Dietary zinc restriction promotes degeneration of the endocrine pancreas in mice.

Zinc is a key component of several proteins, interacting with the pancreatic hormones insulin and amylin. The role of zinc in insulin oligomerization and crystallinity is well established, although the effects of dietary zinc restriction on both energetic metabolism and ß-pancreatic hormonemia and morphology remain unexplored. Here we report the effects of dietary zinc restriction on the endocrine pancreas and metabolic phenotype of mice. Nontransgenic male Swiss mice were fed a low-zinc or control diet for 4 weeks after weanling. Growth, glycemia, insulinemia and amylinemia were lower and pancreatic islets were smaller in the intervention group despite the preserved insulin crystallinity in secretory granules. We found strong immunostaining for insulin, amylin and oligomers in apoptotic pancreatic islet. High production of ß-pancreatic hormones in zinc-restricted animals counteracted the reduced islet size caused by apoptosis. These data suggest that zinc deficiency is sufficient to promote islet ß-cell hormonal disruption and degeneration.

Physico-chemical properties of co-formulated fast-acting insulin with pramlintide.

Since the discovery of amylin its use has been discouraged by the inadequacy of the protocol involving multiple injections in addition to insulin. We aimed here to develop a combined fixed-dose formulation of pramlintide with fast-acting insulin. We have investigated the compatibility of regular and fast-acting insulin analogues (Aspart, AspB28, and LisPro, LysB28ProB29) with the amylin analogue pramlintide by using electrospray ionization - ion mobility spectrometry-mass spectrometry (ESI-IMS-MS), kinetic aggregation assays monitored by thioflavin T, and transmission electron microscopy (TEM) in the evaluation of the aggregation product. Insulin interacts with pramlintide, forming heterodimers as probed by ESI-IMS-MS. While their interaction is likely to delay the amyloid aggregation of pramlintide in phosphate-buffered solution pH 7.0, they do not prevent aggregation at this condition. At acidic sodium acetate solution pH 5.0, combination of pramlintide and the fast-acting insulin analogues become stable against amyloid aggregation. The co-formulated product at high concentration of both pramlintide (600 µg/mL,150 µM) and LisPro insulin (50 IU/mL, 300 µM) showed also stability against amyloid aggregation. These data indicate the physico-chemical short-term stability of the co-formulated preparation of LisPro insulin with pramlintide, which could bring benefits for the combined therapy.

Amyloidogenesis of the amylin analogue pramlintide.

Amylin is a pancreatic peptide hormone co-secreted along with insulin by the ß-cells. It is found in amyloid deposits in both type 2 diabetic individuals and elder non-diabetic. The triple proline amylinomimetic compound (25,28,29-Pro-human amylin) named pramlintide was designed aiming to solve the solubility and amyloid characteristics of human amylin. We have found by using ion mobility spectrometry-based mass spectrometry that pramlintide is able to assembly into multimers. Pramlintide formed amyloid fibrils in vitro in a pH-dependent kinetic process within a few hours, as followed by thioflavin T, quantification of soluble peptide and further characterized by transmission electron microscopy, atomic force microscopy and X-ray diffraction. These data indicate that pramlintide can form amyloid fibers.