Insulin Molecule Research Articles

Cryo-EM structure of the complete and ligand-saturated insulin receptor ectodomain.

Glucose homeostasis and growth essentially depend on the hormone insulin engaging its receptor. Despite biochemical and structural advances, a fundamental contradiction has persisted in the current understanding of insulin ligand-receptor interactions. While biochemistry predicts two distinct insulin binding sites, 1 and 2, recent structural analyses have resolved only site 1. Using a combined approach of cryo-EM and atomistic molecular dynamics simulation, we present the structure of the entire dimeric insulin receptor ectodomain saturated with four insulin molecules. Complementing the previously described insulin-site 1 interaction, we present the first view of insulin bound to the discrete insulin receptor site 2. Insulin binding stabilizes the receptor ectodomain in a T-shaped conformation wherein the membrane-proximal domains converge and contact each other. These findings expand the current models of insulin binding to its receptor and of its regulation. In summary, we provide the structural basis for a comprehensive description of ligand-receptor interactions that ultimately will inform new approaches to structure-based drug design.

Visible-Light-Activated High-Density Materials for Controlled in Vivo Insulin Release.

In this work, we describe the synthesis, characterization, and ultimate in vivo assessment of second-generation insulin photoactivated depot (PAD) materials. These are the first to use visible light to stimulate insulin release and have an in vivo performance that is 28-fold improved relative to first-generation materials. This improvement is due to two major factors linked to the utilized chemistry: (1) we have incorporated the coumarin photocleavable group, which increases the photorelease wavelength into the visible range, enhancing tissue penetration of the light; (2) phototoggling of insulin solubility is produced by linking three insulin molecules to a central bridge via light cleaved groups, and not by bonding to a large polymer. The resulting trimer is, therefore, highly dense (87% insulin dry w/w) but retains the insolubility required of the approach. Only after irradiation with visible light is native, soluble insulin is released from the dermal depot. This high density increases the amount and ease of insulin release, as the density of photolytic groups is 10-20-fold higher than in polymer-based first-generation materials. We have synthesized new azide-terminated coumarin linkers that we react with the amine groups of insulin. Using mass spectrometry methods, we identify the sites of reaction and purify individual isomers, which we demonstrate have in vitro photolysis rates that are within a factor of 2 of each other. We then reacted these terminal azide groups with a tridentate strained alkyne linker. We show that the resulting insulin trimer is highly insoluble, but can be milled into injectable particles that release insulin only in response to light from a 406 nm light source. Finally, we demonstrate that these materials have a significantly improved in vivo performance, releasing 28-fold more insulin on a per energy basis than first-generation materials.

Insulin conformational changes under high pressure in structural studies and molecular dynamics simulations

Abstract To study the mechanisms underlying protein misfolding and aggregation, therapeutic proteins can be successfully used as a model. Currently, insulin is widely tested as a useful model in this field, since it has been proved in both in vivo and in vitro studies that this small protein aggregates. In this article, exploiting the optimal coupling between high pressure protein crystallography and dynamic simulations, we probe the insulin conformations observed under high pressure, namely over the ranges 0–200 MPa for crystallographic experiments and 0–500 MPa for simulations. Crystal structures of insulin determined with diamond anvil cell technique present a step forward in understanding how pressure can modify protein conformation. Obtained results show different responses to volume compression of different fragments of the insulin molecule. For the first time, we have structurally proved that pressure noticeably modifies fragments of insulin molecules, especially terminal fragments of chain B. The observed structural modifications of insulin molecule in crystal state under pressure were compared to the results of insulin pressurization investigated by the molecular dynamic simulations. Comparing the crystallographic results and MD simulations, we were able to draw important considerations about the role of specific amino acids in pressure-induced insulin conformations.

Protein Tyrosine Phosphatase 1B Inhibitors: A Novel Therapeutic Strategy for the Management of type 2 Diabetes Mellitus.

