Recent genome-wide association studies demonstrated that common variants of solute carrier family 30 member 8 gene (SLC30A8) increase susceptibility to type 2 diabetes. SLC30A8 encodes zinc transporter-8 (ZnT8), which delivers zinc ion from the cytoplasm into insulin granules. Although it is well known that insulin granules contain high amounts of zinc, the physiological role of secreted zinc remains elusive. In this study, we generated mice with β cell-specific Slc30a8 deficiency (ZnT8KO mice) and demonstrated an unexpected functional linkage between Slc30a8 deletion and hepatic insulin clearance. The ZnT8KO mice had low peripheral blood insulin levels, despite insulin hypersecretion from pancreatic β cells. We also demonstrated that a substantial amount of the hypersecreted insulin was degraded during its first passage through the liver. Consistent with these findings, ZnT8KO mice and human individuals carrying rs13266634, a major risk allele of SLC30A8, exhibited increased insulin clearance, as assessed by c-peptide/insulin ratio. Furthermore, we demonstrated that zinc secreted in concert with insulin suppressed hepatic insulin clearance by inhibiting clathrin-dependent insulin endocytosis. Our results indicate that SLC30A8 regulates hepatic insulin clearance and that genetic dysregulation of this system may play a role in the pathogenesis of type 2 diabetes.

The Aft1 transcription factor regulates the iron regulon in response to iron availability in Saccharomyces cerevisiae. Aft1 activates a battery of genes required for iron uptake under iron-starved conditions, whereas Aft1 function is inactivated under iron-replete conditions. Previously, we have shown that iron-regulated DNA binding by Aft1 is responsible for the controlled expression of target genes. Here we show that this iron-regulated DNA binding by Aft1 is not due to the change in the total expression level of Aft1 or alteration of DNA binding activity. Rather, nuclear localization of Aft1 responds to iron status, leading to iron-regulated expression of the target genes. We identified the nuclear export signal (NES)-like sequence in the AFT1 open reading frame. Mutation of the NES-like sequence causes nuclear retention of Aft1 and the constitutive activation of Aft1 function independent of the iron status of the cells. These results suggest that the nuclear export of Aft1 is critical for ensuring iron-responsive transcriptional activation of the Aft1 regulon and that the nuclear import/ export systems are involved in iron sensing by Aft1 in S. cerevisiae.Iron is an essential nutrient for virtually all organisms, working as a cofactor for critical proteins that mediate diverse processes such as cellular respiration and synthesis of metabolic intermediates (1). On the other hand, excess iron is extremely toxic, being capable of generating free radicals that damage macromolecules such as proteins, lipids, and DNA (2). The uptake and metabolism of iron must therefore be strictly regulated. In all organisms studied, the rates of iron uptake are tightly coupled to the levels of available iron and the needs of cells (3). In metazoans, the mechanisms that underlie regulation of iron metabolism including uptake, sequestration, and utilization have been well characterized. Two RNA-binding proteins called "iron regulatory protein," IRP1 1 and IRP2, are responsible for this regulation by controlling mRNA translation or mRNA stability by iron regulated mRNA-protein interactions (4). Although these proteins are closely related, IRP1 is a relatively stable protein (5, 6), and its RNA binding activity is inactivated by iron-sulfur cluster formation in iron-replete cells (7). By contrast, IRP2 is degraded by the ubiquitin-proteasome system in iron-replete cells (8).In Saccharomyces cerevisiae, iron deprivation induces activities of a high affinity iron uptake system (9) and siderophoremediated iron uptake system (10, 11). A transcription factor, Aft1, plays a critical role in this process (12). Aft1 protein binds to a regulatory region in the DNA of the genes required for iron uptake and induces the transcription of these genes in ironstarved cells (13). Conversely, Aft1 is not bound to its regulatory target element in iron-replete cells, and the expression of its target genes is not induced. An increasing number of Aft1 target genes have been implicated in iron metabolism, including a putative transporter complex located i...

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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.

The majority of CDF/ZnT zinc transporters form homooligomers. However, ZnT5, ZnT6, and their orthologues form hetero-oligomers in the early secretory pathway where they load zinc onto zinc-requiring enzymes and maintain secretory pathway functions. The details of this hetero-oligomerization remain to be elucidated, and much more is known about homo-oligomerization that occurs in other CDF/ZnT family proteins. Here, we addressed this issue using co-immunoprecipitation experiments, mutagenesis, and chimera studies of hZnT5 and hZnT6 in chicken DT40 cells deficient in ZnT5, ZnT6, and ZnT7 proteins. We found that hZnT5 and hZnT6 combine to form heterodimers but do not form complexes larger than heterodimers. Mutagenesis of hZnT6 indicated that the sites present in transmembrane domains II and V in which many CDF/ZnT proteins have conserved hydrophilic amino acid residues are not involved in zinc binding of hZnT6, although they are required for zinc transport in other CDF/ZnT family homo-oligomers. We also found that the long N-terminal half of hZnT5 is not necessary for its functional interaction with hZnT6, whereas the cytosolic C-terminal tail of hZnT5 is important in determining hZnT6 as a partner molecule for heterodimer formation. In DT40 cells, cZnT5 variant lacking the N-terminal half was endogenously induced during periods of endoplasmic reticulum stress and so seemed to function to supply zinc to zincrequiring enzymes under these conditions. The results outlined here provide new information about the mechanism of action through heterodimerization of CDF/ZnT proteins that function in the early secretory pathway.

A number of enzymes become functional by binding to zinc during their journey through the early secretory pathway. The zinc transporters (ZnTs) located there play important roles in this step. We have previously shown that two zinc transport complexes, ZnT5/ZnT6 heterodimers and ZnT7 homo-oligomers, are required for the activation of alkaline phosphatases, by converting them from the apo-to the holo-form. Here, we investigated the molecular mechanisms of this activation. ZnT1 and ZnT4 expressed in chicken DT40 cells did not contribute to the activation of tissue nonspecific alkaline phosphatase (TNAP). The reduced activity of TNAP in DT40 cells deficient in both ZnT complexes was not restored by zinc supplementation nor by exogenous expression of other ZnTs that increase the zinc content in the secretory pathway. Moreover, we showed that expression of ZnT5/ZnT6 heterodimers reconstituted with zinc transport-incompetent ZnT5 mutant failed to restore TNAP activity but could stabilize the TNAP protein as the apo-form, regardless of zinc status. These findings demonstrate that TNAP is activated not simply by passive zinc binding but by an elaborate two-step mechanism via protein stabilization followed by enzyme conversion from the apo-to the holo-form with zinc loaded by ZnT complexes in the early secretory pathway.

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.

The zinc transporter protein ZIP13 plays critical roles in bone, tooth, and connective tissue development, and its dysfunction is responsible for the spondylocheirodysplastic form of Ehlers-Danlos syndrome (SCD-EDS, OMIM 612350). Here, we report the molecular pathogenic mechanism of SCD-EDS caused by two different mutant ZIP13 proteins found in human patients: ZIP13G64D, in which Gly at amino acid position 64 is replaced by Asp, and ZIP13ΔFLA, which contains a deletion of Phe-Leu-Ala. We demonstrated that both the ZIP13G64D and ZIP13ΔFLA protein levels are decreased by degradation via the valosin-containing protein (VCP)-linked ubiquitin proteasome pathway. The inhibition of degradation pathways rescued the protein expression levels, resulting in improved intracellular Zn homeostasis. Our findings uncover the pathogenic mechanisms elicited by mutant ZIP13 proteins. Further elucidation of these degradation processes may lead to novel therapeutic targets for SCD-EDS.