Insulin Molecule Research Articles

Adsorption of human insulin on single-crystal gold surfaces investigated by in situ scanning tunnelling microscopy and electrochemistry

We have explored the adsorption of zinc-free human insulin on the three low-index single-crystalline Au(111)-, Au(100)- and Au(110)-surfaces in aqueous buffer (KH(2)PO(4), pH 5) by a combination of electrochemical scanning tunnelling microscopy (in situ STM) at single-molecule resolution and linear sweep, LSV, cyclic, CV, and square wave (SQWV) voltammetry.Multifarious electrochemical patterns were observed. Most attention was given to reductive desorption caused by insulin binding to the Au-surfaces via up to three disulfide groups per insulin monomer, presumably converted to single Au-S links. SQWV suggested the Au-S bond strength order Au(111) > Au(110) > Au(100) based on the reductive desorption potentials. The voltammetric diversity was paralleled by different in situ STM insulin adsorption modes on the three surfaces. Single-molecule resolution was achieved in all cases. The coverage followed the order Au(110) > Au(100) > Au(111) and differs from the reductive desorption order that records the Au-S bonding element. Evenly distributed single molecules were scattered over large Au(111)-terraces, with intriguing molecular arrays disclosed near the terrace edges. In comparison, high-density molecular scale structures were observed both over the terraces and across terrace edges on Au(100). Larger rectangular structures also appeared (8-12% coverage). These are Au-islands from the lift of the reconstruction. Notably, 10 x 10 nm(2) patches of highly ordered much smaller structures, possibly from insulin decomposition emerged sporadically within the dense insulin adlayer. Insulin adsorbed in highest coverage on the Au(110) and followed the directional surface topology with insulin molecules aligned in the Au(110)-surface grooves, occasionally "spilling over" and merging into larger structures.Adsorption, Au-S binding, and insulin unfolding are all parts of insulin surface behaviour and reflected in both voltammetry and in situ STM. In spite of these complications, the data show that molecular scale resolution has been achieved and offer other perspectives of insulin surface science such as single-molecule mapping of the insulin monomer/dimer-hexamer interconversion.

A brief account on the study of the insulin crystal structure

Approximately forty years ago when China was undergoing a period of great difficulty, Science in China, previously known as Scientia Sinica, published articles during three consecutive years (Scientia Sinica (test issue) 1972; Scientia Sinica 1973, No. 1, 93; Scientia Sinica 16(1973)136; Scientia Sinica 17(1974)51) on crystal structure analysis of the rhombohedral 2-zinc pork insulin molecule at resolutions of 4.0 A, 2.50 A and 1.80 A, respectively (Figures 1 and 2). These publications delivered rare information on scientific research from a China which, at the height of the Cultural Revolution, was otherwise closed to the outside world. The news made international impact because this was the first protein crystal structure solved in Asia and one of the few rare structures solved worldwide; it heralded the start of intensive biophysical research in China. Nobel Laureate Dorothy Hodgkin of Oxford University traveled to the Institute of Physics in 1972 to visit the Beijing Insulin Structure Research Group (Figure 3). There, she spent one week carrying out a detailed comparison of electron density maps from the Oxford and Beijing labs and met extensively with the Institute’s young Chinese scientists. Later, at the 9th International Conference of Crystallography in Kyoto, she told the story of her visit stating that, “standards there [were] as high as achieved elsewhere.” She further impressed international crystallographers with her comments in a “News and Views” article in Nature (Nature, 1975, 255: 103) that “the present Peking map at 1.80 A resolution is the most accurate map available of the insulin electron density defined by experimental, isomorphous phase angles... and may well remain so.” Additionally, Professor Watanabe, the famous Japanese crystallographer at Osaka University, wrote to the Institute in Beijing seeking permission to translate the 2.50 A article for publication in Japanese journals. Many other scientists of world famous laboratories also poured in to visit. Behind the articles published by Science in China lay a hidden story of dedication, courage and collective wisdom and expertise of intelligent young scientists who worked solidly and tirelessly over many difficult years (Figures 4 and 5). The year 2010 hails the 60th anniversary of this journal and seems to be an exceptionally appropriate opportunity for making a brief recollection of that experience, not only to pay tribute to all the members of the group but also to show gratitude to the journal.