Diabetes is one of the most common endocrine non-communicable metabolic disorders which is mainly caused either due to insufficient insulin or inefficient insulin or both together and is characterized by hyperglycemia. Diabetes emerged as a serious health issue in the industrialized and developing country especially in the Asian pacific region. Out of the two major categories of diabetes mellitus, type 2 diabetes is more prevalent, almost 90 to 95% cases, and the main cause of this is insulin resistance. The main cause of the progression of type 2 diabetes mellitus has been found to be insulin resistance. The type 2 diabetes mellitus may be managed by the change in lifestyle, physical activities, dietary modifications and medications. The major currently available management strategies are sulfonylureas, biguanides, thiazolidinediones, α-glucosidase inhibitors, dipeptidyl peptidase-IV inhibitors, and glucagon-like peptide-1 (GLP-1) agonist. Binding of insulin on the extracellular unit of insulin receptor sparks tyrosine kinase of the insulin receptor which induces autophosphorylation. The phosphorylation of the tyrosine is regulated by insulin and leptin molecules. Protein tyrosine phosphatase-1B (PTP1B) works as a negative governor for the insulin signalling pathways, as it dephosphorylates the tyrosine of the insulin receptor and suppresses the insulin signalling cascade. The compounds or molecules which inhibit the negative regulation of PTP1B can have an inductive effect on the insulin pathway and finally help in the management of diabetes mellitus. PTP1B could be an emerging therapeutic strategy for diabetes management. There are a number of clinical and basic research results which suggest that induced expression of PTP1B reduces insulin resistance. In this review, we briefly elaborate and explain the place of PTP1B and its significance in diabetes as well as a recent development in the PTP1B inhibitors as an antidiabetic therapy.

The Expected Pandemic of Mild-Alzheimer (≈Type 3 Diabetes), How to Combat?

With the increasing aging of the world population, the share in the population with mild Alzheimer's (m-ADs) - also referred to as type-3 diabetes - is increasing. Rough estimates indicate that by 2050 around 140 million people will suffer from this disease, especially in developed countries but with an increasing morbidity in Low- and Middle-Income countries. m-ADs can be characterized by senile plaques that consist of extracellular amyloid beta (Aβ) deposition while neurofibrillary tangles (NFT) -consisting of hyperphosphorylated tau which are located intracellular in Alzheimer Diseased brain- are a second feature. It must be emphasized that these are the consequences of the disease and not the cause. These amyloid plaques have also been observed in other organs of patients with poorly treated type 2 diabetes such as pancreas, liver, kidney and heart, so it must be emphasized that they are not characteristic of the brain alone. But what is then consequently the cause of m-ADs? The idea was first that the brain produced Insulin to a lesser extent, but a landmark study using LC-MS techniques by van Ginneken, et al. indicated that in post mortem Type Diabetes brain donors more saturated bonds occur, making the brain more rigid and the Insulin molecule less likely to reach its destination, leading to neural death. A second hypothesized cause is the formation of plaques in the fluid mosaic membrane of poly unsaturated fatty acids (PUFAs), as a result of which the Insulin molecule is less able to invade the human brain, which also leads to neural death. Finally, the role of Sphingomyelins as lipids in foods such as soy and milk should be mentioned, which can be converted via the Land's cycle in the brain to toxic Ceramide, which also could lead to neural death. In addition, in a small cohort of patients from the Dutch brain bank -where post mortem histopathological the brain disease has been assessed in addition to body characteristics- it appears that m-ADs or also Type-3 Diabetes is independent of Body Mass Index (BMI).

Monitoring Insulin-Aggregated Structures in the Presence of Epigallocatechin-3-gallate and Melatonin by Molecular Dynamics Simulations.

In the present work we illustrate the results of classical molecular dynamics simulations of model systems composed of six insulin molecules in water in the presence and in the absence of either epigallocatechin-3-gallate or melatonin molecules. For each model system, we performed three independent simulations (replicas) to study the aggregate formation dynamics and insulin interaction with epigallocatechin-3-gallate and melatonin. We find that melatonin is less stably close to insulin with respect to epigallocatechin-3-gallate, which interacts more stably with insulin molecules and mainly with insulin's chain B hydrophobic residues. We observe that the shape of the insulin-aggregated structures in the three model systems is different and depends on whether epigallocatechin-3-gallate is present or not. Simulations show that in the absence of epigallocatechin-3-gallate, insulin molecules tend to form linear aggregates, while in the presence of epigallocatechin-3-gallate, aggregates display a globular shape, less prone to form fibril structures.

