Current Insights on the Use of Insulin and the Potential Use of Insulin Mimetics in Targeting Insulin Signalling in Alzheimer's Disease.

Alzheimer's disease (AD) and type 2 diabetes (T2D) are chronic diseases that share several pathological mechanisms, including insulin resistance and impaired insulin signalling. Their shared features have prompted the evaluation of the drugs used to manage diabetes for the treatment of AD. Insulin delivery itself has been utilized, with promising effects, in improving cognition and reducing AD related neuropathology. The most recent clinical trial involving intranasal insulin reported no slowing of cognitive decline; however, several factors may have impacted the trial outcomes. Long-acting and rapid-acting insulin analogues have also been evaluated within the context of AD with a lack of consistent outcomes. This narrative review provided insight into how targeting insulin signalling in the brain has potential as a therapeutic target for AD and provided a detailed update on the efficacy of insulin, its analogues and the outcomes of human clinical trials. We also discussed the current evidence that warrants the further investigation of the use of the mimetics of insulin for AD. These small molecules may provide a modifiable alternative to insulin, aiding in developing drugs that selectively target insulin signalling in the brain with the aim to attenuate cognitive dysfunction and AD pathologies.

The effect of physicochemical factors on the self-association of HMGB1: A surface plasmon resonance study.

HMGB1 triggers proinflammatory reactions by interacting extracellularly with various receptors. HMGB1 also acts in the nucleus by interacting with DNA and controlling DNA transcription, a process which involves its self-association. The self-association of HMGB1 was characterized using surface plasmon resonance (SPR). A dimer/tetramer binding model was developed that provided a good fit to the SPR sensorgrams and enabled the kinetics of self-association of different HMGB1 oligomers to be evaluated under a variety of physicochemical conditions. The formation of HMGB1 tetramers, and not dimers, was strongly influenced by ionic strength. HMGB1 self-association increased as the pH was decreased from 7.4 to 4.8 but was abolished at pH4.0, suggesting the involvement of acidic amino acids of HMGB1 in its self-association. HMGB1 dimers were found to predominate in the absence of zinc, but addition of zinc promoted the formation of HMGB1 tetramers. More reducing conditions favored dimerization but diminished tetramer formation. In contrast, oxidizing conditions favored tetramer formation. Physicochemical factors modulate the extent of self-association of HMGB1. We speculate that HMGB1 dimers may preferentially bind DNA, whereas HMGB1 tetramers may promote inflammatory responses by binding to RAGE and TLRs. The self-association of HMGB1, regulated by variations of physicochemical factors, may influence its roles in DNA rearrangement and regulation of pathophysiological diseases.

Optimization of surface plasmon resonance experiments: Case of high mobility group box 1 (HMGB1) interactions.

Surface plasmon resonance (SPR) is a powerful technique for evaluating protein-protein interactions in real time. However, inappropriately optimized experiments can often lead to problems in the interpretation of data, leading to unreliable kinetic constants and binding models. Optimization of SPR experiments involving "sticky" proteins, or proteins that tend to aggregate, represents a typical scenario where it is important to minimize errors in the data and the kinetic analysis of those data. This is the case of High Mobility Group Box 1 and the receptor of advanced glycation end products. A number of improvements in protein purification, buffer composition, immobilization conditions, and the choice of flow rate are shown to result in substantial improvements in the accurate characterization of the interactions of these proteins and the derivation of the corresponding kinetic constants.

Stripping voltammetric detection of insulin at liquid-liquid microinterfaces in the presence of bovine albumin.

Electrochemistry at the interface between two immiscible electrolyte solutions (ITIES) provides a platform for label-free detection of biomolecules. In this study, adsorptive stripping voltammetry (AdSV) was implemented at an array of microscale ITIES for the detection of the antidiabetic hormone insulin. By exploiting the potential-controlled adsorption of insulin at the ITIES, insulin was detected at 10 nM via subsequent voltammetric desorption. This is the lowest detected concentration reported to-date for a protein by electrochemistry at the ITIES. Surface coverage calculations indicate that between 0.1 and 1 monolayer of insulin forms at the interface over the 10-1000 nM concentration range of the hormone. In a step toward assessment of selectivity, the optimum adsorption potentials for insulin and albumin were determined to be 0.900 V and 0.975 V, respectively. When present in an aqueous mixture with albumin, insulin was detected by tuning the adsorption potential to 0.9 V, albeit with reduced sensitivity. This provides the first example of selective detection of one protein in the presence of another by exploiting optimal adsorption potentials. The results presented here provide a route to the improvement of detection limits and achievement of selectivity for protein detection by electrochemistry at the ITIES.

