Studies on Long-acting Insulin:Crystal Structure of Arg-B31 Human Insulin at 2.0 Resolution

The crystal structure of Arg-B31 human insulin(ABHI), a long-acting insulin derivative, has been determined at 2.0 resolution by using X-ray diffraction analysis. The final crystallographic R factor of the structure model after the refinement is 0.189 with the bond length r. m. s deviation of 0.018 . The refined structure of ABHI showed that the conformation of B-chain C-terminal residues was more stable than that in the native molecule. A striking structural feature of ABHI was an additional ion pair formed between ArgB31 of molecule Ⅰ and Glu-B21 of molecule Ⅱ in a dimer, and three ionic bonds between the neighbouring molecules thereby appeared on the surface of ABHI hexamer.These secondary bonds generated by the insertion of the residue Arg-B31 should make the rate of dissociation of ABHI hexamer slow down when it was injected into the body and the property of protraction should be produced by a 'depot effect'. This ought to be the main structure basis of the prolonged action of ABHI. The results o     

Separation of human,bovine, and porcine insulins,three very closely related proteins,by micellar electrokinetic chromatography

Human, bovine, and porcine insulins are small proteins with very closely related amino acid sequences, which makes their separation challenging. In this study, we took advantage of the high‐resolution power of CE, and more particularly of micellar electrokinetic chromatography, to separate those biomolecules. Among several surfactants, perfluorooctanoic acid ammonium salt was selected. Then, using a design of experiments approach, the optimal BGE composition was found to consist of 50 mM ammonium acetate pH 9.0, 65 mM perfluorooctanoic acid ammonium salt, and 4% MeOH. The three insulins could be separated within 12 min with a satisfactory resolution. This method could be useful to detect possible counterfeit pharmaceutical formulations. Indeed, it would be easy to determine if human insulin was replaced by bovine or porcine insulin.     

THE CRYSTAL STRUCTURE OF SILVER CARP INSULIN AT MEDIUM RESOLUTION

It is an important way of surveying the structure-functionrelationship of insulin tostudy insulins from different species. Based on the structure model of an orthorhombic crys-tal obtained by the molecular replacement method, the crystallographic refinement of a hex-amer of silver carp insulin in an asymmetric unit has been carried out with 2.8A resolutiondata using the restrained least- squares method. The comparisons of insulin structures haveshown that the six silver carp insulin molecules have very similar but not identical three- di-mensional structures which are similar to the known 2Zn pig insulin structure but remarka-bly different in some local conformations.     

Simultaneous quantitative determination of insulin aspart and insulin degludec in human plasma using simple shoot LC method

Combining short-acting and long-acting insulin analogs was a real challenge that was overcome by NovoNordisk through the co-formulation of insulin aspart and insulin degludec in single-dosage form. The proposed study provides a simple, short, and reliable HPLC method with diode array detection that is developed and validated for the simultaneous determination of insulin aspart and insulin degludec in human plasma. The proposed method achieved good separation between the two analytes utilizing a C8 column at 35°C in a very short run time (6 min), with a simple, low-cost, and reliable extraction method through precipitation of plasma protein. Gradient elution was applied using a mobile phase consisting of 0.1 M sodium sulfate (pH 3.4) and acetonitrile. The method was validated according to EMA Guideline on Bioanalytical Validation. The proposed method had a linear range from 3.0 to 300 μg/mL for insulin aspart and from 3.5 to 300 μg/mL for insulin degludec. The intra- and inter-day precision of insulin aspart were 0.36–3.33% and 1.59–8.84%, respectively, and accuracy was between 10.06 and 3.09% The intra- and inter-day precision of insulin degludec were 0.29–1.93% and 0.89–5.14%, respectively, and accuracy was between −5.29 and 3.91%.     