1086-P: Preclinical Development of an Ultralong-Acting Insulin for Once-Weekly Dosing

Basal insulin analogues have been designed to mimic endogenous insulin secretion for maintaining normoglycaemia. Current insulin therapy for type 2 diabetes (T2D) requires at least daily dosing for patients. However, such daily injections can have poor patient compliance. An insulin analogue with longer duration requiring less frequent administration could improve compliance and maintain better glycaemic control. To achieve extended insulin action pharmacology, a panel of recombinant native single chain (SC) insulin molecules, consisting of B chain to A chain-linker variants, were fused to an antibody fragment crystallizable region (Fc). Characterization of the single chain insulin-Fc (SC-Insulin-Fc) variants were conducted in insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) luminescence-linked reporter assays, and with primary rodent and human hepatocyte assays for phosphorylated AKT. SC-Insulin-Fc-1 and -2 retained potency at the human IR within 6-fold of human insulin, without specificity over IGF1R binding, and induced Akt phosphorylation within a range of endogenous insulin. SC-Insulin-Fc variants lowered blood glucose levels in DIO and db/db mice by more than 50% vs. vehicle controls (p<0.05) for 5 days following a single dose, significantly superior to a standard insulin bolus. Continuous glucose monitoring (CGM) telemetry to profile 24 h control of blood glucose in diabetic ZDF rats was also applied. CGM telemetry revealed enhanced basal and postprandial glucose control in both light and dark cycles (p<0.05 vs. vehicle) up to 10 days following a single administration (90 nmol/kg). Repeat dosing of SC-Insulin-Fc variants (10/30/60 nmol/kg, Q1W) over 5 weeks resulted in dose-dependent improvements of glycaemic control (p≤0.05 vs. vehicle) with a long term readout towards decreasing %HbA1c. This dramatic shift in time spent away from hyperglycaemia toward normoglycaemia suggests once-weekly insulin as a novel viable option to treat human T2D. Disclosure V.G. Howard: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. C.D. Church: Employee; Self; AstraZeneca, MedImmune. S. Oldham: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. D.C. Hornigold: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. J. Trevaskis: Employee; Self; Gilead Sciences, Inc., MedImmune. Stock/Shareholder; Self; AstraZeneca. D. Baker: Stock/Shareholder; Self; AstraZeneca. C.J. Rhodes: Employee; Self; AstraZeneca, MedImmune. A. Rossi: Employee; Self; MedImmune. J. Naylor: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca. J. Dhillon: Employee; Self; MedImmune. A. Buchanan: Employee; Self; MedImmune. L. Liang: None.

2465-PUB: Successful Management of Severe Diabetic Ketoacidosis in a Type 2 Diabetes Patient with Insulin Allergy

Objective: To report an episode of severe diabetic ketoacidosis (DKA) in a type 2 diabetes mellitus (DM) patient with insulin allergy treated by continuous intravenous (IV) regular insulin infusion. Methods: We describe the clinical features, laboratory data, and management of severe DKA in a type 2 DM patient with insulin allergy. Results: A 58-year-old man with type 2 DM initiated subcutaneous insulin administration (SIA) after failure of oral antidiabetic therapies. Symptoms of an allergic reaction then developed, especially distinct at the injection site. Pruritic wheals appeared within 10 minutes of injection and lasted over 24 hours. Both skin prick and intradermal tests were positive with different types of insulin. Two days before admission, he stopped SIA because of allergic symptoms, and then experienced weakness and upper abdominal pain. On admission, his heart rate was 130 beats/min, temperature 37°C, blood pressure 150/90 mmHg, and respiratory rate 28 breaths/min. Blood glucose level was 374.6 mg/dL, pH 6.984, bicarbonate 2.5 mmol/L, lactate 1.5 mmol/L, BUN 24.1 mg/dL, creatinine 1.46 mg/dL, and urinary ketone was 66.48 mg/dL. Over 24 hours, the metabolic acidosis was refractory to IV fluids, bicarbonate and potassium replacement, as well as hemodialysis. Ultimately, he received continuous IV regular insulin infusion at an initial rate of 0.1 units/kg/hour, no further allergic reactions were observed. On the day 5, ketonemia and metabolic acidosis completely resolved. He had transition from IV insulin infusion to SIA on day 14. He was discharged on the day 21 with the treatment of SIA. Three months later, he had a good glycemic control, but has still appeared allergic symptoms at the injection sites of insulin. Conclusion: In this patient, SIA caused an allergic reaction in contrast to continuous IV insulin infusion, for which allergic symptoms did not appear. Thus, identical insulin molecules could behave in markedly different ways depending on the route of injection. Disclosure A.D. Nguyen: None. C.Q. Luong: None. H.C. Chu: None. V.K. Nguyen: None. C.V. Nguyen: None. T.A. Nguyen: None. Q.H. Nguyen: None. T.D. Mai: None. D.V. Nguyen: None. B.Q. Nguyen: None. T.H. Tran: None. N.N. Nguyen: None. S.N. Do: None.

