Zrt-Irt-like Proteins (ZIP) Family Zinc Transporters: Emerging Players in Pancreatic β Cell Function and Insulin Regulation

Abstract

Zinc is an essential micronutrient with diverse catalytic, structural, and regulatory roles across various life forms. Its essentiality for human health was recognized in the 1960s, but advancements in understanding the functions of zinc at the tissue, cell, and subcellular levels have accelerated, particularly with the identification of zinc transporters (ZNT). Zinc homeostasis is primarily facilitated by 2 families of transporters, the SLC30A/ZNT (ZNT) and SLC39A/Zrt-Irt-like proteins (ZIP). Among these, the ZNT family transporter ZNT8 has been well-studied for its involvement in insulin production, secretion, and the viability of pancreatic β cells. However, the roles of ZIP family transporters in β-cell insulin-related functions remain less explored. There have been studies implicating regulatory roles of ZIP4, ZIP5, ZIP6, and ZIP7 in β cells and emerging evidence for the involvement of ZIP8 and ZIP14 in β cell function. Despite these insights, the limited number of studies on ZIP family transporters highlights the need to consolidate existing literature to identify gaps and establish targeted, comprehensive research approaches that can further elucidate their critical roles in cellular zinc homeostasis and insulin metabolism. In this review, we first address the role of zinc in insulin production, secretion, and action. Second, we discuss the known ZIP transporters that potentially facilitate zinc delivery to specific cell compartments, focusing on literature addressing zinc and ZNT specifically relevant to insulin and glucose metabolism.

Introduction

Zinc is a fundamental type II essential nutrient integral to numerous aspects of general metabolism and cellular function. Unlike nutrients associated with specific physiological roles, zinc deficiency manifests through a wide array of biochemical dysfunctions, indicating its broad importance in human health. The recognition of zinc's essentiality dates back to 1934 through animal studies [1] and was later confirmed in humans by 1961 [2]. In 2002, the WHO officially acknowledged zinc deficiency as a significant global health risk factor contributing to increased disease-related mortality and morbidity [3]. Currently, an estimated 1.2 billion individuals worldwide, predominantly in developing nations, suffer from inadequate dietary zinc intake [4]. However, zinc deficiency also prevails in industrialized countries, particularly among people with chronic inflammatory and metabolic conditions such as diabetes and obesity [5].

The relationship between zinc and diabetes is especially noteworthy. Patients with diabetes often exhibit elevated urinary zinc levels, indicating excessive zinc loss associated with the disease state. Emerging epidemiological studies further suggest that insufficient dietary zinc intake may elevate risk of developing type 2 diabetes mellitus [6]. Adequate zinc levels are crucial for proper insulin production and secretion, as zinc serves as an essential structural component of insulin crystals and participates in multiple intracellular processes involved in insulin synthesis and release. Consequently, disruptions in zinc homeostasis can have profound implications for glucose metabolism and overall endocrine health. Because zinc homeostasis is often disrupted in metabolic diseases such as obesity, metabolic syndrome, and type 2 diabetes, zinc supplementation has been explored as a potential adjunct therapy, with studies in both animals and humans reporting improvements in fasting blood glucose, insulin levels, and lipid profiles [5,[7], [8], [9], [10]]. However, the effects are inconsistent, as other studies have found no significant improvements [11,12]. These mixed results emphasize the need for more research to better understand how zinc influences insulin and glucose metabolism under normal and disease conditions.

Maintaining cellular zinc homeostasis is facilitated primarily by 2 families of metal transporters: SLC39A/Zrt-Irt-like proteins (ZIP) and SLC30A/ZNT (zinc transporters) (Figure 1) [[13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34]]. To date, 14 ZIP family transporters (ZIP1-14) and 10 ZNT family transporters (ZNT1-10) have been identified. ZIP transporters are responsible for importing zinc into the cytoplasm from extracellular spaces and intracellular compartments, whereas ZNT transporters export zinc from the cytoplasm to extracellular spaces or sequester it within intracellular organelles. These transporters exhibit tissue-specific expression patterns, and their subcellular localization and expression levels can vary based on physiological conditions and the zinc status of the body [34]. Regulation of these transporters is multifaceted, responding not only to zinc levels but also to various cytokines, hormones, secondary messengers, and dietary components, highlighting the dynamic nature of zinc metabolism in both health and disease [13,14,[35], [36], [37], [38], [39], [40], [41], [42]].

FIGURE 1.

Generic depiction of cellular and subcellular distribution of zinc transporters and zinc concentrations [[13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33]]. ∗Unlike other ZNT family transporters that export zinc from the cytosol, ZNT9 functions as a mitochondrial zinc exporter [17]. ER, endoplasmic reticulum; MT, metallothionein; ZIP, Zrt-Irt-like proteins; ZNT, zinc transporters.

