in Human Insulin Is Important for Hormone Functionality

Despite the recent first structural insight into the insulin-insulin receptor complex, the role of the C terminus of the B-chain of insulin in this assembly remains unresolved. Previous studies have suggested that this part of insulin must rearrange to reveal amino acids crucial for interaction with the receptor. The role of the invariant Phe(B24), one of the key residues of the hormone, in this process remains unclear. For example, the B24 site functionally tolerates substitutions to D-amino acids but not to L-amino acids. Here, we prepared and characterized a series of B24-modified insulin analogues, also determining the structures of [D-HisB24]-insulin and [HisB24]-insulin. The inactive [HisB24]-insulin molecule is remarkably rigid due to a tight accommodation of the L-His side chain in the B24 binding pocket that results in the stronger tethering of B25-B28 residues to the protein core. In contrast, the highly active [D-HisB24]-insulin is more flexible, and the reverse chirality of the B24C(α) atom swayed the D-His(B24) side chain into the solvent. Furthermore, the pocket vacated by Phe(B24) is filled by Phe(B25), which mimics the Phe(B24) side and main chains. The B25→B24 downshift results in a subsequent downshift of Tyr(B26) into the B25 site and the departure of B26-B30 residues away from the insulin core. Our data indicate the importance of the aromatic L-amino acid at the B24 site and the structural invariance/integrity of this position for an effective binding of insulin to its receptor. Moreover, they also suggest limited, B25-B30 only, unfolding of the C terminus of the B-chain upon insulin activation.

Endocytosis and Intracellular Dissociation Rates of Human Insulin–Insulin Receptor Complexes by Quantum Dots in Living Cells

Insulin signaling is involved in glucose metabolism, cellular growth, and differentiation. Its function is altered in diabetes and many cancer types. Insulin binding to insulin receptor (IR) triggers diverse signaling pathways. However, signal transduction by IR is not mediated exclusively at the cell surface. Activated ligand-receptor complexes are internalized into endosomes from which the IR recruits adapters acting on substrates that are distinct from those accessible at the membrane. We report the biotinylation of human-recombinant insulin (rhIns) specifically at the position 29 of the B chain. We combined visible fluorescent proteins fused to IR and biotinylated rhIns conjugated with streptavidin-quantum dots to perform extended, quantitative experiments in real time. Modified rhIns bound to the IR and conjugated with the quantum dots was internalized with a rate constant (k) of 0.009 min(-1). Dissociation of insulin-IR complex in endocytosed vesicles occurred with k = 0.006 min(-1).

In Memoriam: Guy Dodson (1937–2012)

In Memoriam: Guy Dodson (1937–2012)