Glucose transporter inhibitor-conjugated insulin mitigates hypoglycemia

Insulin therapy in the setting of type 1 and advanced type 2 diabetes is complicated by increased risk of hypoglycemia. This potentially fatal complication could be mitigated by a glucose-responsive insulin analog. We report an insulin-facilitated glucose transporter (Glut) inhibitor conjugate, in which the insulin molecule is rendered glucose-responsive via conjugation to an inhibitor of Glut. The binding affinity of this insulin analog to endogenous Glut is modulated by plasma and tissue glucose levels. In hyperglycemic conditions (e.g., uncontrolled diabetes or the postprandial state), the in situ-generated insulin analog-Glut complex is driven to dissociate, freeing the insulin analog and glucose-accessible Glut to restore normoglycemia. Upon overdose, enhanced binding of insulin analog to Glut suppresses the glucose transport activity of Glut to attenuate further uptake of glucose. We demonstrate the ability of this insulin conjugate to regulate blood glucose levels within a normal range while mitigating the risk of hypoglycemia in a type 1 diabetic mouse model.

The effect of the chelator characteristics on insulin adsorption in immobilized metal affinity chromatography

Abstract The fact that cryogels have been highly preferred (especially as adsorbent) by scientists in chromatographic processes has made these polymers very popular. In this study, Poly(2-Hydroxyethyl methacrylate-Glycidyl methacrylate), Poly(HEMA-GMA), cryogels were synthesized and then p-aminobenzoic acid, and p-aminopyridine compounds were ligated as a ligand to the structure. In the final stage, a metal ion [Cu(II), Ag(I) or Zn(II)] was immobilized to the polymeric structure, and these structures were used in the adsorption of the insulin molecule from aqueous systems. The highest insulin adsorption performance was observed in cryogel which the p-aminopyridine ligand was bound, and the Cu(II) ions were immobilized. This cryogel was coded as Pr-Cu(II). The insulin adsorption capacity of the Pr-Cu(II) cryogel was tested under varying pH, interaction time, initial insulin concentration, temperature, and ionic strength. On behalf of the characterization works; Fourier Transform Infrared Spectroscopy (FT-IR) analysis, Scanning Electron Microscopy (SEM) analysis, thermal analysis, surface area analysis, elemental analysis, Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES), and Computerized Microtomography (μCT) analysis were performed. The adsorption capacity of the cryogels was calculated to be approximately 242 mg insulin/g cryogel. Additionally, Langmuir isotherm and pseudo-second-order models were found to be suitable for isotherm-kinetic studies of adsorption interaction.

Spectroscopic Evidence of Tertiary Structural Differences Between Insulin Molecules in Fibrils

Protein fibrils are of great interest due to their involvement in several pathologies and their roles in the degradation of many therapeutic protein products. Fibrils share highly similar secondary structural motifs across different proteins and applied stress conditions. However, fibril morphology differs according to the surrounding conditions, with aromatic and hydrophobic amino acids playing important roles in mature fibril formation. In this study, we use Raman microscopy, by means of the aromatic amino acids in insulin molecules as markers, to probe for tertiary structure differences within fibrils. We compared 2 different fibril types, linear fibril bundles and spherulites. Generation of linear fibril bundles was undertaken in an acetic acid–containing formulation, whereas spherulites were generated in a hydrochloric acid–containing formulation. The Raman intensities of tyrosine and phenylalanine side chains suggest that there are significant differences between the fibril bundles. The findings suggest that the insulin components of the fibril strands are not arranged identically in the 2 fibril types and that this gives rise to differences in their tertiary structures. Overall, the work indicates that the physicochemical properties of fibril structures can be altered by changing the formulation and that these alterations can be monitored by Raman spectroscopy.