Genetic mutations affecting ZNT have been linked to several disorders, including acrodermatitis enteropathica (ZIP4) [43], Ehlers-Danlos syndrome (ZIP13) [44], and lethal zinc syndrome (ZNT2) [45]. Notably, mutations in ZNT8 are associated with both type 1 and type 2 diabetes, emphasizing the critical role of zinc transport in maintaining metabolic health. ZNT8 is a member of the SLC30A family zinc transporter, which transports zinc into the secretory insulin granules of β cells [46, 47]. A common polymorphism in the ZNT8 gene (SLC30A8) is associated with an increased risk of type 2 diabetes [48, 49], whereas rare loss-of-function (LOF) mutations provide protective effects [50, 51]. Unexpectedly, high-risk mutant ZNT8 demonstrates significantly enhanced zinc transport activity [52,53], emphasizing the importance of investigating potential counter-regulation by the ZIP family of transporters. However, in sharp contrast to ZNT8, relatively little is known about the functions of ZIP family transporters in β cells and insulin production and secretion. Studies suggest regulatory roles for ZIP4, ZIP5, ZIP6, and ZIP7 in β cells, with potential implications also emerging for ZIP8 and ZIP14 (Figure 2). In addition, ZIP5, ZIP7, ZIP8, ZIP13, and ZIP14-mediated zinc transport is emerging to influence metabolic response in peripheral tissues. Further research is necessary to elucidate these connections fully, which could uncover novel therapeutic targets for managing and preventing diabetes and other zinc-related metabolic disorders. Particular attention should be given to their cellular and subcellular localizations as differential regulation of these transporters and zinc concentration at these localizations provides a mechanistic understanding of their roles in insulin production and secretion by the β cells and their action on the peripheral tissues at normal and pathophysiological conditions. The specific ZNT that are expressed in β cells [54], their roles in the pancreas [55] and potential implications for type 2 diabetes [56] have been reviewed previously. This review focuses on new literature describing mechanistic roles of ZIP family transporters in insulin production, secretion, and β cell function.

FIGURE 2.

Depiction of the cellular and subcellular localizations of zinc transporters in pancreatic β cells during insulin production and secretion. ER, endoplasmic reticulum; GLUT2, glucose transporter 2; GPR39, G protein-coupled receptor 39; INS, insulin; KATP, ATP-sensitive potassium channel; VGCC, voltage-gated calcium channels; ZIP, Zrt-Irt-Like proteins; ZNT, zinc transporters.

Zinc, insulin, and β cells

Pancreatic islets contain the highest concentration of zinc in the entire pancreas. Zinc is the structural component of the insulin hormone. Insulin is a peptide hormone synthesized and secreted by pancreatic β cells. Insulin biosynthesis is a complex, multistep process regulated by various intracellular and extracellular signals and involves transcription, translation, and posttranslational modifications. The first step in insulin biosynthesis is the insulin gene transcription, which is translated into a preproinsulin polypeptide chain. The preproinsulin molecule contains a signal sequence that directs it to the endoplasmic reticulum (ER), where the signal sequence is cleaved from the N-terminus, and the preproinsulin is converted into proinsulin. Proinsulin consists of 3 parts: the A chain and the B chain, which are connected by disulfide bridges, and the C peptide, which is located between the A and B chains. Proinsulin is converted into insulin in the Golgi apparatus, where the C peptide is cleaved by prohormone convertase, converting proinsulin into insulin. As zinc enters the secretory granules, insulin undergoes maturation, and eventually, 2 zinc stabilize the structure of insulin hexamers [[57], [58], [59], [60], [61]]. The insulin is then stored in the secretory granules until it is needed.

Insulin secretion is a multistep process starting with sensing the high blood glucose levels and glucose transport into β cells via glut 1 and glut 2 transporters. Glucokinase controls glucose entry into the glycolytic cycle, glucose oxidation in the tricarboxylic acid cycle, and subsequent ATP production [62]. Glucose metabolism increases the ATP-to-APD ratio, which leads to the closure of potassium ATP (KATP) channels. The 4 sulfonylurea receptor 1 (SUR1) regulatory subunits of KATP channels bind ATP to cause channel closure, membrane depolarization, and opening of voltage-gated calcium channels (VGCC) [63]. This results in the initiation of glucose-stimulated insulin secretion. This stimulus-secretion coupling pathway is termed the "first phase" and occurs over 5–10 min after square-wave glucose stimulation. After the first phase, insulin secretion occurs in the second phase, requiring the recruitment of insulin granules to the plasma membrane. This process is mediated by the rearrangement of cortical filamentous actin (F actin), which facilitates the movement of insulin granules from intracellular storage pools to the plasma membrane. Insulin secretion can occur over the course of hours, the majority of which occurs in the second phase. The first phase of insulin release accounts for roughly 15% of secreted insulin within 1 h, and the rest occurs in the second phase. In addition to the triggering pathway, the amplification pathway serves to optimize insulin secretion. The amplification pathway depends on the triggering pathway and increases the efficacy of Ca2+ on the exocytosis of insulin granules. Extracellular and intracellular zinc ions act as potent, reversible activators of ATP-sensitive potassium (KATP) channels, specifically targeting the SUR subunit. Zinc at micromolar concentrations activates KATP channels composed of Kir6.2 and SUR1 while slightly inhibiting channels containing SUR2A. These findings suggest a role for zinc as a signaling molecule, potentially providing negative feedback to insulin secretion by modulating KATP channel activity. In support of that, secreted extracellular zinc, along with insulin, is suggested to provide autocrine negative regulation for insulin secretion. Studies have shown the inhibitory effect of zinc supplementations on glucose-induced insulin secretion (GSIS) from β cells. Furthermore, recent studies of protective LOF mutation of ZnT8 showed reduced zinc content in islets and enhanced insulin secretion. However, insulin secretion was reduced when zinc was added along with glucose stimulation. The inhibitory effect of zinc on insulin secretion was also confirmed in human islet cultures and human embryonic stem cell-derived β cells [64]. On the basis of these data, we speculate that the modulation of KATP channels by zinc could provide a negative feedback loop during glucose-stimulated insulin secretion.

