Time course of the induced glycemia after injection of the synthetic and wild type cHHs.

<p>a) Time course of hemolymph glycemia after injection of 1.7 pmol/g live weight of D-cHH. The maximum peak of 95.4±8.1 mg/dL glucose is reached after 4 h, high values lasting at 8 h (76±12.1 mg/dL), when the maximum values were also recorded, and the glycemia returning to its nearby basal level, 8.9±1 mg/dL, after 24 h. N = 20. b) Time course of hemolymph glycemia after injection of 1.7 pmol/g live weight of L-cHH. L-cHH induced a slower hyperglycemic response with the maximum peak of 52±6.5 mg/dL glucose after 2 h, and the glycemia returning to its nearby basal level, 5.5±0.4 mg/dL, after 8 h. N = 16. c) Time course of hemolymph glycemia after injection of 1.7 pmol/g live weight of Glp-D-cHH. The time course of Glp-D-cHH shows the stronger hyperglycemic response of 96.1±5.6 mg/dL glucose after 4 h, high values lasting at 8 h (62.9±11.8 mg/dL) and the glycemia returning to its basal level, 2.9±0.5 mg/dL, after 24 h. N = 9. d) Time course of hemolymph glycemia after injection of 1.7 pmol/g live weight of SG-cHH. The hyperglycemic time course after the injection of 1.7 pmol/g live weight of purified SG-cHH was comparable to that of synthetic D-cHH and Glp-D-cHH peptides, but the maximum (44±4.5 mg/dL) was reached after 2 h and the glycemia returning to its basal level, 5±1.1 mg/dL after 24 h. N = 9.</p

Scheme of the SPPS coupled to NCL to obtain the full length peptides.

<p>The leading segments containing a C-terminal thioester, QVF-cHH4-38, QVdF-cHH4-38, pEVdF-cHH4-38, and the cHH39-72 following segment with a free N-terminal cysteine were synthesized by SPPS. The NCL reaction between the leading and following segments returned the full length 72mer cHH isomers, which were further subjected to oxidative folding to obtain the folded peptides.</p

Boxplots of the maximum glucose values recorded for the synthetic and native peptides.

<p>Both D-cHH and Glp-D-cHH induced a strong hyperglycemia with a mean peak glucose concentration of, respectively, 100.5±9.3 and 98.7±6 mg/dL, while the hyperglycemic effect of GS-cHH and of L-cHH was lower, with a mean of max glycemia of 52.7±9.4 mg/dL and of 52.4±6.4 mg/dL respectively.</p

Overview of insulin production and purification process from <i>Pichia pastoris</i> fermentation culture.

<p>Overview of insulin production and purification process from <i>Pichia pastoris</i> fermentation culture.</p

RP-HPLC profiles and ESI-MS spectra of the synthesized cHH isomers.

<p>Reverse Phase HPLC profiles of the purified refolded synthetic polypeptides and their corresponding ESI-MS multiply charged ion and deconvoluted spectra (insets): L-Phe³-cHH (A and B), D-Phe³-cHH (C and D), Glp-D-Phe³-cHH (E and F). Mobile Phase A: 0.1% TFA in water. Mobile Phase B: 0.1% TFA in MeCN. Gradient: 0–100% B over 25 min. Column: Gemini 5 C18 2.0×150 mm.</p