Ultra-high resolution X-ray structures of two forms of human recombinant insulin at 100 K.

The crystal structure of a commercially available form of human recombinant (HR) insulin, Insugen (I), used in the treatment of diabetes has been determined to 0.92 Å resolution using low temperature, 100 K, synchrotron X-ray data collected at 16,000 keV (λ = 0.77 Å). Refinement carried out with anisotropic displacement parameters, removal of main-chain stereochemical restraints, inclusion of H atoms in calculated positions, and 220 water molecules, converged to a final value of R = 0.1112 and Rfree = 0.1466. The structure includes what is thought to be an ordered propanol molecule (POL) only in chain D(4) and a solvated acetate molecule (ACT) coordinated to the Zn atom only in chain B(2). Possible origins and consequences of the propanol and acetate molecules are discussed. Three types of amino acid representation in the electron density are examined in detail: (i) sharp with very clearly resolved features; (ii) well resolved but clearly divided into two conformations which are well behaved in the refinement, both having high quality geometry; (iii) poor density and difficult or impossible to model. An example of type (ii) is observed for the intra-chain disulphide bridge in chain C(3) between Sγ6-Sγ11 which has two clear conformations with relative refined occupancies of 0.8 and 0.2, respectively. In contrast the corresponding S-S bridge in chain A(1) shows one clearly defined conformation. A molecular dynamics study has provided a rational explanation of this difference between chains A and C. More generally, differences in the electron density features between corresponding residues in chains A and C and chains B and D is a common observation in the Insugen (I) structure and these effects are discussed in detail. The crystal structure, also at 0.92 Å and 100 K, of a second commercially available form of human recombinant insulin, Intergen (II), deposited in the Protein Data Bank as 3W7Y which remains otherwise unpublished is compared here with the Insugen (I) structure. In the Intergen (II) structure there is no solvated propanol or acetate molecule. The electron density of Intergen (II), however, does also exhibit the three types of amino acid representations as in Insugen (I). These effects do not necessarily correspond between chains A and C or chains B and D in Intergen (II), or between corresponding residues in Insugen (I). The results of this comparison are reported. Graphical abstract Conformations of PheB25 and PheD25 in three insulin structures: implications for biological activity? Insulin residues PheB25 and PheD25 are considered to be important for insulin receptor binding and changes in biological activity occur when these residues are modified. In porcine insulin and Intergen (II) PheB25 adopts conformation B and PheD25 conformation D. However, unexpectedly PheB25 in Insugen (I) human recombinant insulin adopts two distinct conformations corresponding to B and D, Figure 1 and PheD25 adopts a single conformation corresponding to B not D, Figure 2. Conformations of this residue in the ultra-high resolution structure of Insugen (I) are therefore unique within this set. Figures were produced with Biovia, Discovery Studio 2016.

Influence of glucosamine on the bioactivity of insulin delivered subcutaneously and in an oral nanodelivery system.

The aim of the work reported herein was to study the effect of glucosamine HCl (GlcN·HCl) on the bioactivity (BA) of insulin, administered via subcutaneous (SC) and oral routes, in adult male Sprague Dawley rats. The oral insulin delivery system (insulin-chitosan reverse micelle [IC-RM]) was prepared by solubilizing insulin-chitosan (13 kDa) polyelectrolyte complex in a RM system consisting of oleic acid, PEG-8 caprylic/capric glycerides, and polyglycerol-6-dioleate. The BA of insulin in vivo was evaluated by measuring blood glucose level using a blood glucose meter; the results revealed that the extent of hypoglycemic activity of SC insulin was GlcN·HCl dose dependent when they were administered simultaneously. A significant reduction in blood glucose levels (P<0.05) was found for the insulin:GlcN·HCl at mass ratios of 1:10 and 1:20, whereas lower ratios (eg, 1:1 and 1:4) showed no significant reduction. Furthermore, enhancement of the action of SC insulin was achieved by oral administration of GlcN·HCl for 5 consecutive days prior to insulin injection (P<0.05). For oral insulin administration via the IC-RM system, the presence of GlcN·HCl increased the hypoglycemic activity of insulin (P<0.05). The relative BA were 6.7% and 5.4% in the presence and absence of GlcN·HCl (ie, the increase in the relative BA was approximately 23% due to incorporating GlcN·HCl in the IC-RM system), respectively. The aforementioned findings offer an opportunity to incorporate GlcN·HCl in oral insulin delivery systems in order to enhance a reduction in blood glucose levels.

Influence of glutathione on the bioactivity of subcutaneously or orally administered insulin to rats.

The effect of reduced (GSH) and oxidized (GSSG) glutathione on the bioactivity of insulin was studied. A polyelectrolyte complex (PEC) of insulin with low molecular weight chitosan (13 kDa) was prepared and characterized. The PEC was then solubilized, in the presence and absence of GSH and GSSG, in a reverse micelle consisting of oleic acid and two surfactants (PEG-8 caprylic/capric glycerides and polyglycerol-6-dioleate). The in vitro and in vivo performances of the reverse micelle formulations (RMFs) were evaluated in rats. At pH 6.5 the association efficiency of the PEC was 76.2%. In vitro insulin release from the RMs was negligible at pH 1.2 and was markedly increased at pH 6.8. The hypoglycemic activity of insulin in the PEC was reduced when administered via the subcutaneous route, regardless of the GSH content. On the other hand, the presence of GSSG significantly enhanced hypoglycemia. When the RMF was administered via the oral route, the presence of GSH had no effect on the hypoglycemic activity of insulin compared with the GSH free system. However, the presence of GSSG in the oral preparation increased the hypoglycemic activity of insulin; probably by inhibiting insulin degradation, thereby prolonging its effect. Thus, incorporation of GSSG in the RMF reduces blood glucose levels in rats and protects insulin from degradation.

Low molecular weight chitosan-insulin polyelectrolyte complex: characterization and stability studies.

The aim of the work reported herein was to investigate the effect of various low molecular weight chitosans (LMWCs) on the stability of insulin using USP HPLC methods. Insulin was found to be stable in a polyelectrolyte complex (PEC) consisting of insulin and LMWC in the presence of a Tris-buffer at pH 6.5. In the presence of LMWC, the stability of insulin increased with decreasing molecular weight of LMWC; 13 kDa LMWC was the most efficient molecular weight for enhancing the physical and chemical stability of insulin. Solubilization of insulin-LMWC polyelectrolyte complex (I-LMWC PEC) in a reverse micelle (RM) system, administered to diabetic rats, results in an oral delivery system for insulin with acceptable bioactivity.