MON-145 An Unusual Cause of Hypoglycemia in a Non-Diabetic Elderly Male

Introduction: Hypoglycemia (HYPO) in non-diabetic patients is an unusual scenario and presents a diagnostic challenge, resulting in serious consequences for patients. We describe the case of an elderly non-diabetic male who developed severe persistent HYPO. Case: A 78-year-old male with history of macroprolactinoma taking bromocriptine, Hashimoto's hypothyroidism, initially treated with low dose L-thyroxine, followed by Graves' hyperthyroidism, Parkinson’s disease (PD), osteopenia was noted to have hypoglycemia (54 mg/dL) on routine lab work. When questioned, he admitted to having had years of lightheaded episodes, "feeling jittery", which improved within minutes after eating sugary foods. His wife mentioned that he “loved to eat sweets” and high simple carbohydrate meals(CARBS). Physical exam - orthostatic hypotension and mild resting tremors, both attributed to PD. Continuous glucose monitoring (CGMS) was performed for 2 weeks which documented HYPO overnight while sleeping, fasting and between meals. The HYPO was associated more frequently with sweet bedtime snacks. Laboratory- Glucose tolerance test was stopped due to HYPO with symptoms. ACTH=19 pg/mL (nl 0-47), AM Cortisol 19.4 microg/dL (nl 5.3-22.5), fasting blood glucose 83 mg/dL(nl 70-99), fasting insulin 6.9 mIU/L( nl 0-24.9), proinsulin 16.9 pmol/L ( nl <=18.8 pmol/L), c-peptide 1.39 ng/mL( nl 0.81-3.85), beta-OH butyrate 0.072 mmol/L( nl 0.020-0.270). Islet cell Ab was normal. Gastric emptying study was negative. Although the dose of bromocriptine was reduced due to HYPO, it continued. He was advised to decrease intake of CARBS, eat small, high protein and fat meals with frequent snacks. On subsequent visits, he reported less hypoglycemic episodes with improved energy level and general well-being, which was confirmed by CGMS. Discussion: Although a wide range of diseases can cause HYPO in nondiabetic patients, differences in characteristics between non-diabetic HYPO and diabetic HYPO have not been well studied. Common underlying diseases associated with HYPO include malignancies, cerebrovascular diseases, infection, major organ failure, and alcohol-related disorders. In addition, comorbidities linked with HYPO include sepsis, kidney diseases, and alcohol dependence, pneumonia, liver diseases and insulin abuse. Malnutrition, alcohol, infection, and post gastrectomy are the leading causes of nondiabetic hypoglycemia. In addition, methimazole use has been associated with insulin autoimmune syndrome resulting in HYPO due to interaction of sulfhydryl group with disulfide bond in the insulin molecule Conclusion: Clinicians should be aware of the potential for hypoglycemia in non-diabetic patients, due to other etiologies, which can also result in severe and possibly life-threatening sequelae. An in-depth evaluation, including the use of CGMS can assist in determining underlying causes.

Primárna a sekundárna inzulínová rezistencia

Insulin resistance (IR) is defined as insufficient insulin metabolic effect in target tissues, including glucose utilisation in skeletal muscle, suppression of hepatic glucose production and suppression of lipolysis in fat tissue. Primary IR originates as consequence of rare monogenetic defects of insulin receptor or molecules includes to post-receptor insulin signal cascade. Secondary IR originates mainly as a result of metabolic or hormonal changes, most commonly in visceral obesity by multifactorial postreceptor inhibition of insulin signal and it is associated with metabolic syndrome and type 2 diabetes mellitus. It is also present in endocrinopathies with overproduction of contraregulatory insulin hormones (cortisol, growth hormone, catecholamines) and using of some drugs (mainly steroids, immunosuppressive treatment). In practice IR is usually diagnosed by glycemic parameters with confirmation of prediabetic states and type 2 diabetes mellitus. The healthy life style and physical activity associated with weight loss are the most important for type 2 diabetes prevention. According to actual international guidelines metformin is only antidiabetic drug which is possible to use in prediabetic states with high risk of type 2 diabetes development, mainly in obese subjects with BMI &gt; 35 kg/m2, age under 60 years and in women with history of gestational diabetes.

