Accumulation of cadmium in insulin-producing β cells

Abstract

Evidence suggests that chronic low level cadmium exposure impairs the function of insulin-producing β cells and may be associated with type-2 diabetes mellitus. Herein, we describe the cadmium content in primary human islets and define the uptake kinetics and effects of environmentally relevant cadmium concentrations in cultured β cells. The average cadmium content in islets from 10 non-diabetic human subjects was 29 ± 7 nmol/g protein (range 7 to 72 nmol/g protein). Exposure of the β-cell line MIN6 to CdCl 2 concentrations between 0.1 and 1.0 µmol/L resulted in a dose- and time-dependent uptake of cadmium over 72 h. This uptake resulted in an induction of metallthionein expression, likely enhancing cellular cadmium accumulation. Furthermore, cadmium accumulation resulted in an inhibition of glucose stimulated insulin secretion in MIN6 cells and primary mouse islets. Our results indicate that this impairment in β-cell function is not due to an increase in cell death or due to an increase in oxidative stress. We conclude that mouse β cells accumulate cadmium in a dose- and time-dependent manner over a prolonged time course at environmentally relevant concentrations. This uptake leads to a functional impairment of β-cell function without significant alterations in cell viability, expression of genes important for β-cell function or increase in oxidative stress.

Keywords: cadmium; insulin secretion; metallothionein; zinc transporters; β cells.

Figure 1. (A) Comparison between Cd and Hg accumulation in MIN6 cells following exposure to Cd or Hg 0.1 µmol/L for 48 h (n = 4). (B) Comparison between 3T3 and MIN6 cell Cd accumulation following exposure to 0.1 or 1 µmol/L CdCl2 for 72 h compared with non-exposed control cells (n = 3). (C) Cd accumulation in MIN6 and 3T3 cells following exposure to 0.1 µmol/L CdCl2 for 1, 2, 4, 8, 24, 48 or 72 h (n = 3), (D) MIN6 cell Zn content following Cd exposure for 72 h: Changes in Zn concentration following exposure to 0.1 or 1 µmol/L CdCl2 for 72 h (n = 3). All metal concentrations are expressed as µmol/g protein *, p < 0.05, **, p < 0.01. The Student’s t-test with Bonferroni correction was used for comparison of Cd, Hg and Zn concentrations at 72 h (A, B and D). Two-way ANOVA was used to compare the time dependent rate of Cd accumulation between 3T3 and MIN6 cells (C).

Figure 2. Representative western blot analysis of metallothionein expression in MIN6 cells following exposure or not of MIN6 cells to 0.1µmol/L CdCl2 for 30 min or 1, 2, 4, 8, 24, 48 or 72 h (A, representative of n = 3). Changes in protein levels of total metallothionein (B, representative of n = 3) and mRNA levels of the metallothionein genes MT1 through MT4 (C, n = 5) in dispersed primary mouse islets following exposure to CdCl2 0.1 µmol/L for 48 h relative to control cells. *, p < 0.05 using the Student’s t-test with Bonferroni correction.

Figure 3. (A) GSIS in MIN6 cells following exposure or not to 0.1, 0.5 or 1.0 µmol/L CdCl2 for 48 h (n = 5, each in duplicate). *, p < 0.05 using Student’s t test with Bonferroni correction. (B) Glucose stimulated insulin secretion (GSIS) in primary mouse islet cells following exposure to 0.1 µmol/L CdCl2 for 8 or 48 h compared with control cells (n = 4, each in duplicate).

Figure 4. (A) Viability of dispersed primary mouse islet cells at 48 h: Ratio of viable to dead cells in dispersed primary mouse islets using the Multitox^® assay following exposure to 0.1 µmol/L CdCl2 for 8 or 48 h compared with control (n = 3, each in triplicate). (B) Representative blot of the expression of the heavy metal toxicity marker Heat Shock Protein 70 (HSP70) in dispersed primary mouse islets following exposure to 0.1 µmol/L CdCl2 for 48 h compared with non-exposed control cells.

Figure 5. Oxidative stress and oxidative stress markers following exposure to CdCl2 for 48 h: GSH, GSSG (both normalized to the non-exposed control) and GSSG/GSH ratio in MIN6 cells (A, n = 4, each in triplicate) and dispersed primary mouse islet cells (B, n = 3, each in triplicate). mRNA levels of the oxidative response genes GCLC, HMOX1 and GCLM in MIN6 cells (C, n = 4) and in dispersed primary mouse islets (n = 4). *, p < 0.05 using Student’s t-test with Bonferroni correction.