Probing adsorption and aggregation of insulin at a poly(acrylic acid) brush

A planar poly(acrylic acid) (PAA) brush provides an unusual substrate for the unspecific immobilization of proteins on material surfaces. At neutral pH-values, proteins adsorb at a PAA brush when the ionic strength of the protein solution is low. In contrast, raising the ionic strength to a few 100 mM transforms a PAA brush into a rather protein-resistant surface coating. Moreover, a PAA brush represents a mild environment for adsorbed proteins which preserves their secondary structure and biological activity. In this study, we focus on the biocompatibility of a PAA brush with an insulin solution. Insulin can form amyloid fibrils, which may also be triggered by interfaces. Using neutron reflectometry and attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, the effects of pD value, ionic strength, and glycerol concentration on the density profile and the secondary structure of adsorbed insulin molecules at a PAA brush have been studied. At pD 7, insulin adsorbs at a PAA brush despite its negative net charge. As has been found for other proteins in earlier studies, increasing the ionic strength of the insulin solution to 500 mM decreases the amount of adsorbed insulin drastically. In contrast, at pD 2, addition of salt to the insulin solution induces a thick insulin adsorbate at a PAA brush suggesting both protein-brush and protein-protein interactions, i.e., insulin adsorption and aggregation to be effective. However, in the presence of 2 M glycerol, insulin adsorption is largely suppressed. Furthermore, no major alterations of the secondary structure of adsorbed insulin can be detected by ATR-FTIR spectroscopy under all conditions studied. Hence, the performed experiments demonstrate that a PAA brush does not promote the formation of insulin amyloid structures, which represents a fundamentally new aspect of the biocompatibility of this material surface coating.

Presence of anti-insulin natural autoantibodies in healthy cats and its interference with immunoassay for serum insulin concentrations

A substance interfering with the enzyme-linked immunosorbent assay (ELISA) for feline insulin concentration was investigated in healthy cats. An insulin-binding substance isolated from feline serum showed 2 bands at 25 and 50 kDa in SDS-PAGE, suggesting the presence of immunoglobulin G (IgG). Insulin-binding IgG from healthy cats indeed reduced insulin immunoreactivity in the ELISA for determining insulin concentration. The insulin-binding IgG was polyclonal/polyreactive and showed certain specificity, high affinity, and high binding capacity, which was evaluated by liquid-phase radioimmunoassay with Scatchard plot analysis. Epitope analysis revealed that the insulin-binding IgG showed significant binding at residues A1-5 and B20-30 of the insulin molecule. Removal of the antibodies from serum enabled the determination of serum insulin concentrations by ELISA. Our data indicated that serum from healthy cats contained substantial amounts of natural autoantibodies combined with insulin, and that the antibodies interfered with the heterologous immunoassay for serum insulin concentration.

A brief perspective on insulin production

The preeminent role of the beta cell is to manufacture, store and release insulin. The mature insulin molecule is composed of two polypeptide chains designated as A and B that are joined by two pairs of disulfide bonds with an additional intramolecular disulfide bond in the A chain. However, the two chains of the insulin molecule are not synthesized as separate polypeptide chains but rather are generated by specific proteolytic processing of a larger precursor, proinsulin. This discovery in 1967 and the concept of prohormones changed our view of the biosynthesis of hormones and neuropeptides. It allowed studies of the regulation of insulin biosynthesis that highlighted the key role of glucose. In addition, the C-peptide, the polypeptide that joins the A and B chains in proinsulin and is stored with insulin in the secretory granules and secreted in equimolar amounts, allowed studies of pancreatic beta cell function in vivo including in patients with diabetes. Subsequent studies have identified the specific proteases, prohormone convertases 1/3 and 2 and carboxypeptidase E, that are involved in the conversion of proinsulin to proinsulin intermediates and then to insulin. Disorders of (pro)insulin biosynthesis continue to illuminate important aspects of this pathway, revealing important connections to diabetes pathogenesis. Recent studies of patients with insulin gene mutations that cause permanent neonatal diabetes have identified key residues affecting the folding and structural organization of the preproinsulin molecule and its subsequent processing. These findings have renewed interest in the key role of endoplasmic reticulum function in insulin biosynthesis and the maintainance of normal beta cell health.