KATP channel closure leads to membrane depolarization, the opening of VGCC channels, and insulin exocytosis [63] Several voltage-dependent Ca2+ channels are expressed in the plasma membrane of β cells, including T-, L-, P/Q-, and R-type channels; L-type channels are dominant in both mouse and human β cells. Zinc could act as a competitive inhibitor for Ca2+ permeability in L, N, P/Q, T-type, and store-operated Ca2+ channels in the presence of sufficient Ca2+ levels [[65], [66], [67], [68]]. These findings suggest the presence of a negative feedback mechanism during glucose-stimulated insulin secretion, wherein the large amounts of extracellular zinc cosecreted with insulin may competitively inhibit insulin secretion.

Cav2.3 channels, part of the high-voltage-activated family of VGCCs, are implicated in pancreatic islet hormone secretion and blood glucose homeostasis. Specifically, these channels are involved in β-cell insulin release, a key process in GSIS [[69], [70], [71]]. Cav2.3 channels mediate the majority of native R-type calcium currents and are characterized by their resistance to most organic calcium channel antagonists [72,73]. Importantly, they are highly sensitive to zinc and are recognized as one of the most zinc-responsive molecular targets [74]. Notably, Cav2.3 channels have been shown to specifically contribute to the second phase of insulin secretion, as genetic or pharmacological ablation of these channels selectively attenuates this response [69,70]. These findings suggest that intracellular zinc dynamics, including its redistribution among the cytosol, organelles, and insulin granules, may influence the Cav2.3 channel function, thereby controlling sustained insulin secretion during hyperglycemia.

ZnR/GPR39, a zinc-sensing G-protein-coupled receptor located in the plasma membrane, has emerged as an important regulator of pancreatic function [[75], [76], [77]]. Studies have demonstrated that this receptor is activated by physiological concentrations of zinc [78, 79], and subsequent research has revealed that exogenous, allosteric zinc binding to ZnR/GPR39 triggers calcium release from ER stores [80,81]. GPR39 is predominantly expressed in islet β-cells and ductal cells in pancreatic tissue. Studies using GPR39 knockout mice (KO) have shown impaired insulin secretion, particularly during glucose challenge tests in young adult mice [76,77]. The importance of GPR39 in metabolism is further demonstrated under dietary stress, where GPR39-deficient mice show elevated blood glucose levels on high-fat diets, and older animals exhibit reduced insulin levels on sucrose-rich diets [82]. In contrast, increased GPR39 expression in pancreatic β-cells protects against hyperglycemia [83]. These findings point to a regulatory autocrine feedback mechanism where zinc, coreleased with insulin, may activate GPR39 to influence intracellular calcium release and insulin secretion.

Most zinc research has been focused on GSIS. However, in addition to glucose, pancreatic β cells also produce and secrete insulin in response to nonglucose stimuli, including amino acids [84,85] and fatty acids [86]. Notably, fatty acids are known to boost GSIS. However, chronic exposure to high levels of fatty acids and glucose can compromise β-cell viability and function [86]. The role of zinc in these additional inducers of insulin secretion and related pathways has not been studied and warrants further interesting research.

Zinc, hyperinsulinemia, and pancreatic β cells

Traditional thinking attributes compensatory hyperinsulinemia to insulin resistance and obesity; however, emerging evidence suggests that insulin hypersecretion and hyperinsulinemia precede and contribute to the development of insulin resistance and obesity [87,88]. Studies have shown that high insulin levels in the body, either due to overeating or excessive insulin secretion, lead to significant fat storage and subsequent weight gain [88]. In mice, manipulating genes to prevent chronic hyperinsulinemia reduced the incidence of obesity and insulin resistance caused by a high-fat diet. Similarly, in humans, individuals with high insulin secretion are at a higher risk of developing impaired glucose tolerance and type 2 diabetes [89].

The notion of hyperinsulinemia preceding diabetes and obesity is further supported by the findings from human studies, including the rs13266634 polymorphism and the Eugene study, which demonstrated low peripheral insulin levels in carriers of the SLC30A8 risk allele during early intravenous glucose tolerance test phases and improved glucose metabolism by reducing circulating insulin [90]. Studies where insulin secretion and clearance were investigated using ZnT8 KO (ZnT8-KO) mice exhibit reduced peripheral insulin levels despite unchanged or slightly increased GSIS in islets. Pancreas perfusion experiments confirmed enhanced insulin secretion in ZnT8-KO mice, but dual pancreas-liver perfusion revealed significant insulin degradation during hepatic passage, implicating ZnT8 in regulating hepatic insulin clearance. Mechanistically, ZnT8-mediated zinc counteracts clathrin-mediated endocytosis of the insulin receptor, inhibiting hepatic insulin uptake and optimizing insulin delivery to peripheral organs, thereby influencing whole-body glucose metabolism.

