Down-Regulation of Zinc Transporter 8 (SLC30A8) in Pancreatic Beta-Cells Promotes Cell Survival

Research Article

Austin J Endocrinol Diabetes. 2016; 3(1): 1037.

Down-Regulation of Zinc Transporter 8 (SLC30A8) in Pancreatic Beta-Cells Promotes Cell Survival

Liping Huang1,2* and Catherine P Kirschke¹

¹Obesity and Metabolism Research Unit, USDA/ARS/ Western Human Nutrition Research Center, USA

²Department of Nutrition, University of California Davis, USA

*Corresponding author: Liping Huang, USDA/ARS/ Western Human Nutrition Research Center, Davis, CA 95616, USA

Received: November 03, 2015; Accepted: February 23, 2016; Published: March 16, 2016


The pancreatic islet contains high levels of zinc in granular vesicles of β-cells where insulin is matured, crystallized, and stored before secretion. Zinc is an essential co-factor for insulin crystallization forming dense cores in secretory granules. In insulin-containing secretory granules, zinc is mainly brought in by ZNT8 (SLC30A8), a zinc transporter predominantly expressed in pancreatic β-cells in the body. A recent study in humans has revealed that haploinsufficiency of ZNT8 may reduce the risk of Type 2 Diabetes (T2D). The mechanism by which ZNT8 haploinsufficiency protects individuals from T2D is not understood. The aim of this study was to investigate expression levels of ZNT8 in human normal and T2D pancreases and in pancreases from mice with diet-induced insulin resistance using immunohistochemistry and to understand the molecular mechanism underlying the protection from T2D by allelic deficiency of ZNT8. Our results showed that ZNT8 expression was upregulated in islets of both diabetic and insulin resistant conditions. shRNA knockdown of Znt8 expression to ~50% of the normal level (a condition which mimicked the allelic deficiency in humans) in MIN6 β-cells stimulated activations of Akt, p70S6K and p38, key kinases involved in cell proliferation and survival. Consistent with these observations, we showed that Znt8KD MIN6 β-cells had increased cell proliferation and were resistant to inflammation-induced cell death. Our results suggest that ZNT8 upregulation due to β-cell compensation during insulin resistance could be harmful for β-cell survival whereas down regulation could be beneficial, pointing to a potential target for T2D prevention and/or treatment.

Keywords: Zinc transporter 8; SLC30A8; ZNT8; MIN6 β-cells; Glucose metabolism; β-cell survival


SLC30A8: Human Solute Carrier Family 30 Member 8; Slc30a8: Mouse Solute Carrier Family 30 Member 8; ZNT8: Human Zinc Transporter 8; ZnT8: Mouse Zinc Transporter 8; ZnT7: Mouse Zinc Transporter 7; ZIP, ZRT: IRT-Like Protein; MIN6: Mouse Insulinoma Cells; TNFα: Tumor Necrosis Factor α; Akt: V-Akt Murine Thymoma Viral Oncogene Homolog; p38 MAPK: Mitogen-Activated Protein Kinase; p70S6K: S6 Ribosomal Protein Kinase; KD: Knockdown; KO: Knockout


Zinc is an essential trace metal for life. Dietary zinc deficiency causes stunted growth, impaired immune function, dermatitis, and hypogonadism. Severe zinc deficiency caused by rare genetic diseases, such as acrodermatitis enteropathica in humans and lethal milk in mice, is life threatening without treatment [1-3]. Cellular zinc homeostasis is largely maintained by two families of zinc transporters, namely SLC30A (ZNT) and SLC39A (ZIP) in humans. The ZNT and ZIP zinc transporter families contain 10 and 14 members, respectively. ZNT and ZIP proteins coordinate each other to maintain cellular zinc levels in a narrow physiological range with ZNTs controlling efflux of excess cytoplasmic zinc into the extracellular space or into intracellular organelles and/or vesicular compartments when zinc is replete and with ZIP functioning in an opposite direction.

