Characterisation of an important enzymatic component in collagenase that is essential for the effective digestion of the human and porcine pancreas

Recent clinical results from Edmonton have demonstrated the feasibility of achieving normoglycemia in type I diabetic patients by islet transplantation. One of the key issues in obtaining this success was transplanting sufficient numbers of islets by sequential transplants. Although the development of semipurified endotoxin-free Clostridium histolyticum-derived collagenase (Liberase) has improved islet yields from the human pancreas, batch-to-batch variation and loss of activity with time still hampers progress in obtaining consistent islet preparations. In order to define key components of crude collagenase, a panel of monoclonal antibodies (McAbs) was raised against crude collagenase. Monoclonal antibodies were generated by fusions between splenocytes of BALB/c mice immunized with Boheringer P collagenase and the myeloma cell line NS-0. These monoclonal antibodies were used as probes to study molecular differences between effective and ineffective collagenase batches using Western blotting. Two monoclonal antibodies (LDS71 and LDS81) were raised and characterized as recognizing separate epitopes on a 125-kDa component. Western blotting indicated that the 125-kDa band was rapidly broken down by storage or by dialysis in the presence of dithiothreitol. However, this breakdown could be prevented by the addition of leupeptin (a protease inhibitor) to the dialysis buffer. On testing fractions at 5-min intervals from the “Ricordi” digestion circuit during porcine and human pancreas digestion, the 125-kDa component was rapidly broken down in relatively ineffective collagenase batches but in effective batches was present throughout the digestion process. The correlation between the presence of the 125-kDa band and effectiveness of pancreas digestion suggests that this may be a key component in the formulation of C. histolyticum collagenase

Inhibitory Effect of Unsaturated Fatty Acids on Saturated Fatty Acid-Induced Apoptosis in Human Pancreatic β-Cells: Activation of Caspases and ER Stress Induction

Aims: In this study we have tested the effect of unsaturated fatty acids on the proapoptotic effects of saturated fatty acids in the human pancreatic β-cells NES2Y. Results: We found that unsaturated palmitoleic and oleic acid at a concentration of 0.2 mM and higher are able to completely inhibit the proapoptotic effect of their counterpart saturated palmitic and stearic acid at a concentration of 1 mM. Apoptosis induced by stearic acid was associated with significant activation of caspase-6, -7, -9, -2 and -8, but not with significant activation of caspase-3. The activation of caspases was blocked by coincubation with oleic acid. Stearic acid treatment was not associated with a significant change in mitochondrial membrane potential, reactive oxygen species level and with cytochrome c release from mitochondria. Furthermore, stearic acid treatment was not associated with changes in p21[superscript WAF1/CIP1], PIDD, Fas receptor and Fas ligand expression. However, we detected endoplasmic reticulum (ER) stress markers, i.e. a significant upregulation of BiP and CHOP expression as well as XBP1 mRNA splicing. These changes were inhibited by coincubation with oleic acid. Conclusion: Presented data indicate that oleic acid inhibits apoptosis induction by stearic acid in NES2Y cells upstream of caspase activation and ER stress induction. It does not involve an interference with the mitochondrial pathway of apoptosis induction, with p53 activation and PIDD expression as well as with Fas receptor and Fas ligand expression