ZIP family ZNT, insulin production/secretion, and pancreatic β cells

SLC39A4/ZIP4

ZIP4 is a ZIP family zinc transporter that influences zinc homeostasis in pancreatic β cells and the intestine [91]. It has been established that mice ZIP4 colocalizes with insulin under immunofluorescence imaging [92]. Zip4 expression in pancreatic β cells is further validated by qPCR analysis of Zip4 mRNA in mouse β cells. The functional link between ZIP4 and zinc transport in β cells is established by in vitro overexpression of Zip4 and the resulting increase in insulin secretion. Surprisingly, immunofluorescence imaging revealed that ZIP4 was not explicitly localized to plasma membranes, but instead was diffused throughout the mouse β cells. Despite its diffuse staining, ZIP4 overexpression resulted in an 18% increase in intracellular zinc concentration. ZIP4 overexpression in vitro resulted in increased insulin secretion, but did not affect the expression level of insulin mRNA or the insulin processing enzymes Pc1 and Cpe. ZIP4 overexpression, however, may have an impact on insulin processing, as the processing enzyme PC2 exhibits increased mRNA expression.

Paradoxically, the insulin secretion level in β cell-specific Zip4 KO (Zip4-βKO) mice remained unchanged. Although Zip4-βKO mice demonstrated improved glucose homeostasis, insulin secretion, and blood glucose levels remained unchanged. In Zip4-βKO mice, ZnT8 expression is slightly elevated above control. Whether this elevation could functionally compensate for the loss of Zip4 is unknown. However, it is noteworthy that different ZNT can have potential compensatory mechanisms. It is hypothesized that elevated expression of ZnT8 in Zip4-βKO might lead to extra zinc being transported to insulin granules, thus offsetting the impact of Zip4 KO. Taken together, it appears that ZIP4-mediate Zn transport in β cells can supply Zn to increase insulin processing and secretion, but is not necessary for proper β cell function in vivo.

SLC39A5/ZIP5

Measurement of SLC39A family zinc transporter mRNA expression level in β cell islets of diabetic mouse models reveals that Zip5 is consistently expressed at lower levels across all tested models: High-fat diet-fed (HFD) mice, leptin mutant mice (ob/ob), and leptin receptor mutant mice (db/db) [93]. Whole-body KO of Zip5 mice demonstrated significantly reduced GSIS compared with the wild type. Pancreatic β cell-specific Zip5 KO (Zip5-βKO) mice demonstrate significantly reduced glucose-mediated zinc uptake, suggesting a critical role for ZIP5 in β cell zinc uptake and insulin secretion upon glucose stimulation.

Another connection between ZIP5 and diabetes is the ability of ZIP5 to regulate the expression level of glucose transporter GLUT2 and corresponding blood glucose levels. GLUT2 is an important glucose transporter in the pancreas, kidney, intestine, and nervous system. Genome-wide association studies (GWAS) have associated GLUT2 variations with hyperglycemia and type 2 diabetes. In Zip5-βKOmice, GLUT2 protein expression level is markedly reduced. As a result, Zip5-βKOmice demonstrated impaired glucose uptake and increased blood glucose levels after glucose treatment.

In pancreatic islets from Zip5-βKO mice, Sirt1, Ppargc1a, and Mtf1 genes have significantly reduced levels of mRNA expression. Sirt 1 regulates mitochondrial biogenesis and mitophagy and is a regulator of Pgc1-a. Pgc1-a is a regulator of glucose metabolism and mitochondrial biogenesis. Overexpression of Pgc1-a in MIN6 pancreatic β cell line rescues Glut2 expression, and Pgc 1-a activator ZLN005 treatment rescues insulin in pancreatic islets of Zip5-βKOmice. Similarly, Sirt1 activator SRT1720 also rescues secretion in Zip5-βKO islets. The regulatory roles of SIRT 1 and PGC 1-a in mitochondrial biogenesis might potentially lead to mitochondrial differences between Zip5-βKO and control islets. Mitochondria swellings and altered membrane potential are observed in Zip5-βKO β cells, but the expression levels of mitochondrial complexes I through V remain unaltered.

SLC39A6/ZIP6 AND SLC39A7/ZIP7

ZIP6 and ZIP7 are the most highly expressed ZIP proteins in human and mouse β cells [94]. Interestingly, both ZIP6 and ZIP7 colocalize with insulin in mice pancreatic β cells and display lower mRNA expression in islets of patients with type 2 diabetes [54]. ZIP6 has been shown to colocalize with the ER, whereas ZIP7 has been shown to colocalize with the ER and the Golgi. In dispersed pancreatic islets of mice, both ZIP6 and ZIP7 colocalize with the ER, and ZIP6 migrates toward the plasma membrane whereas ZIP7 remains in the ER after glucose stimulation. siRNA knockdown of Zip6 leads to increased expression of ZIP7, but the reverse is not observed. Simultaneous overexpression of ZIP6 and ZIP7 leads to an increase in zinc uptake and sustained elevation of intracellular zinc content. siRNA knockdown of both Zip6 and Zip7 leads to a significant decrease in glucose-stimulated zinc uptake, but not basal zinc uptake, in dispersed mouse pancreatic islet cells. In β cells, siRNA knockdown of both Zip6 and Zip7, but not Zip6 or Zip7 individually, impairs insulin secretion upon glucose and KCl stimulation, indicating a possible compensatory mechanism between ZIP6 and ZIP7. In the double knockdown of Zip6 and Zip7, the insulin granule exocytosis rate is significantly reduced during the first phase of insulin secretion. A significant rise in Reactive Oxygen Species (ROS) level is also observed in the double knockdown of Zip6 and Zip7 β cells, indicating increased oxidative stress. However, apoptosis is not observed in the double knockdown of Zip6 and Zip7 β cells, given unaltered caspase 3/7 activity.

