Glucose controls metallothionein 1 gene expression in pancreatic islets via a mechanism involving cellular metabolism, granular zinc and the zinc transporter ZnT8 in rat and mouse.

AIMS/HYPOTHESIS: Metallothionein 1 (MT1) negatively regulates insulin secretion in pancreatic beta cells, and is upregulated by low non-stimulating glucose levels compared with physiological glucose stimulation in cultured rodent and human islets. Whether this regulation also occurs in vivo and its underlying mechanisms remain unclear. Here we examine whether physiological changes in blood glucose levels in vivo control islet Mt1 gene expression similarly to ex vivo observations, and identify the mechanisms involved.

METHODS: Rats fasted for 1, 2 or 3 days and rats fasted for 2 days and refed for 1 day were compared with rats fed throughout. Mice implanted with subcutaneous insulin mini-osmotic pumps were compared with control mice. Rat and mouse islets were cultured in the presence of optimal glucose levels (10 mmol/l; G10 medium) or low non-stimulating glucose levels (2 or 5 mmol/l; G2 or G5 medium) in the absence or presence of various modulators of mitochondrial metabolism, protein synthesis, Ca2+ influx, autophagy and insulin secretion. Islets from Znt8 (Slc30a8) knockout (KO) mice and corresponding control mice were studied. Gene mRNA levels were assessed by reverse-transcription followed by real-time PCR, plasma and secreted insulin levels were assessed using RIA, intracellular free Zn2+ ([Zn2+]c) was assessed using FluoZin-3, metal regulatory transcription factor 1 (MTF1) activity was assessed using an MTF1-luciferase lentiviral reporter system, and beta cell morphology was assessed by transmission electron microscopy.

RESULTS: Among glucose-affected zinc transporters/importers in rat islets, only the zinc efflux transporter Znt1 (Slc30a1) was upregulated under low non-stimulating glucose levels (G2 and G5 medium) vs physiological glucose stimulation (G10 medium), in parallel with Mt1. In vivo, Mt1 mRNA levels were upregulated approximately 3-4-fold in the islets of fasted rats and mice implanted with insulin pumps, with both conditions inducing sustained hypoglycaemia. This effect was fully reversed after normalisation of blood glucose levels. Interestingly, Znt1 mRNA levels changed in parallel with those of Mt1. Hypoglycaemia also markedly altered the expression of beta cell-enriched genes (downregulated), adaptive unfolded protein response (UPR) genes (downregulated), integrated stress response (ISR)/proapoptotic UPR genes (upregulated) and oxidative stress-responsive genes (upregulated). Normalisation of blood glucose levels largely reversed these alterations. In cultured islets, addition to G2 medium of mitochondrial metabolic substrates that bypass glycolysis inhibited Mt1 expression to the same extent as G10 medium. Conversely, the reduction of Mt1 expression in G10 medium was prevented when mitochondrial function or protein synthesis were inhibited. In comparison with fed mice, beta cells of fasted mice had degradative autophagic vacuoles containing secretory granule components. In agreement, islet cells cultured in G2 vs G10 medium showed an increase in [Zn2+]c that was prevented by chloroquine treatment. Co-staining with nuclear and cytosolic markers confirmed the cytosolic and perinuclear subcellular localisation of increased [Zn2+]c in G2 medium. This effect was reproduced in MIN6 cells cultured in G5 vs G25 medium (containing 25 mmol/l glucose, which is the optimal concentration for culture of MIN6 cells) and was paralleled by a significant increase in MTF1 activity, which is a robust indicator of an increase in cytosolic free Zn2+. In Znt8-KO islets, the depletion of zinc from secretory granules prevented the increase in [Zn2+]c and the upregulation of islet Mt1 and other MTF1-regulated genes (Mt2 and Znt1) by low glucose levels.

CONCLUSIONS/INTERPRETATION: Physiological changes in glucose levels dynamically regulate islet Mt1 gene expression in vivo as previously shown ex vivo. Mt1 downregulation by glucose depends on active mitochondrial metabolism and protein synthesis, and correlates with reduced [Zn2+]c and MTF1 activity. Its upregulation under low glucose is associated with autophagic degradation of insulin granules, release of intragranular Zn2+ into the cytosol, an increase in [Zn2+]c, and increased MTF1 activity. This upregulation is absent in Znt8-KO islets, thereby demonstrating that it requires a functional ZnT8 transporter. Our findings establish a novel link between MT1 and ZnT8, and shift our understanding of Mt1 regulation from a simple stress-dependent mechanism to a more complex model involving the modulation of cellular metabolism and intracellular zinc dynamics.