Essential Role of the Zinc Transporter ZIP9/SLC39A9 in Regulating the Activations of Akt and Erk in B-Cell Receptor Signaling Pathway in DT40 Cells

The essential trace element zinc is important for all living organisms. Zinc functions not only as a nutritional factor, but also as a second messenger. However, the effects of intracellular zinc on the B cell-receptor (BCR) signaling pathway remain poorly understood. Here, we present data indicating that the increase in intracellular zinc level induced by ZIP9/SLC39A9 (a ZIP Zrt-/Irt-like protein) plays an important role in the activation of Akt and Erk in response to BCR activation. In DT40 cells, the enhancement of Akt and Erk phosphorylation following BCR activation requires intracellular zinc. To clarify this event, we used chicken ZnT5/6/7-gene-triple-knockout DT40 (TKO) cells and chicken Zip9-knockout DT40 (cZip9KO) cells. The levels of Akt and ERK phosphorylation significantly decreased in cZip9KO cells. In addition, the enzymatic activity of protein tyrosine phosphatase (PTPase) increased in cZip9KO cells. These biochemical events were restored by overexpressing the human Zip9 (hZip9) gene. Moreover, we found that the increase in intracellular zinc level depends on the expression of ZIP9. This observation is in agreement with the increased levels of Akt and Erk phosphorylation and the inhibition of total PTPase activity. We concluded that ZIP9 regulates cytosolic zinc level, resulting in the enhancement of Akt and Erk phosphorylation. Our observations provide new mechanistic insights into the BCR signaling pathway underlying the regulation of intracellular zinc level by ZIP9 in response to the BCR activation

Characterisation of Ppy-lineage cells clarifies the functional heterogeneity of pancreatic beta cells in mice

Aims/hypothesis Pancreatic polypeptide (PP) cells, which secrete PP (encoded by the Ppy gene), are a minor population of pancreatic endocrine cells. Although it has been reported that the loss of beta cell identity might be associated with beta-to-PP cell-fate conversion, at present, little is known regarding the characteristics of Ppy-lineage cells. Methods We used Ppy-Cre driver mice and a PP-specific monoclonal antibody to investigate the association between Ppy-lineage cells and beta cells. The molecular profiles of endocrine cells were investigated by single-cell transcriptome analysis and the glucose responsiveness of beta cells was assessed by Ca2+ imaging. Diabetic conditions were experimentally induced in mice by either streptozotocin or diphtheria toxin. Results Ppy-lineage cells were found to contribute to the four major types of endocrine cells, including beta cells. Ppy-lineage beta cells are a minor subpopulation, accounting for 12–15% of total beta cells, and are mostly (81.2%) localised at the islet periphery. Unbiased single-cell analysis with a Ppy-lineage tracer demonstrated that beta cells are composed of seven clusters, which are categorised into two groups (i.e. Ppy-lineage and non-Ppy-lineage beta cells). These subpopulations of beta cells demonstrated distinct characteristics regarding their functionality and gene expression profiles. Ppy-lineage beta cells had a reduced glucose-stimulated Ca2+ signalling response and were increased in number in experimental diabetes models. Conclusions/interpretation Our results indicate that an unexpected degree of beta cell heterogeneity is defined by Ppy gene activation, providing valuable insight into the homeostatic regulation of pancreatic islets and future therapeutic strategies against diabetes

Detailed analyses of the crucial functions of Zn transporter proteins in alkaline phosphatase activation

Numerous zinc ectoenzymes are metalated by zinc and activated in the compartments of the early secretory pathway before reaching their destination. Zn transporter (ZNT) proteins located in these compartments are essential for ectoenzyme activation. We have previously reported that ZNT proteins, specifically ZNT5-ZNT6 heterodimers and ZNT7 homodimers, play critical roles in the activation of zinc ectoenzymes, such as alkaline phosphatases (ALPs), by mobilizing cytosolic zinc into these compartments. However, this process remains incompletely understood. Here, using genetically-engineered chicken DT40 cells, we first determined that Zrt/Irt-like protein (ZIP) transporters that are localized to the compartments of the early secretory pathway play only a minor role in the ALP activation process. These transporters included ZIP7, ZIP9, and ZIP13, performing pivotal functions in maintaining cellular homeostasis by effluxing zinc out of the compartments. Next, using purified ALP proteins, we showed that zinc metalation on ALP produced in DT40 cells lacking ZNT5-ZNT6 heterodimers and ZNT7 homodimers is impaired. Finally, by genetically disrupting both ZNT5 and ZNT7 in human HAP1 cells, we directly demonstrated that the tissue-nonspecific ALP-activating functions of both ZNT complexes are conserved in human cells. Furthermore, using mutant HAP1 cells, we uncovered a previously-unrecognized and unique spatial regulation of ZNT5-ZNT6 heterodimer formation, wherein ZNT5 recruits ZNT6 to the Golgi apparatus to form the heterodimeric complex. These findings fill in major gaps in our understanding of the molecular mechanisms underlying zinc ectoenzyme activation in the compartments of the early secretory pathway

