Exogenous Metallothionein Potentiates the Insulin Response at Normal Glucose Concentrations in INS‐1E Beta‐Cells without Disturbing Intracellular ZnT8 Expression

Abstract

As a consequence of the global epidemic of obesity, the incidence of type 2 diabetes (T2D) is increasing worldwide. T2D is characterized by hyperglycaemia, hyperinsulinaemia and a reduced insulin response in muscular and fatty tissue. Over time, an increased insulin demand leads to cellular fatigue and death of the insulin-producing b-cells. In response, the patients with T2D become insulin-dependent and subjected to the boundaries of lifelong insulin treatment. Preservation of b-cell insulin secretion and a sufficient b-cell mass is thus a corner stone in optimal T2D treatment.Physiologically, b-cell function and survival are closely related to zinc homeostasis. Zinc is essential for the primary functions of b-cells; insulin biosynthesis, insulin storage and insulin secretion and zinc are co-secreted with insulin in response to glucose stimulation. Hypozincaemia accompanied by hyperzincuria is often present in patients with diabetes [1]. On the other hand, fluctuations in extracellular zinc levels can become toxic for b-cells if high concentrations arise locally during stress or increased insulin secretion [2].Regulation of cellular-free zinc homeostasis is orchestrated by two categories of zinc-carrying proteins; the zinc-buffering metallothioneins (MTs) and the zinc transporters also known as Zinc Transporters (ZnTs) and Zrt-and Irt-like proteins (ZIPs). The ZnTs are responsible for Zn 2+ efflux from the cytoplasm to the extracellular matrix and into intracellular organelles like insulin-containing vesicles, whereas the ZIPs transport Zn 2+ in the opposite direction [3]. MTs tightly regulate the intracellular level of free zinc, and the levels of MTs are relatively high in the pancreas [3,4], suggesting that MTs are involved in the normal function of the gland. New studies link dysregulation or dysfunction of zinc-transporting proteins with impaired insulin processing and impaired glucose metabolism [4,6]. Furthermore, polymorphisms in genes encoding for isoforms of MTs have been related to the development of type 2 diabetes and to the extent of diabetic complications [7]. Transgenic mice, with b-cell-specific over-expression of MT-2 display, significantly reduced b-cell death, and the mice have a better preservation of insulin production when exposed to bcell toxic Streptozotocin [8] . Moreover, a specific neuroprotective role has been postulated for extracellular MT [9]. Here, we suggest that increasing MT levels by means of exogenous Zn 7 -MT-2A will be beneficial for b-cell function by contributing to an optimal zinc supply and regulation, testing this hypothesis in the glucose-sensitive, insulin-producing INS-1E b-cell culture. MethodsCell culture. INS-1E (rat), originally kindly provided by Prof. C.B. Wollheim, Switzerland, was employed in this study. The INS-1E cells are cultured in a CO 2 atmosphere in complete RPMI 1640 supplemented with 11 mM glucose, 10% (v/v) heat-inactivated foetal bovine serum, 50 lM b-mercaptoethanol, 2 mM L-Glutamine, 100 U/ ml penicillin and 100 lg/ml streptomycin as previous...

Identification of early biological changes in palmitate-treated isolated human islets

“…Transgenic mice, with a beta-cell-specific over-expression of MT-2 displayed a significantly reduced beta-cell death and a better preserved insulin production when exposed to streptozotocin [ 30 ]. Also, addition of extracellular Zn7-MT-2A potentiated insulin production and secretion from insulin-producing INS-1E beta-cell culture [ 31 ]. The abovementioned suggest that an initial increase in MTs transcript level in palmitate-treated human islets is an adaptive mechanism to support insulin synthesis during the insulin hypersecretion.…”

RETRACTED ARTICLE: Metallothionein ameliorates airway epithelial apoptosis upon particulate matter exposure: role of oxidative stress and ion homeostasis

“…HBE cells were treated with 1 μg/mL metallothioneins (rabbit liver, ALX-202-070, ENZO, USA)) dissolved in 0.9% saline for 6 h (Nygaard et al 2015;Leung et al 2018), then exposed to 0, 100, or 500 μg/mL PM for 24 h. Gene expression levels, ROS generation, caspase-3, -8, -9 ELISA assay, and apoptosis were evaluated in HBE cells as described above.…”

The zinc transporter Zip14 (SLC39a14) affects Beta-cell Function: Proteomics, Gene expression, and Insulin secretion studies in INS-1E cells

“…Using PrimeFlow-based flow cytometry, we measured the effect of glucose concentration on Zip14 , Znt8 , and insulin (Ins) mRNA expression, to determine whether Zip14 is regulated by glucose and whether its expression is co-regulated with insulin mRNA levels. Znt8 was used as a positive control for glucose dependence, as we have previously found decreased expression levels upon high glucose stimulation 3,45 .…”