THE CHARACTERISTICS AND MOTION MODEL OF INSULIN MONOMER

The extensive conformational comparisons among the determined structures of the differeat species and crystal forms of insulin and the varied insulin derivatives were performed by using the least-squares superimposition technique and the graphics technique. The results of the investigation showed that the structure of molecule I in 2Zn insulin was closer to that of the natural monomer; the conformational difference between two molecules of a dimer came out during dimerization and it was further improved and stabilized during the hexamerization and packing of hexamers in crystal; through the hinge peptides, such as A10, B4, B8, B24, B20 and B23, there was a flexible relative motion among the structural segments in the insulin molecule, and the residues at the B-chain C-terminal might have a shift of more than 10; the mobility for each residue side-chain was very different due to the different surroundings.     

油酸酰化胰岛素的制备及飞行质谱分析

采用二环己基碳二亚胺缩合法由油酸和苯并三唑合成了油酰苯并三唑,并利用FT-IR和1H NMR对产物进行了表征;利用油酰苯并三唑对胰岛素侧链氨基在非保护下进行化学修饰,制备得到油酰胰岛素,飞行时间质谱分析显示该修饰作用是成功的,并且单修饰是主要的,有少量二修饰物,没有三修饰产物.     

STUDIES ON THE CRYSTAL STRUCTURE OF Al-(D-TRYPTOPHAN) INSULIN

We have determined the crystal structure of Al-(D-Trp) insulin and discovered that it belongs to the trigonal system with space group R3. The parameters of the unit cell are a=b=78.6, c=50.0. A set of data for half a sphere reciprocal space to a spacing of 2.2 were collected. The model was adjusted and refined by using a step-by-step approach and a stereochemically-restrained least squares program, assisted by manual revision based on the difference Fourier maps, to a final R-factor of 0.218. The main and side chains of both Al-D-Trp residues in the asymmetric unit are well ordered. The packing of Al-(D-Trp) insulin in the unit cell, the conformational differences with other insulin structures and its structure and function relationship bave also been discussed.     

CRYSTAL STRUCTURE DETERMINATION OF DESHEPTAPEPTIDE (B24—B30)-INSULIN

Desheptapeptide (B24—B30)-insulin (DHPI), an essentially inactive insulin analog, is crystallized in space group P2_12_12_1 with two molecules in an asymmetric unit. The orientations of the molecules in the crystal cell have been determined by using Patterson search method at 6 resolution and the positions of the molecules are deduced from translation function calculation and R search at 3. resolution. After using the rigid body refinement (CORELS) further to refine the orientational and positional parameters as well as the initial energy restrained refinement (EREF) for the model, the crystallographic R valueis reduced to 0.384 at 3 resolution. The initial Fourier map shows that the B-chain N-terminal (B1—B8) and C-terminal (B20—B22)segments, compared with the native 2 zinc insulin, exhibit drastic conformational changes, but the three helices of B- and A-chains and their relative arrangement are essentially kept in DHPI.     

亚油酸酰化胰岛素的制备及飞行质谱分析

胰岛素侧链氨基的酰化是胰岛素化学修饰较常用的方法。天然胰岛素分子中有三个裸露的氨基,即G lyA1、PheB1的α-氨基和LysB29的ε-氨基,都可作为胰岛素化学修饰的位点。酰化试剂通常经由N-羟基琥珀酰亚胺酯对胰岛素进行修饰。N-羟基琥珀酰亚胺酯可由羧酸与N-羟基琥珀酰亚胺(HOS     

An Aldehyde Responsive,Cleavable Linker for Glucose Responsive Insulins

A glucose responsive insulin (GRI) that responds to changes in blood glucose concentrations has remained an elusive goal. Here we describe the development of glucose cleavable linkers based on hydrazone and thiazolidine structures. We developed linkers with low levels of spontaneous hydrolysis but increased level of hydrolysis with rising concentrations of glucose, which demonstrated their glucose responsiveness in vitro. Lipidated hydrazones and thiazolidines were conjugated to the LysB29 side-chain of HI by pH-controlled acylations providing GRIs with glucose responsiveness confirmed in vitro for thiazolidines. Clamp studies showed increased glucose infusion at hyperglycemic conditions for one GRI indicative of a true glucose response. The glucose responsive cleavable linker in these GRIs allow changes in glucose levels to drive the release of active insulin from a circulating depot. We have demonstrated an unprecedented, chemically responsive linker concept for biopharmaceuticals.     