SLC39A8/ZIP8

Expression of ZIP8 in human β cell-enriched pancreatic samples and islets was shown [54]. Furthermore, 2 independent human datasets demonstrated differential regulation of ZIP8 in type 2 diabetic islets compared with nondiabetic islets, suggesting a role for ZIP8 in β cell biology and insulin production and secretion.

The potential role of ZIP8 in pancreatic β cells was first identified in isolated mouse islets, where glucose stimulation increased intracellular zinc concentrations and upregulated Zip8, along with Zip6 and Zip7 [95]. These changes were attributed to the necessity of zinc uptake in response to glucose to support new insulin synthesis. Our recent study confirmed a significant increase in ⁶⁵Zinc uptake in MIN6 cells after glucose-only treatment, supporting these findings [15]. Furthermore, combined glucose and KCl treatment resulted in greater insulin and ⁶⁵Zinc efflux, confirming zinc loss accompanying insulin secretion. These findings are in alignment with decreased zinc in MIN6 cells in response to prolonged stimulation of insulin by KCl treatment [96]. Additional studies further support the role of ZIP8 in β-cell zinc transport. In rat islets, intermittent hypoxia downregulated ZIP8 in the β-cell membrane, accompanied by reduced zinc uptake [97]. Similarly, siRNA-mediated knockdown of Zip8 in rat islets led to decreased zinc uptake, highlighting its zinc transport function in islets.

The role of ZIP8 in β-cell differentiation and function was further investigated using stem cells from human exfoliated deciduous teeth (SHED). During differentiation into insulin-secreting β cell-like cells (SHED-β cells), ZIP8 expression increased significantly [98]. Zinc supplementation (50 μM) enhanced the expression of pancreatic β cell markers, including insulin and GLUT2, elevated ZIP8 protein levels, and boosted insulin secretion by ∼25%. Immunofluorescence and western blot analyses confirmed higher levels of insulin and ZIP8 in SHED-β cells after zinc supplementation.

Zip8 expression appears to be influenced by ZnT8 activity. In haplodeficient ZnT8 MIN6 cells, used as a model for low type 2 diabetes risk, LOF variants of ZnT8, only Zip8 and Zip14 expression were reduced among 24 transporters measured, along with lower cellular zinc levels, suggesting a particular impact of ZnT8 knockdown on ZIP8 and Zip14 expression compared with other ZIP family transporters [99]. The iron and manganese levels were also measured because these transporters may transport those metals. However, no change was observed in the cellular levels of these metals. These findings emphasize the importance of the potential counteraction of ZIP family transporters to maintain optimum zinc concentrations in pancreatic β cells. Interestingly, lower hepatic ZnT8 expression in ZIP8 knock-in (ZIP8-KI) mice carrying single-nucleotide polymorphism (SNP) variant A391T (rs13107325) in human ZIP8 was shown [100], suggesting a potential regulatory link between these 2 transporters.

Collectively, ZIP8-mediated Zn transport appears to be involved in β-cell differentiation and function, as its expression is stimulated by glucose and differentiation of SHED-β cells. However, it remains to be seen what specific mechanistic roles ZIP8 may play in insulin production and secretion, and whether or not it is necessary for β-cell function.

SLC39A14/ZIP14

ZIP14 is expressed and studied in the liver, small intestine, and adipose tissue. The function of ZIP14 in those tissues and others has been reviewed elsewhere [101]. In the pancreas, expression of ZIP14 in human β cell-enriched pancreatic samples and islets was shown [54]. Furthermore, ZIP14 is downregulated in type 2 diabetes islets compared with nondiabetic islets, indicating a possible role for ZIP14 in regulating zinc homeostasis in β cells.