High-fat diet induced alterations in plasma membrane cholesterol content impairs insulin receptor binding and signalling in mouse liver but is ameliorated by atorvastatin.

A high-fat diet (HFD) impairs insulin binding and signalling and may contribute to the development of insulin resistance. In addition, in vitro studies have shown that alterations in plasma membrane cholesterol influence ligand binding and downstream signalling for several receptor-tyrosine kinases (RTKs), including the insulin receptor. Using an ex vivo approach, we explored the effects of a HFD on insulin binding and signalling in mouse liver and relate these to observed changes in plasma membrane cholesterol. Mice fed a HFD demonstrated decreased insulin signalling compared to mice fed a normal chow diet (ND), indicated by a 3-fold decrease in insulin binding (P < 0.001) and a similar decrease in insulin receptor phosphorylation (~2.5-fold; P < 0.0001). Interestingly, we also observed a marked decrease in the cholesterol content of liver plasma membranes in the HFD fed mice (P < 0.0001). These effects of the HFD were found to be ameliorated by atorvastatin treatment (P < 0.0001). However, in ND mice, atorvastatin had no influence on membrane cholesterol content or insulin binding and signalling. The influence of membrane cholesterol on insulin binding and signalling was also corroborated in HepG2 cells. To the best of our knowledge, this is the first demonstration of the effects of a HFD and atorvastatin treatment on changes in plasma membrane cholesterol content and the consequent effects on insulin binding and signalling. Collectively, these findings suggest that changes in membrane cholesterol content could be an important underlying reason for the long-known effects of a HFD on the development of insulin resistance.

The acidic tail of HMGB1 regulates its secondary structure and conformational flexibility: A circular dichroism and molecular dynamics simulation study.

High mobility group box 1 (HMGB1) is a damage-associated molecular pattern (DAMP) molecule that triggers the progression of several pro-inflammatory diseases such as diabetes, Alzheimer's disease and cancer, by inducing signals upon interaction with the receptors such as the receptor for advanced glycation end-products (RAGE) and toll-like receptors (TLRs). The acidic C-terminal tail of HMGB1 is an intrinsically disordered region of the protein which is known to determine the interaction of HMGB1 to DNA and histones. This study characterizes its structural properties using a combination of circular dichroism (CD) and molecular dynamics (MD) simulations. The full-length and tail-less forms of HMGB1 were compared to rationalise the role of the acidic tail in maintaining the stability of the entire structure of HMGB1 in atomistic detail. Consistent with experimental data, the acidic tail was predicted to adopt an extended conformation that allows it to make a range of hydrogen-bonding and electrostatic interactions with the box-like domains that stabilize the overall structure of HMGB1. Absence of the acidic tail was predicted to increase structural fluctuations of all amino acids, leading to changes in secondary structure from α-helical to more hydrophilic turns along with increased exposure of multiple amino acids to the surrounding solvent. These structural changes reveal the intrinsic conformational dynamics of HMGB1 that are likely to affect the accessibility of its receptors.

Use of virus-like particles as a native membrane model to study the interaction of insulin with the insulin receptor.

There is emerging evidence of the utility of virus-like particles (VLPs) as a novel model for the study of receptor-ligand interactions in a native plasma membrane environment. VLPs consist of a viral core protein encapsulated by portions of the cell membrane with membrane proteins and receptors expressed in their native conformation. VLPs can be generated in mammalian cells by transfection with the retroviral core protein (gag). In this study, we used Chinese hamster ovary (CHO T10) cells stably overexpressing the insulin receptor (IR) to generate IR bearing VLPs. The diameter and size uniformity of VLPs were estimated by dynamic light scattering and morphological features examined by scanning electron microscopy. The presence of high affinity IR on VLPs was demonstrated by competitive binding assays (KD: 2.3 ±â€¯0.4 nM, n = 3), which was similar to that on the parental CHO T10 cells (KD: 2.1 ±â€¯0.4 nM, n = 3). We also report that increases or decreases in membrane cholesterol content by treatment with methyl-ß-cyclodextrin (MBCD) or cholesterol pre-loaded methyl-ß-cyclodextrin (cMBCD), respectively, substantially decreased insulin binding (> 30%) to both VLPs and cells, and we speculate this is due to a change in receptor disposition. We suggest that this novel finding of decreases in insulin binding in response to changes in membrane cholesterol content may largely account for the unexplained decreases in insulin signalling events previously reported elsewhere. Finally, we propose VLPs as a viable membrane model for the study of insulin-IR interactions in a native membrane environment.