A neutron crystallographic analysis of T6 porcine insulin at 2.1 A resolution.

Neutron diffraction data for T(6) porcine insulin were collected to 2.1 A resolution from a single crystal partly deuterated by exchange of mother liquor. A maximum-likelihood structure refinement was undertaken using the neutron data and the structure was refined to a residual of 0.179. The hydrogen-bonding network of the central core of the hexamer was observed and the charge balance between positively charged Zn ions and their surrounding structure was interpreted by considering the protonation and/or deprotonation states and interactions of HisB10, water and GluB13. The observed double conformation of GluB13 was essential to interpreting the charge balance and could be compared with the structure of a dried crystal of T(6) human insulin at 100 K. Differences in the dynamic behaviour of the water molecules coordinating the upper and lower Zn ions were observed and interpreted. The hydrogen bonds in the insulin molecules, as well as those involving HisB10 and GluB13, are discussed. The hydrogen/deuterium (H/D) exchange ratios of the amide H atoms of T(6) porcine insulin in crystals were obtained and showed that regions highly protected from H/D exchange are concentrated in the centre of a helical region of the B chains. From the viewpoint of soaking time versus H/D-exchange ratios, the amide H atoms can be classified into three categories.

Dual behavior of sodium dodecyl sulfate as enhancer or suppressor of insulin aggregation and chaperone-like activity of camel αS 1-casein

Sodium dodecyl sulfate (SDS) at low concentrations considerably enhanced insulin aggregation and reduced the chaperone-like activity of purified camel αS 1-casein (αS 1-CN). These observed changes were the result of repulsive electrostatic interactions between both negative charged head groups of SDS and αS 1-CN, and the net negative charge of insulin molecules, resulting in the greater exposure of hydrophobic patches of insulin and its enhanced aggregation. In contrast, enhanced hydrophobic interactions were primarily responsible for the conformational changes observed in insulin and αS 1-CN at high SDS concentrations, resulting in increased binding of SDS and αS 1-CN to insulin and its reduced aggregation.

Crystallization and structure of two bovine insulin derivatives

Insulin is a polypeptide hormone, produced by the  -cells in the pancreas, that regulates carbohydrate metabolism and it also takes part in the metabolism of fat and proteins. Insulin is structured as two polypeptide chains, chain A consists of 21 and chain B of 30 amino acids. Bovine insulin differs in sequence from human insulin at residues A8, A10 and B30. There are three forms of insulin hexamers. These forms of insulin are used in therapeutc preparations for the control of diabetes. The crystal structure of T6 bovine insulin was described by Smith et al in 2005 [1]. It has been known for many years that native insulin is in the T3R3 form in the crystals grown in high chloride ion concentrations [2]. This work presents crystallization of two bovine insulin derivatives: in high iodine and bromine concentrations. Structural investigations on bovine bromo- and iodo-derivatives show that these halogen atoms are bound to the zinc ions. Crystals were grown by the hanging drop vapour diffusion crystallisation method. Single crystal diffraction data were collected on laboratory instrument at 100 K. The investigated insulin derivatives belong to the R3 rhombohedral space group with cell parameters a = 79.73 A ; , c = 36.34 A ; and a = 79.25 A ; , c = 36.97 A ; for the bromo- and iodo-derivative, respectively. Conformation of the insulin molecule and coordination of Zn- ions will be discussed.

The role of insulin and proinsulin in the regulation of triglyceride metabolism.

