Metallothionein 1 inhibits glucose-stimulated insulin secretion and is differentially regulated in conditions of beta cell compensation and failure

Background and aims: The mechanisms responsible for β cell compensation in obesity and for β cell failure in type 2 diabetes (T2D) are poorly defined. Metallothioneins play a role in both Zn2+ homeostasis and the regulation of cellular redox state. The mRNA levels of several metallothionein genes are upregulated in islets from subjects with T2D, but their role in β cells is not clear. Here we examined: 1) the temporal changes of islet Mt1 and Mt2 gene expression in models of β cell compensation and failure, and 2) the role of Mt1 and Mt2 in β cell function and glucose homeostasis. Materials and methods: Mt1 and Mt2 expression was assessed in islets from control lean (chow diet) and diet-induced obese (DIO) mice (8 weeks high fat diet), and pre-diabetic (6-week-old) and diabetic (16-week-old) db/db mice and age-matched db/+ (control) mice. Mt1-Mt2 double knockout (KO) mice, Mt1 overexpressing transgenic mice (Tg-Mt1) and corresponding control mice were studied. Mt1 and Mt2 were inhibited in MIN6 cells by small interfering RNAs. mRNA levels were assessed by real-time RT-PCR, plasma insulin and islet metallothionein levels by ELISA, glucose tolerance by i.p. glucose tolerance tests (ipGTT) and fasting-1h refeeding tests, insulin secretion by RIA, cytosolic free Ca2+ with Fura-2 LR, NAD(P)H by autofluorescence and cytosolic redox state using roGFP1 ratiometric thiol redox probe. Results: Increased plasma insulin levels (β cell compensation) correlated with marked downregulation of Mt1 and Mt2 mRNA levels in islets of DIO mice (Mt1: ~4-fold, p<0.01 and Mt2: ~4.5-fold, p<0.05), and prediabetic db/db mice (both by ~2-fold, p<0.01). These findings were confirmed in β cells of DIO mice (β cell-specific translating ribosome affinity purification model). In contrast, β cell failure in islets from diabetic db/db mice correlated with a tendency for increased Mt1 (~1.3-fold) and significantly upregulated Mt2 (~1.6-fold, p<0.05) mRNA levels. Ex vivo treatment of islets for 18-48h in high glucose (10-30 mM vs. 2-5 mM) strongly downregulated Mt1 and Mt2 mRNA and protein levels in parallel with increased insulin secretion. Interestingly, KO mice displayed markedly improved glucose tolerance during ipGTT (p<0.01) and fasting-refeeding tests (p<0.01), in association with increased plasma insulin levels (30 min following ipGTT, p<0.05). Glucose-stimulated insulin secretion (GSIS) was potentiated in islets isolated from KO mice vs. control islets while insulin content was unchanged. In MIN6 cells, knockdown of Mt1, but not Mt2, potentiated GSIS by ~1.8-fold (p<0.01). The potentiation of GSIS in KO islets occurred despite similar rises in intracellular Ca2+ and NAD(P)H levels, and the mRNA levels of β cell enriched genes preproinsulin, Pdx1, Glut2 and Pc and stress response genes Hmox1, Hspa5 and Ddit3 were unchanged. Nevertheless, basal and acute H2O2-induced cytosolic roGFP1 oxidation was slightly lower in KO islets. On the other hand, overexpression of Mt1 in islets from Tg-Mt1 inhibited GSIS by ~1.5-fold (p