THE POSSIBLE MECHANISM OF BINDING INTERACTION OF INSULIN MOLECULE WITH ITS RECEPTOR

Detailed structural comparisons and investigation of DPI, 2Zn insulin and some other derivatives of insulin were performed by the least-squares superimposition technique and the graphics technique. It is pointed out in this paper that the binding interaction with the receptor molecule should take place mainly on an amphipathic surface of the insulin molecule. In the middle, there is a hydrophobic surface with an area of about 150 consisting of many hydrophobic residues; while the polar or charged groups distributing around the hydro. phobic surface construct a hydrophilic zone. The hydrophobic surface is usually covered by the extended B-chain C-terminal peptides with great mobility and protected from the solvent molecules. The angle between the amphipathic surface and the surface of dimerization is about 20 degrees. The results from the detailed structural comparison between A1-(L-Trp) insulin and A1-(D-Trp) insulin have provided a very good explanation to their great difference in biological activity,     

MOLECULAR CLOSE-PACKING METHOD AND ITS APPLICATION TO CRYSTAL STRUCTURE DETERMINATION OF DESHEXAPEPTIDE (B25--B30) INSULIN

Based on the molecule-packing theory, we defined a molecule-packing function express-ing the compatibility of packing among the symmetry-related molecules in a unit cell. Acomputer program imitating the close-packing of molecules in the objective crystal latticeand giving the function value of each rotation and translation of the molecule in the unitcell was performed, and it therefore made the close-packing of molecules expressquantitatively. This method not only could judge a correct solution from several peaks ofthe rotation or translation function but it may also independently, quantitatively and quicklysolve some specific problems of rotation and translation. Using known structure of despenta-peptide (B26--B30) insulin as an example, the effectiveness of this method and its programwas inspected, and this method was successfully applied to solving the translation problem ofthe unknown structure of deshexapeptide (B25--B30) insulin. The molecular close-packingmethod proved by the results of R--search     

Inositols in PCOS

(1) Background: Myoinositol (MI) and D-chiro-inositol (DCI) are involved in a number of biochemical pathways within oocytes having a role in oocyte maturation, fertilization, implantation, and post-implantation development. Both inositols have a role in insulin signaling and hormonal synthesis in the ovaries. (2) Methods: Literature search (with key words: inositols, myo-inositol, d-chiro-inositol, PCOS) was done in PubMed until Sept. 2020 and 197 articles were identified, of which 47 were of clinical trials (35 randomized controlled trials). (3) Results: Many studies have demonstrated that in patients with polycystic ovarian syndrome (PCOS) MI treatment improved ovarian function and fertility, decreased the severity of hyperandrogenism including acne and hirsutism, positively affected metabolic aspects, and modulated various hormonal parameters deeply involved in the reproductive axis function and ovulation. Thus treating with MI has become a novel method to ameliorate PCOS symptoms, improve spontaneous ovulation, or induce ovulation. The current review is focused on the effects of MI and DCI alone or in combination with other agents on the pathological features of PCOS with focus on insulin resistance and adverse metabolic outcomes. (4) Conclusions: The available clinical data suggest that MI, DCI, and their combination in physiological ratio 40:1 with or without other compound could be beneficial for improving metabolic, hormonal, and reproductive aspects of PCOS.     

新颖的双胰岛素取代钴(III)卟啉的制备和表征

A novel insulin-disubstituted Co(Ⅲ)protoporphyrin IX,CoPI,where I is insulin and CoP is Co(Ⅲ)protopor-phyrin IX,was prepared by covalently coupling the propionate groups on the porphyrin ring to the ε-amino groupsof B29-Lys on insulin via amide linkages.The FTIR spectra in the amide I region(1600—1700 cm~(-1))and circulardichroism study show that CoPI has a conformation similar to the insulin at pH 6.9,whereas it exhibits significantconformational changes in structure as compared with the insulin self at pH 8.2.The pH absorption titration indi-cates that the alkaline conditions(pH≥8.0)are required for the formation of complexes between the free CoP andthe insulin.The thermodynamic and kinetic data reveal that free CoP is bound to either the zinc-insulin or free insu-lin with a dissociation constant of(2.0±0.3)×10~(-5)or(2.2±0.3)×10~(-5)mol/L.     