The mechanisms underlying the associations between abnormal glucose tolerance and myocardial infarction are poorly understood. It has often been suggested that an increased plasma insulin concentration is causally linked to many of the metabolic abnormalities that are associated with abnormal glucose tolerance, although this suggestion remains controversial. Recently it has been proposed that proinsulin and proinsulin-like molecules may also be involved in the atherogenic process. Both hyperinsulinaemia and insulin resistance are associated with fasting hypertriglyceridaemia and both increased VLDL production and increased plasma triglyceride concentrations commonly occur in association with abnormal glucose tolerance and atheromatous vascular disease. In order to study the effects of insulin, proinsulin and proinsulin-like molecules on hepatic triglyceride secretion we have undertaken experiments in vitro using the liver cell line HepG2. In conjunction with these in vitro experiments we have also studied, in vivo, the associations between insulin, proinsulin, proinsulin-like molecules and plasma triglyceride concentrations in subjects with both normal and abnormal glucose tolerance. Our results in vitro show that proinsulin and proinsulin-like molecules have similar and not different effects to insulin but are less biologically active. In vivo, our results show that concentrations of insulin, proinsulin and proinsulin-like molecules per se are not an important determinant of plasma triglyceride concentrations. Both abnormal NEFA suppression during an oral glucose tolerance test and increased central adiposity are closely linked to poor glucose tolerance and are the most important determinants of plasma triglyceride concentrations. Taken together these results suggest that it is not insulin nor proinsulin concentrations per se that are causally linked to hypertriglyceridaemia. We suggest that abnormal NEFA suppression plays an important part in the increase in risk of vascular disease associated with insulin resistance.

New developments in the treatment of type 1 diabetes mellitus.

Treatment of type 1 diabetes mellitus has made tremendous advances within the last decades. With concern to insulin delivery there are two promising new approaches. One is the intrapulmonary insulin delivery which has become feasible by the development of new inhalation devices which provide a sufficient degree of intrapulmonary drug retention. Also oral insulin delivery seems feasible when surface active substances are used to cross the mucosal membrane in the gut. Clinical research has also focussed on coatings for the insulin molecules to solve the problem raised by the proteolytic activity of the digestive system. A very new agent produced by a fungus called Pseudomassaria has been demonstrated to reverse the clinical signs of diabetes mellitus in mice. The compound diffuses through the cell membrane, binds to the inner part of the insulin receptor and activates the insulin typical biological effects. Nowadays a variety of insulin analogs are designed and tested for their clinical use. By shifting the isoelectric point towards to a slightly acidic pH, HOE 901 precipitates at physiologic pH resulting in a constant and peakless insulin delivery. NN 304 is a 14-carbon aliphatic fatty acid acylated analog that binds to serum albumin resulting in a flatter time-action profile than NPH insulin. Also rapid acting insulin analogs are or will be launched in the near future aiming to ensure an improved postprandial glucose regulation. Glucagon-like peptide-1 (GLP-1) improves metabolic control by a variety of effects, e.g. the enhancement of insulin secretion and inhibition of glucagon secretion. Moreover, GLP-1 reduces food and water intake controlled by the brain, and inhibits gastric emptying. A disadvantage of GLP-1 is its very short half-life. Novel derivatives with the beneficial effects of GLP-1 but a better resistance against degradation have been designed. In addition substances have been developed inhibiting GLP-1 degradation or augmenting GLP-1 release from its abundant endogenous pool. Finally, there is a variety of interesting approaches aiming to improve or ease blood glucose self-monitoring. One is the development of subcutaneous catheters for continuous blood glucose control. In another system reverse iontophoresis is used for sampling interstitial fluid which reflects capillary blood glucose levels. Instead of using an electric current, a brandnew system creates micropores in the skin by a laser ablation system. Through these micropores a specific device performs a mild suction to obtain intersitial fluid. Further systems which measure blood glucose by near infrared spectroscopy are still investigated in order to improve their technical function and to reduce their weight. This article intends to give an overview over the new developments in the treatment and management of type-1-diabetes mellitus.