We have previously shown that whole-body Zip14 KO mice displayed phenotypes that are relevant to β cell insulin-related functions, including increased intestinal permeability with elevated serum endotoxin levels (metabolic endotoxemia), altered body composition with a shift toward fat mass, a shift in energy substrate preference toward glucose, and enlarged pancreatic islets with mildly increased levels of serum insulin (hyperinsulinemia) [[101], [102], [103]]. One of the consequences of metabolic endotoxemia and low-grade chronic inflammation is compensatory hyperinsulinemia. With prolonged inflammation, hyperglycemia may develop due to exhaustion and failure of β cells to secrete insulin. Surprisingly, Zip14 KO mice do not develop hyperglycemia at a steady state despite the presence of low-grade chronic inflammation and insulin resistance in adipose tissue. We hypothesized that additional metabolic stress to hyperinsulinemic whole-body (WB) Zip14 KO mice by 16 weeks of HFD feeding might cause β cell dysfunction and impaired insulin secretion. However, in our studies, the highest levels of insulin were found in the plasma of HFD-fed Zip14 KO mice [13,15], suggesting that β cells in the Zip14 KO mice were protected from chronic inflammation-induced damage. Recently, we have characterized the role of ZIP14-mediated intracellular zinc trafficking in β cells in insulin secretion and subsequent metabolic responses (Figure 3) [15]. In the mouse pancreas, ZIP14 was found to be highly expressed in islets and colocalized with insulin. This was in alignment with data from single-cell RNA-seq data from human pancreatic tissues showing that SLC39A14, along with SLC30A8, was highly expressed in β cells [54,102]. On stimulation with glucose, ZIP14 expression was strongly decreased in mouse pancreas tissue and isolated islets. This, however, is in contrast to studies in INS-1E cells in which Zip14 mRNA was found to be increased after treatment with high glucose [104]. A key difference is that our studies utilized cotreatment with glucose and KCl. This is in alignment with previous work showing that prolonged Potassium chloride (KCl) stimulation could decrease intracellular zinc levels in pancreatic β cells, resulting in downregulation of Zip14 mRNA, collectively indicating a possible role for ZIP14 in regulating zinc homeostasis in β cells. Using a β cell-specific KO of Slc39a14 (β-Zip14 KO), we found that loss of ZIP14 in β cells resulted in greater insulin secretion. Studies in INS-1E cells have also linked siRNA silencing of Zip14 to greater insulin production [104]. β-Zip14 KO mice also experienced greater loss of pancreatic zinc after glucose stimulation compared with control mice. Islets isolated from β-Zip14 KO secreted more insulin than islets from control animals during GSIS assays, in alignment with in vivo findings. This was accompanied by a decrease in intracellular zinc content, again in alignment with findings in mouse pancreatic tissue. Experiments in MIN6 cells showed that overexpression of ZIP14 resulted in increased intracellular zinc and a slight decrease in insulin secretion, collectively suggesting a causal role for ZIP14 in β cell zinc dynamics and insulin secretion. More specifically, ZIP14 was found to be localized on the ER in MIN6 cells but reduced in expression upon stimulation of insulin production and secretion, indicating that the reduction of ZIP14 on the ER in β cells acts to retain zinc within the organelle for use in insulin production and secretion. Proteomic analysis after the knockdown of Zip14 in INS-1E cells revealed that the loss of Zip14 leads to the downregulation of proteins involved in insulin secretion pathways, ribosomal and mitochondrial proteins, metal-binding proteins, and proteins associated with oxidative phosphorylation pathways. In Zip14 siRNA-treated β cells, there was reduced expression of proliferation marker Ki67 and reduced active apoptosis and necrotic β cell death, as indicated by intracellular histone-associated DNA fragments [104]. This is consistent with previous findings that ZIP14 can offer protection against sustained apoptosis under pancreatic ER stress [40] and supports our findings of ZIP14 localization on the ER in β cells.

FIGURE 3.

ZIP14 contributes to the regulation of insulin secretion by modulating the zinc pool within the ER/Golgi system. The figure is modified from [15]. (A) Colocalization of immunofluorescent staining for ER membrane (KDEL) and ZIP14 was performed in MIN6 cells. Red: ZIP14; green: KDEL. Arrows highlight the colocalization of ZIP14 and KDEL. Scale bar = 10 μm. (B) Quantification of ER membrane positive for ZIP14 and the level of secreted insulin in MIN6 cells with and without a cotreatment of glucose and KCl. (C) Depiction of the proposed model for the role of ZIP14 in insulin secretion. ER, endoplasmic reticulum; Glu, glucose; KCl, potassium chloride; KDEL, lysine (K), aspartic acid (D), glutamic acid (E), and leucine (L); MIN6, mouse insulinoma cell line.

The loss of ZIP14 in β-Zip14 KO not only impacted β cells but also altered systemic metabolism. In a Comprehensive Laboratory Animal Monitoring System, the respiratory exchange ratio of β-Zip14 KO was significantly lower than control mice, suggesting that β-Zip14 KO mice had shifted fuel source preference toward fatty acid utilization. When fed a chronic HFD (16 weeks), β-Zip14 KO mice had a greater body weight, less lean mass, an ∼3-fold increase in islet size, and more severe hyperinsulinemia compared with control mice. These metabolic and morphological changes suggest that loss of ZIP14 in β cells resulted in a predisposition to symptoms of diet-induced obesity.

In addition to zinc, ZIP14 has also been shown to contribute to nontransferrin-bound iron (NTBI) uptake into β cells, which may impair islet function in cases of iron overload. siRNA knockdown of Zip14 in both βLox5 cells as well as in primary human islets reduced NTBI uptake by ∼50%. Overexpression of ZIP14 resulted in increased NTBI uptake into βLox5 cells [105]. This work suggests that ZIP14 contributes to, but is not solely responsible for, NTBI uptake into β cells.