The self-association of HMGB1 and its possible role in the binding to DNA and cell membrane receptors.

High mobility group box 1 (HMGB1), a chromatin protein, interacts with DNA and controls gene expression. However, when HMGB1 is released from apoptotic or damaged cells, it triggers proinflammatory reactions by interacting with various receptors, mainly receptor for advanced glycation end-products (RAGE) and toll-like receptors (TLRs). The self-association of HMGB1 has been found to be crucial for its DNA-related biological functions. It is influenced by several factors, such as ionic strength, pH, specific divalent metal cations, redox environment and acetylation. This self-association may also play a role in the interaction with RAGE and TLRs and the concomitant inflammatory responses. Future studies should address the potential role of HMGB1 self-association on its interactions with DNA, RAGE and TLRs, as well as the influence of physicochemical factors in different cellular environments on these interactions.

jAMVLE, A new integrated molecular visualization learning environment*.

A new computer-based molecular visualization tool has been developed for teaching, and learning, molecular structure. This java-based jmol Amalgamated Molecular Visualization Learning Environment (jAMVLE) is platform-independent, integrated, and interactive. It has an overall graphical user interface that is intuitive and easy to use. The application can be downloaded free from the internet at wabri.org.au/jamvle. A cohort of 28 third year undergraduate molecular biotechnology degree students evaluated the new application through an essay-style project. These were analyzed to identify themes expressed by students in the content of their evaluations. Most students were positive about the new jAMVLE learning environment, and five major benefits emerged from the analysis. In particular, the students perceived that jAMVLE has an appealing interface, is interactive, provides a useful integrated environment, is user friendly, and is an excellent learning tool. Overall, students found that the jAMVLE application stimulated their interest, was a more active learning environment, provided better guidance, and made learning fun.

Alzheimer's beta-amyloid peptides compete for insulin binding to the insulin receptor.

The amyloid-beta (Abeta) peptide is neurotoxic and associated with the pathology of Alzheimer's disease (AD). We investigated the effect of Abeta peptides on insulin binding to the insulin receptor because it is known that (1) Abeta and insulin are both amyloidogenic peptides sharing a common sequence recognition motif, (2) Abeta and insulin are substrates for the same insulin degrading enzyme, and (3) impaired glucose metabolism is a characteristic event in the pathology of AD. We discovered that Abeta(1-40) and Abeta(1-42,) the main physiological forms, reduced insulin binding and receptor autophosphorylation. The reduction in binding was caused by a decrease in the affinity of insulin binding to the insulin receptor. This reduction was independent of the receptor concentration. The reverse, control peptide Abeta(40-1) did not reduce insulin binding or insulin receptor autophosphorylation. These results demonstrate that Abeta is a direct competitive inhibitor of insulin binding and action. We speculate that the increased levels of Abeta in Alzheimer's disease may be linked to the associated insulin resistance that has been observed previously in this disease.

Correlation between inactive cathepsin D expression and retinal changes in mcd2/mcd2 transgenic mice.

PURPOSE: To investigate the correlation between the presence of the inactive cathepsin D (CatD) and retinal changes in mcd2/mcd2 transgenic mice. METHODS: Computational modeling was used to examine whether CatD mutants maintain competitive substrate binding. D407 cells were transfected with pcDNACatDM1 or pcDNACatDM2, containing procathepsin D (pro-CatD) with 6-bp (CatDM1) or 12-bp (CatDM2) deletions, respectively, flanking the pro-CatD cleavage site, and the aspartic protease activity of the transfected cells was measured. Subsequently, transgenic mice (mcd2/mcd2) containing CatDM2 were generated. Relative transgene copy number and transcript levels in the previously produced mcd/mcd (carrying CatDM1) and mcd2/mcd2 mice were measured by quantitative real-time PCR. Western blot analysis and aspartic protease activity were used to characterize the mutated proteins. Retinal changes were described by using color fundus photography and fluorescein angiography, histology, immunohistochemistry, and electron microscopy. RESULTS: Computational modeling of the CatDM1 and CatDM2 structures indicated that the substrate binding site was not altered. There was limited or no aspartic protease activity associated with CatDM1 and CatDM2 proteins, respectively. Mcd2/mcd2 animals contained a higher amount of inactive CatD than mcd/mcd or wild-type mice. Retinal abnormalities in mcd2/mcd2 mice developed at 3 months of age, earlier than in mcd/mcd mice. These changes included hypopigmentation, hyperfluorescence, retinal pigment epithelial (RPE) cell depigmentation or clumping, cell proliferation, and pleomorphism. Proliferating cells were identified as being of RPE origin. CONCLUSIONS: This study demonstrated a correlation between the presence of the inactive CatD in RPE cells and the development of ophthalmoscopic, cellular, and histologic changes in the retina.