Type 2 Diabetes (T2D) affects about 26 million people in the U.S. according to the NIH AMP factsheet of T2D. Diabetes is a leading cause of kidney failure, lower limb amputations, and blindness. Failure to produce adequate insulin from pancreatic β-cells to maintain euglycemia is one of the hallmarks of T2D. The pancreas contains high levels of zinc in the endocrine component of the organ, mostly in the β-cell of the islet of Langerhans [4,5]. Insulin is secreted from β-cells in response to increased blood glucose levels, such as after a meal. It stimulates glucose uptake, glycogenesis, and lipogenesis in insulin-sensitive tissues, such as skeletal muscle, liver, and fat [6]. It is known that zinc co-resides with insulin in secretory granules of β-cells [7] where it is integrated into insulin to form insulin-zinc crystals [8]. The importance of zinc and its roles in insulin synthesis, processing, and secretion has been demonstrated by studying zinc transporters in insulin-secreting cell lines and in knockout animal models. For example, over-expressing Zinc Transporter 7 (ZnT7) in rat insulin-secreting cells stimulates insulin transcription leading to an increase in basal insulin secretion [9]. Consistent with the function of ZnT7 in basal insulin secretion, Znt7 Knockout (KO) mice have abnormalities in fasting insulin secretion [10]. The process of converting proinsulin to insulin requires zincdependent endopeptidases (PC1 and PC2) and carboxypeptidase E [11,12]. ZNT8 has been shown to be associated with the availability of zinc ions for these zinc-dependent enzymes [13]. Inadequate zinc supply to these enzymes along the secretory pathway due to mutations in ZNT8 may have detrimental effects on proinsulin maturation [14]. Additionally, in the secretory granules, processed insulin is crystallized in the presence of zinc ions and stored before secretion [15]. ZNT8 is implicated in this process by transporting zinc ions into the secretory granule [16]. Other zinc transporters, such as ZIP14 may also play roles in insulin and glucose metabolism in the body. Mice with a Zip14-null mutation display hypoglycemia and hyper insulinemia due to decreased gluconeogenesis in the liver and enlarged islets with increased cell mass in the pancreas [17].

A recent study has demonstrated that haploinsufficiency of ZNT8 in humans reduces the risk of T2D [18]. How the haploinsufficiency of ZNT8 protect individuals from T2D is currently not understood. The aim of this study was to understand the molecular mechanism underlying the protection of T2D development from allelic deficiency of ZNT8. Our results demonstrated that the protective effect of allelic ZNT8 deficiency from T2D development could be likely via an Akt-dependent survival mechanism due to increase in β–cell proliferation and/or decrease in β-cell death.

Material and Methods

Human normal and diabetic pancreatic tissue sections

Paraffin embedded human pancreatic tissue sections were purchased from BioChain Inc. (Newark, CA). According to the manufacturer, the normal pancreatic tissue sections were prepared from pancreases of male donors at 66- and 71-year-old. The diabetic diseased pancreatic sections were prepared from a 67-year-old diabetic male donor.

Animals and preparation of mouse pancreas sections

Normal pancreases were isolated from 16-week-old male C57BL/6 (B6) mice fed a standard laboratory chow diet (Laboratory Rodent Diet 5001, Lab Diet, St. Louis, MO). Pancreases with insulin resistance were isolated from 16-week-old male B6 mice fed a high fat diet (45% kcal, Research Diets, New Brunswick, NJ) from 5- to 16-week-old (insulin resistant phenotypes were reported previously) [10]. Mice were housed in a temperature-controlled room at 22-24°C with a 12-h light: dark cycle. All animal experiments were conducted in accordance with National Institutes of Health Guidelines for the Care and Use of Experimental Animals and were approved by the Institutional Animal Care and Use Committee of the University of California Davis. The harvested pancreases were rinsed, fixed (4% paraformaldehyde), embedded, and sectioned (5 μm) as previously described [19].