Role of Zinc Homeostasis in the Pathogenesis of Diabetes and Obesity

Zinc deficiency is a risk factor for obesity and diabetes. However, until recently, the underlying molecular mechanisms remained unclear. The breakthrough discovery that the common polymorphism in zinc transporter SLC30A8/ZnT8 may increase susceptibility to type 2 diabetes provided novel insights into the role of zinc in diabetes. Our group and others showed that altered ZnT8 function may be involved in the pathogenesis of type 2 diabetes, indicating that the precise control of zinc homeostasis is crucial for maintaining health and preventing various diseases, including lifestyle-associated diseases. Recently, the role of the zinc transporter ZIP13 in the regulation of beige adipocyte biogenesis was clarified, which indicated zinc homeostasis regulation as a possible therapeutic target for obesity and metabolic syndrome. Here we review advances in the role of zinc homeostasis in the pathophysiology of diabetes, and propose that inadequate zinc distribution may affect the onset of diabetes and metabolic diseases by regulating various critical biological events

Role of Zinc Homeostasis in the Pathogenesis of Diabetes and Obesity

Zinc deficiency is a risk factor for obesity and diabetes. However, until recently, the underlying molecular mechanisms remained unclear. The breakthrough discovery that the common polymorphism in zinc transporter SLC30A8/ZnT8 may increase susceptibility to type 2 diabetes provided novel insights into the role of zinc in diabetes. Our group and others showed that altered ZnT8 function may be involved in the pathogenesis of type 2 diabetes, indicating that the precise control of zinc homeostasis is crucial for maintaining health and preventing various diseases, including lifestyle-associated diseases. Recently, the role of the zinc transporter ZIP13 in the regulation of beige adipocyte biogenesis was clarified, which indicated zinc homeostasis regulation as a possible therapeutic target for obesity and metabolic syndrome. Here we review advances in the role of zinc homeostasis in the pathophysiology of diabetes, and propose that inadequate zinc distribution may affect the onset of diabetes and metabolic diseases by regulating various critical biological events

Proposed action sites of intracellular zinc release by ZIP9 in DT40 cells for activation of B cell receptor signaling.

<p>It is the proposed mechanism of Zn-induced PTPase inhibition by ZIP9, which leads to the activation of B cell receptor signaling in DT40 cells. Intracellular zinc is incorporated into the Golgi by ZnT5/6/7. Zinc is released as induced by ZIP9 into the cytosol from the Golgi, which in turn inhibits PTPase activity and induces the phosphorylation of Akt and ERK probably indirectly by regulating upstream components of the signal transduction.</p

Effect of overexpression of human Zip9 on phosphorylation levels of Akt and Erk in response to zinc treatment and anti-IgM antibody stimulation.

<p>(A) Overexpression of hZip9 restored the phosphorylation of Akt and Erk. Western blot analysis was performed using exponentially growing WT (lane 1), cZip9KO (lane 2), and cZip9KO+hZip9HA (lane 3) cells. (B) Overexpression of hZip9 in cZip9KO cells by ZyPy treatment stimulated the phosphorylation of both proteins. Serum-starved WT (lanes 1–3), cZip9KO (lanes 4–6), and cZip9KO+hZip9HA (lanes 7–9) cells were treated with 10 µM ZnPy for 10 min (lanes 2, 5 and 8) and 30 min (lanes 3, 6 and 9). The abbreviation, “unt.” was defined the untreated sample. (C) Analysis of total PTPase activity. Serum-starved WT (lanes 1–3), cZip9KO (lanes 4–6), and cZip9KO+hZip9HA (lanes 7–9) cells were treated with 10 µM ZnPy for 10 min (lanes 2, 5 and 8) and 30 min (lanes 3, 6 and 9). Values are expressed as the mean ± standard deviations. Significant difference at the level of *<i>P</i><0.01 for the columns linked by a line. (D) Overexpression of hZip9 restored the response to anti-IgM antibody-stimulated BCR activation. Serum-starved WT (lanes 1 and 2), cZip9KO (lanes 3 and 4), and hZip9-HA-overexpressing cZip9KO (lanes 5 and 6) cells were treated with 0.5 mg/mL anti-IgM antibody for 10 min. All data are representative of three independent experiments.</p

Akt and Erk phosphorylation in zinc-transporter-knockout DT40 cells.

<p>(A) Suppression of Akt and Erk phosphorylation in cZip9KO cells. Western blot analysis was performed using exponentially growing WT (lane 1), TKO (lane 2), and cZip9KO (lane 3) cells. (B) Analysis of total PTPase activity. WT (column 1), TKO (column 2), and cZip9KO (column 3) cells were subjected to PTPase assay. Values are expressed as the mean ± standard deviations. Significant difference at the level of *<i>P</i><0.01 against the activity of WT cells (column 1). (C) ZnPy failed to induce Akt and Erk phosphorylation in cZip9KO cells. Serum-starved WT (lanes 1 and 2), TKO (lanes 3 and 4), and cZip9KO (lanes 5 and 6) cells were treated with (+) or without (−) 10 µM ZnPy for 10 min. (D) Analysis of PTPase activity in serum-starved DT40 cells. After treatment of serum-starved WT (columns 1 and 2), TKO (columns 3 and 4), and cZip9KO (columns 4 and 5) cells treated with (columns 2, 4 and 6) or without (columns 1, 3 and 5) 10 µM ZnPy for 10 min, and subjected to PTPase assay. Values are expressed as the mean ± standard deviations. Asterisk represents significant difference at the level of *<i>P</i><0.01 for the columns linked by a line. All data are representative of three independent experiments.</p