Effects of running on adiponectin, insulin and cytokines in cerebrospinal fluid in healthy young individuals

Exercise can prevent the sedentary lifestyle-related risk of metabolic and cognitive decline, but mechanisms and mediators of exercise effects on human brain are relatively unexplored. We measured acute exercise-induced changes in adiponectin, insulin and other bioactive molecules in cerebrospinal fluid (CSF) and serum from young lean individuals. Samples of serum and CSF were obtained before and 1-h after the 90-min run (75–80% HRmax; maximal heart rate), additional serum was taken at finish-line. Body composition, physical fitness, metabolic rate, cognitive functions, food preference, glucose, insulin and albumin were measured. The spectrum of 174 cytokines was assessed by protein arrays, adiponectin was also determined by ELISA and immunoblotting. CSF adiponectin decreased post-exercise by 21.3% (arrays) and 25.8% (ELISA) (p < 0.009). Immunoblotting revealed reduction in a low-molecular-weight-adiponectin (p < 0.005). CSF adiponectin positively correlated with CSF/serum albumin ratio (p < 0.022), an indicator of blood-brain-barrier permeability. CSF and serum adiponectin were positively associated with memory and running-induced changes in insulinemia and CSF insulin. Additionally, running modulated CSF levels of 16 other cytokines. Acute running reduced CSF adiponectin and modulated insulin and albumin in CSF and serum. Associations of adiponectin with memory and metabolism indicate the potential role of this bioactive molecule in mediating exercise-induced adaptive response in human brain.

Identification and Study of Polymorphic Structures of Hierarchically Twisted Amyloid Fibrils by Atomic Force Microscope

Uncontrolled protein folding often results in amyloid fibrillogenesis associated with many neurodegenerative diseases. Nowadays the interest to this phenomenon is driven also by the observation that some protein fibrils can be functional in vivo, and by exciting perspectives to use them in (nano)technology and for new materials synthesis. At different conditions, numerous proteins might be subject to aggregation, and rather different aggregation mechanisms and polymorphic structures of aggregated proteins were reported. Well-characterized insulin molecules early emerged as a kind of a “model object” to study the process, and the conclusions obtained when investigating insulin fibrillization proved to be important for numerous other proteins. Here, we present the results of systematic Atomic Force Microscopy (AFM)- based studies of insulin molecules fibrillization. We fully exploited the ultahigh z-resolution of AFM, and introduce a new parameter, named Integrated Maximal Height (IMH), which is the sum of average height of a filament and half of its height fluctuation amplitude (i.e. the difference of maximal and minimal height), to classify the observed both twisted and ribbon-like fibril structures. The structures involving up to six mutually twisted protofilaments were observed and quantified. All possible arrangements were recorded for those composed by up to four protofilaments, while ribbon-like structures including five and six protofilaments were not observed, clearly indicating that the formation of such ribbon-like structures are less favorite in our experimental conditions. We discuss the possible relation between the interfilament forces and the peculiarities and statistics of the structures observed, and its meaning for the general fibrillization mechanism.

Molecularly imprinted polymer based quartz crystal microbalance sensor for the clinical detection of insulin

In this study, quartz crystal microbalance sensors based on molecular imprinting technology were fabricated for real-time detection of insulin in aqueous solution and artificial plasma. This study describes the preparation of insulin imprinted poly(hydroxyethyl methacrylate)-N-methacryloyl-(l)-histidine methyl ester based quartz crystal microbalance sensor for insulin determination. Poly(hydroxyethyl methacrylate)-N-methacryloyl-(l)-histidine methyl ester based film on chip surface was synthesized by ultra violet (UV) polymerization for the detection of insulin at low concentrations. At the first step, N-methacryloyl-(l)-histidine methyl ester complex was formed with insulin and then, the insulin imprinted film has been prepared. The characterization of the polymeric film has been conducted with ellipsometry, contact angle, Fourier transform infrared-attenuated total reflectance and atomic force microscopy measurements. Langmuir, Freundlich and Langmuir-Freundlich adsorption isotherm models were applied for this system. The best fitted model to explain the interactions between molecular imprinted chip and insulin molecules was the Langmuir adsorption isotherm (R2: 0.999). The repeatability of insulin imprinted chip was investigated by using of equilibration-binding-regeneration cycles for four times. The detection limit was found as 0.00158 ng/mL. According to the results, the QCM sensor has showed low-detection limit, high selectivity and sensitivity for insulin assay.

The Autoantigenic Proinsulin B-Chain Peptide B11-23 Synergises with the 70 kDa Heat Shock Protein DnaK in Macrophage Stimulation.