ZIP family ZNT and metabolic responses by peripheral tissues

SLC39A5/ZIP5

Rare LOF variants in SLC39A5 have been associated with elevated serum zinc levels and a reduced risk of type 2 diabetes in humans [106]. Studies in mice with Zip5 KO confirmed elevated serum zinc levels, allowing further investigation of zinc redistribution across tissues. These studies revealed increased hepatic zinc levels in KO animals. To assess glycemic traits, Zip5 KO mice were subjected to congenital [double knockouts (DKO) with Zip5 KO or Lepr KO (leptin receptor deficiency)] and diet-induced obesity through a high-fat, high-fructose diet (HFFD). DKO and Zip5 KO mice on HFFD exhibited significantly lower fasting blood glucose levels without changes in fasting insulin levels compared with littermate controls.

Interestingly, no differences in insulin production, insulin clearance, or blood glucose levels were observed between individuals with LOF variants and controls, suggesting that the protective metabolic effects of ZIP5 mutation occur in peripheral tissues, specifically in the liver. These findings in humans with LOF mutation [106] do not align with those in whole-body and β cell-specific Zip5 KO mice discussed earlier [93], reinforcing the careful consideration of species-specific differences. These considerations, however, should not eliminate the possibility that different experimental settings and goals in these studies might have been underlying reasons for the differences observed.

SLC39A7/ZIP7

The zinc transporter ZIP7 plays a critical role in cellular signaling pathways and zinc homeostasis. Located in the ER membrane, ZIP7 regulates the release of zinc from organelles like the ER and Golgi apparatus into the cytosol [[107], [108], [109]], a process modulated by casein kinase II (CK2)-mediated phosphorylation [110]. Genetic studies have revealed that the ablation of ZIP7 leads to reduced cytosolic zinc levels, impaired ER function, and abnormal cell proliferation [108], as seen in human osteosarcoma cell lines [37]. Dysregulated ZIP7 expression has also been linked to tamoxifen-resistant Michigan Cancer Foundation-7 (MCF-7) breast cancer cell proliferation [110].

Recent research has extended the role of ZIP7 into glucose metabolism and glycemic control in skeletal muscle cells. ZIP7 ablation in muscle cells resulted in a substantial reduction of key genes and proteins involved in glucose homeostasis, including Akt phosphorylation, insulin receptor signaling molecules (Irs1 and Irs2), glucose transporter Glut4, and glycogen branching enzyme (Gbe) [108]. Additionally, ZIP7 expression was significantly downregulated in the skeletal muscle of HFD mice compared with normal feed pellets-fed controls [38]. This reduction corresponded with lower Glut4 protein levels, impairing glucose uptake and contributing to diminished glucose tolerance and insulin responsiveness, suggesting a link between ZIP-7-mediated zinc transport and insulin response in muscle.

SLC39A8/ZIP8

GWAS have identified the SNP variant A391T (rs13107325) in ZIP8 as being associated with metabolic health traits, including reduced arterial blood pressure, increased BMI, and hyperlipidemia [[111], [112], [113]]. Using ZIP8 knock-in (ZIP8-KI) mice carrying the corresponding polymorphism (ZIP8 A393T), researchers observed reduced arterial blood pressure, consistent with GWAS findings [100]. To further investigate the metabolic phenotype, insulin sensitivity and glucose tolerance were evaluated under normal and high-sucrose dietary conditions. When challenged with dietary sucrose supplementation, ZIP8-KI mice exhibited significant insulin resistance but were protected from hyperglycemia. This protection was attributed to elevated circulating insulin levels and increased urinary glucose excretion. The mechanism underlying the elevated circulating insulin remains unclear. It is yet to be determined whether this results from enhanced pancreatic β-cell function and insulin secretion or impaired insulin clearance. Notably, lower hepatic ZnT8 expression was observed in ZIP8-KI mice, which is intriguing given that improved hepatic insulin clearance has been reported in ZnT8 KO mice. This raises the possibility that reduced ZnT8 expression may contribute to altered insulin clearance in ZIP8-KI mice.

SLC39A13/ZIP13

Humans with Ehlers-Danlos syndrome who carry an LOF mutation in SLC39A13 have been reported to have a significantly decreased white fat mass [44]. Recent studies investigating ZIP13 KO mice revealed significant metabolic improvements, including resistance to diet-induced obesity and enhanced glucose tolerance and insulin sensitivity [114]. Notably, these mice exhibited increased energy expenditure and oxygen consumption without changes in food intake or physical activity. The metabolic benefits were attributed to enhanced beige adipocyte biogenesis, particularly in inguinal white adipose tissue, and increased thermogenic capacity, suggesting ZIP13 as a negative regulator of adipocyte browning and energy expenditure.

SLC39A14/ZIP14

ZIP14 has been shown to play a crucial role in regulating metabolic pathways, particularly in the liver and adipose tissue. Our previous studies demonstrated that during insulin-mediated glucose uptake, ZIP14 translocates from the plasma membrane to early and late endosomes in hepatocytes, along with the insulin receptor [13]. In Zip14 KO mice, hepatic endosomes were zinc depleted, resulting in impaired activity of zinc-dependent enzymes, including insulin-degrading enzyme and cathepsin D, essential for insulin receptor inactivation and recycling. This impairment led to prolonged insulin receptor activation and increased hepatic glucose uptake. Additionally, Zip14 KO mice exhibited increased liver glycogen content due to disrupted negative feedback regulation of glycogen synthesis mediated by ZIP14-dependent zinc transport. The KO mice also displayed impaired gluconeogenesis and glycolysis, attributed to reduced cytosolic zinc concentrations. Notably, zinc supplementation effectively rescued these metabolic defects, partially or entirely, highlighting the critical role of zinc and zinc transport pathways in maintaining hepatic metabolic homeostasis and their potential implications in metabolic disorders.