Mouse anti-human ZNT8 polyclonal antibody was purchased from R&D Systems (Minneapolis, MN). Guinea pig anti-insulin polyclonal antibody was purchased from Abcam (Cambridge, MA). A guinea pig anti-mouse ZnT8 polyclonal antibody was raised against a synthetic peptide (CASRDSQVVRREIAKALSKSFTM) and affinitypurified (Thermo Fisher Scientific, Waltham, MA). The peptide used to generate the mouse ZnT8 antibody came from the same region used for generating the human ZNT8 antibody (R&D Systems). The Actβ antibody was purchased from Sigma-Aldrich (St. Louis, MO). Antibodies against phosphor-AKT (Ser473), phosphor-70S6 kinase (Thr389) and phosphor-p38 MAPK (Thr180/Tyr182) were purchased from Cell Signaling Technology (Danvers, MA). Horseradish peroxide-conjugated secondary antibodies were also purchased from Cell Signaling Technology.

Immunohistochemical Analysis

Immunohistochemical analysis of human and mouse pancreases was performed as previously described [20]. Briefly, following the rehydration steps, tissue sections were incubated in 5% H2O2 to inhibit endogenous peroxidase activity. Non-specific binding was blocked by treating the tissue sections with avidin-biotin blocking solution followed by 3% goat serum treatment (Vector Labs, Burlingame, CA). Primary antibodies were diluted in 1x PBS containing 2% goat serum and incubated at 4°C overnight. Dilution factors for the primary antibodies used in this study were as follows: human ZNT8, 1:100 and mouse ZnT8, 1:2000. Secondary biotinylated goat anti-mouse or anti-guinea pig antibody (Vector Labs) was diluted at 1:200 in 1x PBS containing 2% goat serum. The immunoreactivity was developed in tissue sections using VECTASTAIN elite ABC and HRP substrate ImmPACT DAB kits (Vector Labs) which produced brown deposits within cells.

Cell culture

MIN6 β-cells were purchased from AddexBio (San Diego, CA). Cells were grown in DMEM containing 15% (v/v) Fetal Bovine Serum (FBS), 4.5 g/L glucose, 1.0 mM sodium pyruvate, 50 μM β- Mercaptoethanol (BME), 50 U/mL penicillin, and 50 μg/mL streptomycin (complete medium, Thermo Fisher Scientific). Cells were cultured in a humidified atmosphere at 37 °C with 5% CO2.

Znt8 knockdown in insulin-secreting MIN6 β-cells

MissionTM shRNA lentiviral particles were purchased from Sigma-Aldrich. Lentiviral particles for shRNAs targeting Znt8 (TRCN000979767) or scrambled non-target negative control (SHC002V) were transducted to MIN6 β-cells according to the manufacturer’s protocol. Briefly, 18 h before transduction, MIN6 β-cells were seeded in 6-well plates at ~50% confluence. The next day, hexadimethrine bromide (8 μg/mL, Sigma-Aldrich) was added to the culture medium. Viral particles were subsequently added into the medium at ~2.5 particles/cell. After overnight incubation, the medium containing lentiviral particles were removed. The cells were then cultured in the complete medium plus 450 μg/mL G418 (Znt8KD) or 1 μg/mL puromycin (control). After 5 days, individual cell colonies were selected by the limiting dilution method and expanded by culturing in the complete medium under selection [21]. Cell lines were tested for Znt8 mRNA expression. Two cell lines with a ~50% reduction in Znt8 mRNA expression relative to the controls (also 2 cell lines) were selected for subsequent experiments.

RNA isolation, cDNA synthesis and quantitative PCR

Znt8KD and control MIN6 β-cells were lysed in TRIzol® reagent (Thermo Fisher Scientific). Total RNA were isolated according to the manufacturer’s protocol. cDNA was synthesized using an iScriptTM reverse transcription kit (BioRad, Hercules, CA) following the manufacturer’s protocol. cDNA samples were diluted 10-fold with double-distilled water before being used in quantitative PCR (qPCR). Gene expression for Znt8 and Ins was performed using specific primers (Table 1) with Sso Advanced SYBR Green Supermix (BioRad) and normalized to the expression of Actβ. Fold differences were calculated using the ΔCt method as described previously [22].