Human insulin and desamido human insulin isotherms in ethanol-water reversed phase systems

The multi-component isotherms for human insulin (HI) and desamido human insulin (dHI) over reversed phase packing (C18) and with 29.8% (w/w) ethanol-water as mobile phase have been determined experimentally. The isotherms of HI in ethanol-water differ from those obtained with the more commonly applied methanol-water and acetonitrile-water mobile phase, as described in this paper. The isotherm exhibits anti Langmuirian behavior and can be very well modeled by an anti Langmuir isotherm presented in this paper. The HI and dHI anti Langmuir isotherm are determined as: qHI = (8.4C(HI) + 3C(HI)CdHI)/(1 - 0.05C(HI) - 0.14CdHI + 0.04C(HI)CdHI) and qdHI = (11.4CdHI + 2C(HI)CdHI)/ (1 - 0.05C(HI) - 0.14CdHI + 0.04C(HI)CdHI)     

Signaling mechanisms of glucose-induced F-actin remodeling in pancreatic islet β cells

The maintenance of whole-body glucose homeostasis is critical for survival, and is controlled by the coordination of multiple organs and endocrine systems. Pancreatic islet β cells secrete insulin in response to nutrient stimuli, and insulin then travels through the circulation promoting glucose uptake into insulin-responsive tissues such as liver, skeletal muscle and adipose. Many of the genes identified in human genome-wide association studies of diabetic individuals are directly associated with β cell survival and function, giving credence to the idea that β-cell dysfunction is central to the development of type 2 diabetes. As such, investigations into the mechanisms by which β cells sense glucose and secrete insulin in a regulated manner are a major focus of current diabetes research. In particular, recent discoveries of the detailed role and requirements for reorganization/remodeling of filamentous actin (F-actin) in the regulation of insulin release from the β cell have appeared at the forefront of islet function research, having lapsed in prior years due to technical limitations. Recent advances in live-cell imaging and specialized reagents have revealed localized F-actin remodeling to be a requisite for the normal biphasic pattern of nutrient-stimulated insulin secretion. This review will provide an historical look at the emergent focus on the role of the actin cytoskeleton and its regulation of insulin secretion, leading up to the cutting-edge research in progress in the field today.     

Smart Protein-Based Formulation of Dendritic Mesoporous Silica Nanoparticles: Toward Oral Delivery of Insulin

Oral insulin administration still represents a paramount quest that almost a century of continuous research attempts did not suffice to fulfill. Before pre-clinical development, oral insulin products have first to be optimized in terms of encapsulation efficiency, protection against proteolysis, and intestinal permeation ability. With the use of dendritic mesoporous silica nanoparticles (DMSNs) as an insulin host and together with a protein-based excipient, succinylated β-lactoglobulin (BL), pH-responsive tablets permitted the shielding of insulin from early release/degradation in the stomach and mediated insulin permeation across the intestinal cellular membrane. Following an original in vitro cellular assay based on insulin starvation, direct cellular fluorescent visualization has evidenced how DMSNs could ensure the intestinal cellular transport of insulin.     