As mentioned earlier, Zip14 KO mice displayed a phenotype including low-grade chronic inflammation due to impaired intestinal integrity, mild hyperinsulinemia, and a greater adipose/body fat ratio [[101], [102], [103]]. Notably, these are also common clinical findings in patients with metabolic disorders, particularly type 2 diabetes and obesity. Adipose tissue is important for insulin and glucose metabolism because it is highly responsive to both insulin and inflammation. Accordingly, we have shown in the white adipose tissue of Zip14 KO mice enhanced inflammatory pathway activation, including MyD88, NF-κB, and STAT3, and consequent increase in proinflammatory cytokines, such as IL-6, TNF-α, and IL-1β, and reduced insulin receptor phosphorylation, indicative of insulin resistance [39]. Importantly, bioavailable cytosolic zinc was reduced in these animals. Collectively, these findings highlighted the complex interplay between zinc metabolism, inflammation, insulin response, and the function of the adipose tissue.

Perspective

Zinc plays essential biological roles that can be classified into 3 functional categories: catalytic, structural, and regulatory [34]. Emerging research indicates that these roles are interconnected, often coexisting within complex multistep cellular processes facilitated by targeted zinc transport via specialized ZNT [13,14,38,40,44,93,106,108,114,115]. Metabolic diseases are multifactorial conditions influenced by both genetic predisposition and environmental factors such as diet and inflammation. Because ZNT are responsive not only to zinc status but also to cytokines, hormones, and signaling molecules, further mechanistic studies are crucial [13,14,[35], [36], [37], [38], [39], [40], [41], [42]]. Investigations into changes in zinc transporter expression, subcellular localization, metallomic analysis of affected cellular sites, and their physiological outcomes could significantly enhance our understanding of the interplay between zinc and ZNT in both healthy and pathological states. Such insights hold promise for advancing the use of zinc and its transporters in the prevention and treatment of metabolic disorders.

Advancements in single-cell RNA sequencing have opened new avenues for analyzing cell-type-specific expression patterns of ZNT in human samples from both healthy individuals and patients with diabetes [[116], [117], [118], [119]]. This technology holds immense promise for uncovering previously unrecognized links between zinc transporter dysfunction and insulin-related metabolic traits. Emerging evidence also suggests more genetic variants in ZIP family transporters may be associated with insulin metabolism and diabetes susceptibility [106, 112], highlighting the importance of targeted investigations into specific transporters relevant to glucose homeostasis and β cell function. To address these gaps, future research should prioritize the identification of ZIP transporters linked to human metabolic health and insulin regulation. Mechanistic studies utilizing genetically modified preclinical animal models will be particularly crucial, as they allow for in-depth investigations into the roles of ZIP transporters in β-cell function and insulin signaling while facilitating metallomics analyses to establish accurate reference values for zinc distribution across tissues, cells, and organelles. Additionally, the emerging technique of spatial transcriptomics and imaging [[120], [121], [122]]—which integrates advanced imaging technologies with omics analyses—offers an innovative approach to spatially profile zinc transporter expression. These approaches could be used to detect changes in pancreatic cell type abundance, cellular pathways, gene regulatory networks, and cell–cell signaling, enabling the identification of ZNT that modulate pivotal pathways and gene networks implicated in metabolic dysfunction. Such multidisciplinary efforts could provide invaluable insights into the causal roles of ZNT in diabetes pathophysiology and the development of effective therapeutic interventions.

Traditional perspectives have long attributed compensatory hyperinsulinemia to insulin resistance and obesity; however, emerging evidence suggests that insulin hypersecretion and hyperinsulinemia may precede and actively contribute to the development of insulin resistance and obesity [[87], [88], [89], 123]. Although much of the focus in recent research has been on hyperglycemia-induced alterations in ZIP family transporters, the direct relationship between insulin and ZIP transporter regulation remains unclear. Considering the central role of ZNT in cellular zinc homeostasis and their involvement in glucose metabolism, exploring how insulin itself regulates ZIP family transporters is a critical area of unmet need. Future studies should prioritize investigating the regulation and dysregulation of ZIP transporters under hyperinsulinemic conditions to uncover potential mechanisms linking insulin signaling to zinc flux. Addressing this knowledge gap may help clarify the role of ZIP transporters in the early stages of insulin resistance and associated metabolic disorders, paving the way for targeted interventions aimed at zinc transporter modulation as a therapeutic strategy.

Author contributions

Both authors wrote and reviewed the manuscript and gave the final decision for publication, and also read and approved the final manuscript.

Funding

This project was supported by Cornell University Division of Nutritional Sciences funds to TBA.

Conflict of interest

The authors report no conflicts of interest.