Self‐Assembled Nanogel of Hydrophobized Dendritic Dextrin for Protein Delivery

Highly branched cyclic dextrin derivatives (CH-CDex) that are partly substituted with cholesterol groups have been synthesized. The CH-CDex forms monodisperse and stable nanogels with a hydrodynamic radii of approximately 10 nm by the self-assembly of 4-6 CH-CDex macromolecules in water. The CH-CDex nanogels spontaneously trap 10-16 molecules of fluorescein isothiocyanate-labeled insulin (FITC-Ins). The complex shows high colloidal stability: no dissociation of trapped insulin is observed after at least 1 month in phosphate buffer (0.1 M, pH 8.0). In the presence of bovine serum albumin (BSA, 50 mg . mL(-1)), which is a model blood system, the FITC-Ins trapped in the nanogels is continuously released ( approximately 20% at 12 h) without burst release. The high-density nanogel structure derived from the highly branched CDex significantly affects the stability of the nanogel-protein complex.

Shear-Induced Deformation of Bovine Insulin in Couette Flow

We have applied a uniform, shear-driven flow field (Couette flow) to study the effect of shear on the structure and conformation of aqueous bovine insulin, in situ and in real time, using intrinsic Tyr fluorescence and circular dichroism (CD) spectroscopy. The morphology of post-shear insulin samples was analyzed using atomic force microscopy (AFM). Both fluorescence and CD data show a shear-dependent deformation of bovine insulin in Couette flow. The shear effect is more pronounced with increasing shear rate. AFM images show large aggregates for insulin samples sheared at 200 and 400 s(-1), whereas samples sheared at 600 s(-1) contained fibrillar forms. We hypothesize that helical segments unfold upon extensional strain in the deformation flow field, resulting in unstructured, aggregation-prone insulin molecules. The occurrence of rotational diffusion in the direction of flow facilitates the coalescence of deformed insulin molecules into oligomeric aggregates. The size of the insulin aggregates diminished with increasing shear rate. This shows that the deformation cycle in fast flow fields retards the formation of large aggregates and promotes the ordering of deformed insulin molecules into the more stable fibrillar forms.

Noncooperative Dimethyl Sulfoxide-Induced Dissection of Insulin Fibrils: Toward Soluble Building Blocks of Amyloid

The enormous molecular weight complicates detailed structural studies of amyloid fibrils and obscures identification of biologically active forms of protein aggregates in amyloid-related diseases. Here we show that aqueous solutions of dimethyl sulfoxide (DMSO) solubilize insulin fibrils while maintaining their beta-pleated structure. This is accompanied by a marked decrease in the fluorescence of thioflavin T. According to atomic force microscopy images and dynamic light scattering measurements, the partial DMSO-induced dissection of insulin fibrils favors formation of smaller soluble oligomers, which retain a limited capacity to induce daughter generation of fibrils through seeding to the native insulin, as well as the ability to reassemble into fibrils upon removal of DMSO through dialysis against water. These findings suggest that the DMSO-induced ensembles of insulin molecules are closely related to elementary building blocks of amyloid fibrils.

Molecular Basis of Catalytic Chamber-assisted Unfolding and Cleavage of Human Insulin by Human Insulin-degrading Enzyme

Insulin is a hormone vital for glucose homeostasis, and insulin-degrading enzyme (IDE) plays a key role in its clearance. IDE exhibits a remarkable specificity to degrade insulin without breaking the disulfide bonds that hold the insulin A and B chains together. Using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to obtain high mass accuracy, and electron capture dissociation (ECD) to selectively break the disulfide bonds in gas phase fragmentation, we determined the cleavage sites and composition of human insulin fragments generated by human IDE. Our time-dependent analysis of IDE-digested insulin fragments reveals that IDE is highly processive in its initial cleavage at the middle of both the insulin A and B chains. This ensures that IDE effectively splits insulin into inactive N- and C-terminal halves without breaking the disulfide bonds. To understand the molecular basis of the recognition and unfolding of insulin by IDE, we determined a 2.6-A resolution insulin-bound IDE structure. Our structure reveals that IDE forms an enclosed catalytic chamber that completely engulfs and intimately interacts with a partially unfolded insulin molecule. This structure also highlights how the unique size, shape, charge distribution, and exosite of the IDE catalytic chamber contribute to its high affinity ( approximately 100 nm) for insulin. In addition, this structure shows how IDE utilizes the interaction of its exosite with the N terminus of the insulin A chain as well as other properties of the catalytic chamber to guide the unfolding of insulin and allowing for the processive cleavages.

