Characterisation of insulin analogues therapeutically available to patients

The structure and function of clinical dosage insulin and its analogues were assessed. This included ‘native insulins’ (human recombinant, bovine, porcine), ‘fast-acting analogues’ (aspart, glulisine, lispro) and ‘slow-acting analogues’ (glargine, detemir, degludec). Analytical ultracentrifugation, both sedimentation velocity and equilibrium experiments, were employed to yield distributions of both molar mass and sedimentation coefficient of all nine insulins. Size exclusion chromatography, coupled to multi-angle light scattering, was also used to explore the function of these analogues. On ultracentrifugation analysis, the insulins under investigation were found to be in numerous conformational states, however the majority of insulins were present in a primarily hexameric conformation. This was true for all native insulins and two fast-acting analogues. However, glargine was present as a dimer, detemir was a multi-hexameric system, degludec was a dodecamer (di-hexamer) and glulisine was present as a dimer-hexamer-dihexamer system. However, size-exclusion chromatography showed that the two hexameric fast-acting analogues (aspart and lispro) dissociated into monomers and dimers due to the lack of zinc in the mobile phase. This comprehensive study is the first time all nine insulins have been characterised in this way, the first time that insulin detemir have been studied using analytical ultracentrifugation and the first time that insulins aspart and glulisine have been studied using sedimentation equilibrium. The structure and function of these clinically administered insulins is of critical importance and this research adds novel data to an otherwise complex functional physiological protein

The hypoglycaemic effect of pumpkins as anti-diabetic and functional medicines

Diabetes mellitus is considered as a common, growing, serious, costly, and potentially preventable public health problem. In 2030, the number of people with diabetes is estimated to increase from 117 million in 2000 to 366 million. The prevalence of diabetes has and will continue to have burden on the health and finances of economic climates, which in turn, will impact on individuals, families and nations. There are many different types of insulins available to treat diabetes, but there are still physiological consequences for such use. Alternatives are, therefore, required and this includes herbal preparations as well as dietary plants in the form of curcubitaceae (pumpkin). Pumpkin is widely considered to have active hypoglycaemic properties. Pumpkin is a plant, which has been used frequently as functional food or medicine and belongs to the family Cucubitaceae, and consists of succulent stem with numerous seeds. Based on previous evidence of its fruit pulp, it is reported to have anti-diabetic effects. This review has focused on the main medicinal properties of pumpkin and how this has been used in animal models, and point out areas for future research to further elucidate mechanisms whereby this compound may reduce disease risk. © 2011 Elsevier Ltd. All rights reserved

c(s) vs. sedimentation coefficient distributions measured using AUC-SV at 45k rpm.

<p>(a) Native insulins; (b) Rapid-acting analogues; (c) Slow-acting analogues. Monomers (M), Dimers (D), Hexamers (H), and Di and Tri Hexamers (Di/TriH) were identified by molar mass calculated through the sedimentation coefficient and the frictional ratio.</p

Weight-average molar mass estimates from AUC-SV, AUC-SE and SEC-MALS results.

<p>Estimates uncorrected for non-ideality are labelled ‘apparent’ (_app). All AUC-SV estimates were made on data from 45k rpm and based upon estimates of the frictional ratio fitted from boundary spread, and are weight averaged in the case of multiple sedimenting species. AUC-SE analysis was made at the indicated rotor speeds in parentheses (k rpm). SEC-MALS estimates are shown as consensus values from PBS and TRIS mobile phases. IDeg was unanalysable using MSTAR and MULTISIG. IGla was not assayed using SEC-MALS due to pI/pH incompatibilities.</p

Molar mass distributions measured against concentration using MULTISIG-RADIUS from AUC-SE data, with superimposed n-, w- and z-average molar masses.

<p>Insets are fits from INVEQ analysis. (a) IHr; (b) IBov; (c) IPor; (d) IAsp; (e) IGlu; (f) ILis; (g) IGla; (h) IDet; (i) IDeg. MULTISIG-RADIUS was unable to adequately fit data from IDeg, but INVEQ was able.</p

Elution from SEC plots of insulin and analogues.

<p>Black line represents PBS as the mobile phase, grey line represents TRIS. (a) IHr; (b) IBov; (c) IPor; (d) IAsp; (e) IGlu; (f) ILis; (g) IDet; (h) IDeg. IGla was not injected due to pI/pH incompatibilities. Monomers (M), Dimers (D), Hexamers (H), Dihexamers (DiH), multihexamers (multiH) and Excipients (Ex) were identified by molar mass.</p

Primary structure of human insulin and its analogues.

<p>Differences highlighted and numbered.</p

c(M) vs. molar mass distributions measured using SEDFIT-MSTAR from AUC-SE data, with superimposed M*(r) extrapolations to the cell base (relative radius = 1), and c(M) fits over raw AUC-SE data in insets.

<p>(a) IHr; (b) IBov; (c) IPor; (d) IAsp; (e) IGlu; (f) ILis; (g) IGla; (h) IDet; (i) IDeg. SEDFIT-MSTAR was unable to adequately fit data from IDeg.</p

Hydrodynamic parameters measured using AUC-SV and AUC-SE.

<p>Sedimentation coefficients are weight-averaged in the cases of multi-species distributions. IGlu and IDet yielded fits with BM<0. All insulins were measured at ~3.5mg/mL, except for IDet which was measured at 14.2mg/mL.</p