Background Heat shock proteins (Hsp) act as intracellular chaperones and in addition are used as adjuvant in vaccines of peptides complexed with recombinant Hsp. By interacting with autologous peptides, Hsp may promote the induction of autoimmune reactivity. Objective Here, we analysed whether the effect of Hsp on macrophages is modulated by insulin peptides known to interact with Hsp. Results Combinations of the 70 kDa Hsp DnaK with peptide B11-23 from the core region of the proinsulin B-chain induced the release of the inflammatory mediators interleukin-6, tumor necrosis factor α, and interleukin-1β from cells of human and murine macrophage lines. In parallel, there was high-affinity binding of B11-23 to DnaK. DnaK mixed with peptides from other regions of the insulin molecule did not stimulate cytokine secretion. DnaK alone induced little cytokine production, and peptides alone induced none. Conclusion The macrophage-stimulating potential of Hsp70 family proteins when combined with the proinsulin B-chain peptide B11-23 may contribute to the immunodominance of this peptide in the development of beta cell-directed autoimmunity in type 1 diabetes.

Optimization of Infrared Microscopy to Assess Secondary Structure of Insulin Molecules Within Individual Subvisible Particles in Aqueous Formulations

The analysis of subvisible particles is currently challenging but pivotal to the understanding and control of the quality of protein therapeutics. While a range of characterization methods is available for subvisible particles, information on the protein conformation in a particle—considered a possible parameter in eliciting unwanted immunogenicity of protein therapeutics—is especially challenging in the lower micrometer range using existing analytical technologies. Using 6 different protein particle populations, we show that transmission Fourier transform infrared (FTIR) microscopy can determine protein secondary structure in single particles down to 10 μm. The analytical setup presented here is able to immobilize protein particles and obtain transmission FTIR spectra on individual protein particles in their intact aqueous environment. Spectra of dried particles, on the other hand, were found to occasionally differ from spectra of particles in aqueous environment. In summary, using the analytical setup described in this study, transmission FTIR microscopy uniquely provides information on single protein particles in particle populations in their aqueous environment without interference from the background protein solution.

In Silico Study of Phospholipids as An Oral Insulin Delivery System

Phospholipids have been applied as a material for an oral insulin delivery system that either in the form of solid lipid nanoparticles, liposomes, or microemulsions. However, the administration of this delivery system for diabetic treatment requires twenty times higher of dosage than the injection one. In the present study, we performed molecular dynamics simulation to evaluate how phospholipids can protect insulin at the molecular level and why its efficiency for the diabetic treatment is low. Phosphatydilcholine group, such as DPPC, OPPC, LPC and combine of DPPC-LPC, was used as the phospholipid material to model the delivery system in our simulations because in vitro and in vivo studies of these phospholipids as the delivery system have been well-conducted. In these simulations, the waters-phospholipid-insulin system was prepared by randomly mixing phospholipids molecules inside the solvent box containing an insulin molecule. NPT ensemble was used in all simulations with the setting temperature at 310 K and 1 atm for the pressure. The simulation results showed that DPPC and LPC delivery systems were able to maintain both secondary and tertiary structures of the encapsulated insulin within the time range of simulation. However, all phospholipid delivery system models failed to fully encapsulate insulin surface within 150 ns simulation. This study may explain the reason for the low success rate of using phospholipid as the delivery system of oral insulin in the experiment.

Investigation of manganese(II)-insulin complexes using electrospray ionization mass spectrometry

Manganese is a trace element in human nutrition. It is involved in many enzymes, proteins and biological activities. Mn(II) ion has the capable of binding to protein or peptides. Insulin is a blood glucose-lowering peptide hormone and it is secreted by the pancreatic β-cells. In this study, the binding capability of Mn(II) ions to insulin was studied using ESI-MS, nano-ESI-MS and MS/MS methods. The binding of Mn(II) ions to insulin molecule was studied by examining the effect of pH, the molar ratio of Mn(II) ions to insulin, the flow rate with nano-ESI system and MS/MS spectrometry. The ESI-MS measurements showed that the Mn(II)-insulin complexation mostly produces ML and M2L type complexes. The highest binding ratio was found at pH 7. The complex formation equilibrium constants of Mn(II)-insulin were calculated as Kf1: 1.03 ± 0.12 × 104 and Kf2: 1.93 ± 0.17 × 103. The nano-ESI-MS and MS/MS measurements exhibited strong and specific binding of Mn(II) ions to insulin molecule. It was concluded from all the ESI-MS measurements that Mn(II) ion has a high affinity to insulin molecule to form stable complexes.