Bovine Insulin Filaments Induced by Reducing Disulfide Bonds Show a Different Morphology, Secondary Structure, and Cell Toxicity from Intact Insulin Amyloid Fibrils

Amyloid fibrils are associated with more than 20 diseases, including Alzheimer's disease and type II diabetes. Insulin is a 51-residue polypeptide hormone, with its two polypeptide chains linked by one intrachain and two interchain disulfide bonds, and has long been known to self-assemble in vitro into amyloid fibrils. We demonstrate here that bovine insulin forms flexible filaments in the presence of a reducing agent, Tris (2-carboxyethyl) phosphine. The insulin filaments, possibly formed due to partial reduction of S-S bonds in insulin molecules, differ from intact insulin fibrils in terms of their secondary structure. The insulin filaments were determined to have an antiparallel β-sheet structure, whereas the insulin fibrils have a parallel β-sheet structure. Of importance, the cell toxicity of the insulin filaments was remarkably lower than that of the insulin fibrils. This finding supports the idea that cell toxicity of amyloids correlates with their morphology. The remarkably low toxicity of the filamentous structure should shed new light on possible pharmacological approaches to the various diseases caused by amyloid fibrils.

Docking of Insulin to its Receptor

The human insulin receptor (IR) is a homodimeric membrane-spanning ligand-activated tyrosine kinase. Although a detailed theory describing how binding of the hormone insulin triggers kinase activity of IR remains elusive, the recently published crystal structure of the IR ectodomain offers the unique opportunity to use physical modeling to begin to construct this theory. We present here the results of ∼100 ns of explicit-solvent all-atom molecular dynamics (MD) simulations of IR in both the apo and putative T and R-state insulin docked states. Our simulations confirm the large interdomain flexibility of IR and the stability of its dimeric interfaces. More importantly, however, our simulations demonstrate the evolution of large-scale asymmetry in IR relative to the crystal structure, a result that reflects the structural requirements of a “see-saw” mechanism that guarantees negative homeotropic allostery in insulin binding. This asymmetry also manifests itself in the opening of one of the two equivalent insulin binding pockets and closing of its partner. This result is significant because it allows for the first time computational docking of an intact molecule of insulin into its binding pocket on an intact IR ectodomain. We use a Monte-Carlo docking algorithm followed by MD equilibration to predict bound states of insulin on IR. These simulations allow us to identify unambiguously the residues on IR that form the “site-2” binding epitope which recognizes residues on insulin responsible for its hexamerization.

Biomarkers of oxidant stress, insulin sensitivity and endothelial activation in rheumatoid arthritis: a cross-sectional study of their association with accelerated atherosclerosis

BackgroundWomen with rheumatoid arthritis (RA) have increased morbidity and mortality due to coronary heart disease. Chronic systemic inflammation is known to accelerate atherosclerosis and increase arterial stiffness in patients, but other mechanisms may also be involved. Biomarkers of oxidant stress, inflammation, insulinaemia and endothelial dysfunction were measured in blood and urine from 46 RA patients and 48 age-matched controls. Plaque formation and intima-medial thickness (IMT) were measured using B-mode carotid Doppler scan.FindingsThe prevalence of plaque was increased (p = 0.042) in RA patients between 50–59 years old compared to the same age group in controls. 8-isoprostane (p = 0.004), C-reactive protein (p < 0.001), interleukin-6 (p < 0.001), insulin (p = 0.035), adiponectin (p = 0.012), vascular cell adhesion molecule (VCAM) (p = 0.029) and E-selectin (p < 0.001) were all increased while selenium (p = 0.003) and LDL-cholesterol (p = 0.025) were both decreased in all RA patients. 8-isoprostane correlated with 10 year cardiac risk (r = 0.55, p < 0.001), VCAM with IMT (r = 0.37, p = 0.012) and E-selectin with rheumatoid factor titre (r = 0.43, p = 0.003) in RA patients. In the control group, age, carotid IMT, VCAM, systolic blood pressure and smoking status were all associated with plaque development whereas in RA patients only age was associated with plaque.ConclusionThe burden of atherosclerosis is particularly increased in middle-aged women with RA. Patients with RA have increased levels of oxidant stress, inflammation, insulin and soluble adhesion molecules. As the association between classical risk factors was much weaker in RA patients compared to controls, these additional factors may be more important in the accelerated development of atheroma in RA.