两种胰岛素方案治疗门诊初诊2型糖尿病患者的疗效及低血糖发生率比较

目的研究两种胰岛素方案治疗门诊初诊2型糖尿病患者的疗效及低血糖发生率。方法选取孝感市中心医院2013年3月—2014年8月84例门诊初诊2型糖尿病患者,抽签随机分为两组,其中42例采用餐前皮下注射门冬胰岛素治疗记为对照组,剩下42例采用餐前注射门冬胰岛素联合晚间注射甘精胰岛素强化治疗记为观察组。结果两组治疗后血糖相关指标比较无明显差异,不具有统计学意义(P0.05);观察组低血糖率11.90%较对照组30.95%显著较低,具有统计学意义(P0.05)。结论两种胰岛素方案治疗初诊2型糖尿病均较为有效,但早期采用强化治疗方案能有效稳定平衡血糖代谢、预防低血糖发生,可作为临床治疗初诊2型糖尿病的首选参考方法。     

重组人胰岛素注射液中B3和B3iso脱氨人胰岛素的分析与鉴定

建立了有效分离重组人胰岛素注射液中B3和B3iso脱氨人胰岛素的反相高效液相色谱(RP-HPLC)法,并采用高效液相色谱-四极杆-飞行时间质谱联用技术(HPLC-MS/MS)对其进行氨基酸序列的鉴定。在《中国药典》2010版重组人胰岛素注射液有关物质检查分析方法的基础上,优化流动相p H值,以0.2mol/L硫酸盐缓冲液(p H 3.6)-乙腈(90∶10)作为A相,乙腈-水(50∶50)为B相,梯度洗脱,分别收集杂质峰1和杂质峰2,脱盐浓缩后,用丝氨酸蛋白酶(V8酶)酶切,取部分酶切溶液经三(2-羧乙基)膦盐酸盐(TCEP)还原,分别得到还原和非还原肽段溶液,利用LC-MS/MS鉴定氨基酸序列,未知杂质分别为重组人胰岛素B3位发生脱氨和异构化的产物,即B3和B3iso脱氨人胰岛素。将优化后的方法与《中国药典》方法对市售重组人胰岛素注射液进行有关物质的对比分析,已上市厂家的重组人胰岛素注射液均有不同程度的B3和B3iso脱氨人胰岛素存在。该方法不影响重组人胰岛素其它有关物质的检测,可用于监控人胰岛素注射液产品储存过程中脱氨类杂质的增长趋势。     

Stevioside Attenuates Insulin Resistance in Skeletal Muscle by Facilitating IR/IRS-1/Akt/GLUT 4 Signaling Pathways: An In Vivo and In Silico Approach

Type-2 diabetes mellitus (T2DM), the leading global health burden of this century majorly develops due to obesity and hyperglycemia-induced oxidative stress in skeletal muscles. Hence, developing novel drugs that ameliorate these pathological events is an immediate priority. The study was designed to analyze the possible role of Stevioside, a characteristic sugar from leaves of Stevia rebaudiana (Bertoni) on insulin signaling molecules in gastrocnemius muscle of obesity and hyperglycemia-induced T2DM rats. Adult male Wistar rats rendered diabetic by administration of high fat diet (HFD) and sucrose for 60 days were orally administered with SIT (20 mg/kg/day) for 45 days. Various parameters were estimated including fasting blood glucose (FBG), serum lipid profile, oxidative stress markers, antioxidant enzymes and expression of insulin signaling molecules in diabetic gastrocnemius muscle. Stevioside treatment improved glucose and insulin tolerances in diabetic rats and restored their elevated levels of FBG, serum insulin and lipid profile to normalcy. In diabetic gastrocnemius muscles, Setvioside normalized the altered levels of lipid peroxidase (LPO), hydrogen peroxide (H₂O₂) and hydroxyl radical (OH*), antioxidant enzymes (CAT, SOD, GPx and GSH) and molecules of insulin signaling including insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and Akt mRNA levels. Furthermore, Stevioside enhanced glucose uptake (GU) and oxidation in diabetic muscles by augmenting glucose transporter 4 (GLUT 4) synthesis very effectively in a similar way to metformin. Results of molecular docking analysis evidenced the higher binding affinity with IRS-1 and GLUT 4. Stevioside effectively inhibits oxidative stress and promotes glucose uptake in diabetic gastrocnemius muscles by activating IR/IRS-1/Akt/GLUT 4 pathway. The results of the in silico investigation matched those of the in vivo study. Hence, Stevioside could be considered as a promising phytomedicine to treat T2DM.