Real-time and Label-free Bio-sensing of Molecular Interactions by Surface Plasmon Resonance: A Laboratory Medicine Perspective.

Radioactive, chromogenic, fluorescent and other labels have long provided the basis of detection systems for biomolecular interactions including immunoassays and receptor binding studies. However there has been unprecedented growth in a number of powerful label free biosensor technologies over the last decade. While largely at the proof-of-concept stage in terms of clinical applications, the development of more accessible platforms may see surface plasmon resonance (SPR) emerge as one of the most powerful optical detection platforms for the real-time monitoring of biomolecular interactions in a label-free environment.In this review, we provide an overview of SPR principles and current and future capabilities in a diagnostic context, including its application for monitoring a wide range of molecular markers of disease. The advantages and pitfalls of using SPR to study biomolecular interactions are discussed, with particular emphasis on its potential to differentiate subspecies of analytes and the inherent ability for quantitation through calibration-free concentration analysis (CFCA). In addition, recent advances in multiplex applications, high throughput arrays, miniaturisation, and enhancements using noble metal nanoparticles that promise unprecedented sensitivity to the level of single molecule detection, are discussed.In summary, while SPR is not a new technique, technological advances may see SPR quickly emerge as a highly powerful technology, enabling rapid and routine analysis of molecular interactions for a diverse range of targets, including those with clinical applicability. As the technology produces data quickly, in real-time and in a label-free environment, it may well have a significant presence in future developments in lab-on-a-chip technologies including point-of-care devices and personalised medicine.

A comparative structural bioinformatics analysis of the insulin receptor family ectodomain based on phylogenetic information.

The insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R) and the insulin receptor-related receptor (IRR) are covalently-linked homodimers made up of several structural domains. The molecular mechanism of ligand binding to the ectodomain of these receptors and the resulting activation of their tyrosine kinase domain is still not well understood. We have carried out an amino acid residue conservation analysis in order to reconstruct the phylogeny of the IR Family. We have confirmed the location of ligand binding site 1 of the IGF1R and IR. Importantly, we have also predicted the likely location of the insulin binding site 2 on the surface of the fibronectin type III domains of the IR. An evolutionary conserved surface on the second leucine-rich domain that may interact with the ligand could not be detected. We suggest a possible mechanical trigger of the activation of the IR that involves a slight 'twist' rotation of the last two fibronectin type III domains in order to face the likely location of insulin. Finally, a strong selective pressure was found amongst the IRR orthologous sequences, suggesting that this orphan receptor has a yet unknown physiological role which may be conserved from amphibians to mammals.

Insulin decreases the secretion of apoB-100 from hepatic HepG2 cells but does not decrease the secretion of apoB-48 from intestinal CaCo-2 cells.

We compared the acute effect of insulin on the human colonic intestinal epithelial cell line CaCo-2 and the transformed human hepatic cell line HepG2. Over 24 h, 100 nM and 10 microM insulin significantly inhibited the secretion of apolipoprotein (apo) B-100 from HepG2 cells to 63 and 49% of control, respectively. Insulin had no effect on the secretion of apoB-48 from CaCo-2 cells. There was no effect of insulin on the cholesterol ester or free cholesterol concentrations in HepG2 or CaCo-2 cells. HepG2 and CaCo-2 cells bound insulin with high affinity, leading to similar stimulation of insulin receptor protein tyrosine kinase activation. Protein kinase C or mitogen-activated protein kinase activity in the presence or absence of insulin was not correlated with apoB-48 production in CaCo-2 cells. Therefore, insulin acutely decreases the secretion of apoB-100 in hepatic HepG2 cells, but does not acutely modulate the production or secretion of apoB-48 from CaCo-2 intestinal cells.