Water/O2-Plasma-Assisted Treatment of PCL Membranes for Biosignal Immobilization

The main purpose of this study was to obtain COOH functionalities on the surface of poly-ε-caprolactone (PCL) membranes using low-pressure water/O2-plasma-assisted treatment. PCL membranes were prepared using the solvent-casting technique. Then, low-pressure water/O2 plasma treatments were performed in a cylindrical, capacitively coupled RF-plasma-reactor in three steps: H2O/O2-plasma treatment; in situ (oxalyl chloride vapors) gas/solid reaction to convert –OH functionalities into –COCl groups; and hydrolysis for final –COOH functionalities. Optimization of plasma modification processes was done using the DoE software program. COOH and OH functionalities on modified surfaces were detected quantitatively using the fluorescent labeling technique and an UVX 300G sensor. Chemical structural information of untreated, plasma treated and oxalyl chloride functionalized PCL membranes were acquired using pyrolysis GC/MS and ESCA analysis. High-resolution AFM images revealed that nanopatterns were more affected than micropatterns by plasma treatments. AFM images recorded with amino-functionalized tips presented increased size of the features on the surface that suggests higher density of the carboxyls on the nanotopographical elements. Low-pressure water/O2-plasma-treated and oxalyl chloride functionalized samples were biologically activated with insulin and/or heparin biosignal molecules using a PEO (polyoxyethylene bis amine) spacer. The success of the immobilization process was checked qualitatively by ESCA analysis. In addition, fluorescent labeling techniques were used for the quantitative determination of immobilized biomolecules. Cell-culture experiments indicated that biomolecule immobilization onto PCL scaffolds was effective on L929 cell adhesion and proliferation, especially in the presence of heparin.

Nuevas insulinas. Actualización

Standard human insulin preparations such as regular soluble insulin and neutral protamine Hagedorn (NPH) insulin have shortcomings in terms of their pharmacokinetic and pharmacodynamic properties that limit their clinical efficacy. In the last decade, due to advances in genetic engineering, structurally modified insulin molecules have been developed with the aim of changing their galenic properties to make regular soluble insulin faster and NPH insulin slower. Five insulin analog preparations have become commercially available: two long-acting analogs (insulin glargine and detemir) and three rapid or fast-acting analogs (insulin lispro, aspart and glulisine). This article highlights the specific properties of these new insulin analog preparations and focuses on their potential advantages and disadvantages when used in children with diabetes mellitus.

Insulin analogues: an example of applied medical science

Insulin analogues were developed to try and achieve more physiological insulin replacement from injection in the subcutaneous site. Their pharmacokinetics and pharmacodynamics differ from human insulin when injected subcutaneously because of alterations in the amino acid sequence of the insulin molecule. The rapid-acting insulin analogues, lispro, aspart and glulisine, have a rapid onset of action and shorter duration of action because of changes to the B26-30 portion of insulin inhibiting formation of dimers and hexamers. They appear to improve postprandial glucose, incidence of hypoglycaemia and patient satisfaction and, when used in combination with basal insulin analogues, improve glycosylated haemoglobin in comparison to conventional insulin therapy. Additionally, they have been successfully used in children, pregnant women, in pump therapy and as part of premixed biphasic regimens. The two basal insulin analogues, glargine and detemir, developed by adjusting the isoelectric point and adding a fatty acid residue, respectively, have a protracted duration of action and a relatively smooth profile. Their pharmacokinetic and pharmacodynamic profiles have been assessed using euglycaemic clamp protocols. Both analogues have a longer duration of action, less of a peak of activity and a reduced variability with repeated injection. There is some evidence to suggest that detemir may have a slight hepatoselective effect. Clinical studies have shown a lower relative risk of hypoglycaemia and detemir appears to have a weight-sparing action. Insulin analogues represent a successful example of applied medical science.