Protective Antioxidant and Antiapoptotic Effects of ZnCl2 in Rat Pancreatic Islets Cultured in Low and High Glucose Concentrations

Abstract

Aim/HypothesisRat pancreatic islet cell apoptosis is minimal after prolonged culture in 10 mmol/l glucose (G10), largely increased in 5 mmol/l glucose (G5) and moderately increased in 30 mmol/l glucose (G30). This glucose-dependent asymmetric V-shaped profile is preceded by parallel changes in the mRNA levels of oxidative stress-response genes like Metallothionein 1a (Mt1a). In this study, we tested the effect of ZnCl2, a potent inducer of Mt1a, on apoptosis, mitochondrial oxidative stress and alterations of glucose-induced insulin secretion (GSIS) induced by prolonged exposure to low and high vs. intermediate glucose concentrations.MethodsMale Wistar rat islets were cultured in RPMI medium. Islet gene mRNA levels were measured by RTq-PCR. Apoptosis was quantified by measuring islet cytosolic histone-associated DNA fragments and the percentage of TUNEL-positive β-cells. Mitochondrial thiol oxidation was measured in rat islet cell clusters expressing “redox sensitive GFP” targeted to the mitochondria (mt-roGFP1). Insulin secretion was measured by RIA.ResultsAs observed for Mt1a mRNA levels, β-cell apoptosis and loss of GSIS, culture in either G5 or G30 vs. G10 significantly increased mt-roGFP1 oxidation. While TPEN decreased Mt1a/2a mRNA induction by G5, addition of 50–100 µM ZnCl2 to the culture medium strongly increased Mt1a/2a mRNA and protein levels, reduced early mt-roGFP oxidation and significantly decreased late β-cell apoptosis after prolonged culture in G5 or G30 vs. G10. It did not, however, prevent the loss of GSIS under these culture conditions.ConclusionZnCl2 reduces mitochondrial oxidative stress and improves rat β-cell survival during culture in the presence of low and high vs. intermediate glucose concentrations without improving their acute GSIS.

Glucokinase activation is beneficial or toxic to cultured rat pancreatic islets depending on the prevailing glucose concentration

“…We (9,40) have previously shown that the glucose-induced changes in rat ␤-cell apoptosis are preceded by parallel changes in the oxidation of mt-roGFP1, a good indicator of mitochondrial thiol oxidation (32). Similar changes in mtroGFP1 fluorescence ratio were observed in the present study, with a large increase after culture at G5 vs. G10 and a small, significant increase after culture at G30 vs. G10 (Fig.…”

“…Histone-associated DNA fragments were measured in islet cell cytosolic extracts using the Cell Death Detection ELISA PLUS kit (Roche Diagnostics) exactly as described in (36). The percentage of apoptotic ␤-cells was determined on islet sections by TUNEL, using the In Situ Cell Death Detection Kit (Roche Diagnostics) followed by insulin immunostaining (9).…”

“…The mitochondrial redox status was assessed by measuring the thiol/disulfide equilibrium with the redox-sensitive green fluorescent protein targeted to the mitochondria (mt-roGFP1) (14), as described (9,40). The mtroGFP1 fluorescence ratio was normalized to the difference between the ratio measured after addition of 10 mM DTT (set to 0%) and that measured after addition of 1 mM H2O2 (set to 100%).…”

“…Total glutathione was measured with the DetectX Glutathione Fluorescent Detection Kit (Arbor Assays, Ann Arbor, MI) and normalized for differences in islet protein content (9,40).…”

“…4). Prolonged culture at a nonstimulating or very high vs. intermediate glucose concentration not only triggers ␤-cell apoptosis but also reduces the islet insulin content and induces marked alterations of the ␤-cell secretory responses to subsequent acute glucose simulation (9,21,40). In agreement with previous studies, after 1 wk of culture at G10 alone, rat islets displayed typical changes in NAD(P)H autofluorescence, [Ca 2ϩ ] i , and insulin secretion upon acute stepwise glucose stimulation: a concentration-dependent increase in NAD(P)H autofluorescence (Fig.…”