Metallothionein in isolated pancreatic islets of mice: Induction by zinc and streptozotocin, a naturally occurring diabetogen

Abstract

Metallothionein (MT) concentrations were determined in the cytosol of isolated pancreatic islets of mice, using both the cadmium (Cd)-heme and the Cd-Chelex assay. Both constitutive MT levels and significant MT induction were detected in islet cells. For MT induction, mice were injected intraperitoneally (i.p.) with either zinc sulfate (Zn) or the diabetogen streptozotocin (STZ). Following an i.p. injection of 15 mg Zn.kg-1 body weight (body wt), the mean index of MT induction found in islets was comparable to that found in liver tissue, which was used as control. After i.p. injection of 30 mg Zn.kg-1 or a single high dose of STZ (100 or 200 mg.kg-1), the indices of MT induction in islets exceeded those in liver by a factor of 1.3, 2.5, or 1.5. After multiple low doses of STZ (3 or 5 x 40 mg.kg-1 given on consecutive days), in contrast, the MT induction indices in islets exceeded those in liver by a factor of 3.5 and 3.9 for 3 x 40 respectively 5 x 40 mg STZ.kg-1. In conclusion, our results demonstrate constitutive MT levels in isolated pancreatic islets and significant MT induction after i.p. injection with Zn or STZ, in particular after repeated low doses of STZ.

Zinc sulphate induces metallothionein in pancreatic islets of mice and protects against diabetes induced by multiple low doses of streptozotocin

“…By immunohistochemistry, an antibody reacting with both MT-1 and MT-2 isoforms failed to stain islets of normal and of transgenic mice overexpressing MT-1 [31]. Our laboratory applying quantitative measurements [32] found, however, constitutive concentrations and induction of MT with Zn sulphate (Zn 2+ ) in islets of mice in vitro [33] and ex vivo [34].…”

“…Mice whose water had no additive served as controls. Islets were isolated from individual mice [34] and prepared for measurement of MT concentrations [33].…”

“…Cd-haemoglobin saturation assay as described previously [32,34]. Briefly, isolated islets from individual mice were washed three times with PBS, covered with 50 ml double-distilled water and stored at ±80 C. For measurement of total protein and MT in cytosolic preparations, the deep-frozen islets were thawed, kept at 4 C, lyophilised and resuspended in 300 ml TRIS/HCl (10 mmol/l, 85 mmol/l NaCl, pH 7.4).…”

Generation of hydrogen peroxide and failure of antioxidative responses in pancreatic islets of male C57BL/6 mice are associated with diabetes induced by multiple low doses of streptozotocin

“…Pancreatic islets were isolated from solvent-injected mice on day 3 after the final injection and from MLD-STZ-injected mice on day 1 and/or day 3 after the final injection. These time points were chosen because islets isolated from comparable treated groups did not differ significantly in number [24] and in total protein levels (µg/islet) [14]. An essential contribution of contaminating leukocytes as an H 2 O 2 donor is excluded by using islets isolated on day 1 and/or day 3 after the final STZ injection.…”

“…Pancreatic islets were isolated by collagenase digestion as described previously [24], and cultured free-floating in RPMI 1640 medium containing 5.6 mmol/l D-glucose, 10% FCS, 100 U/ml penicillin and 0.1 mg/ml streptomycin in an atmosphere of 95% air and 5% CO 2 at 37°C for 2 h. To analyse H 2 O 2 generation induced by MLD-STZ in vivo or STZ in vitro, groups of 150 islets were kept in 2700 µl KRB without BSA at 37°C. To compare STZmediated H 2 O 2 levels with the potent H 2 O 2 -generating compound, alloxan, islets were incubated with the latter diabetogen using concentrations equimolar to STZ.…”