| Background Diabetes mellitus is characterized by chronic elevation of blood glucose concentration （hyperglycemia） as a consequence of decreased blood levels or decreased action of insulin. Since it was shown in 1934 that zinc is a component of insulin crystals, a relationship between zinc and diabetes mellitus has been proposed. From then on, numerous studies trying to elucidate the role of zinc in diabetes mellitus have been conducted with the aim of identifying new causal mechanisms and new therapeutical options. In fact, the zinc level in pancreatic islets is amongst the highest in the body and reduction in its levels in the pancreas has been associated with diabetes.Zinc homeostasis is regulated by the ZnT and ZIP zinc transporters in cells. In general, the ZnTs coded by SLC30A gene transport zinc from the cytoplasm to extracellular spaces or to intracytoplasmic vacuoles, such as secretory granules, while the ZIPs coded by SLC39A gene are thought to increase cytoplasmic zinc. At least 15 members of ZIP and 10 members of ZnT transporters are described to date.ZnT8 （zinc transporter 8）, a new member of zinc transporter family, coded by the gene SLC30A8, is a 369 amino acids protein. It has been identified as a pancreaticβ-cell -specific secretory granule zinc transporter. Recent works have shown that ZnT8 also expressed in human adipose tissue, blood lymphocytes, the cubical epithelium that lines thyroid follicles, and the adrenal cortex. The strong linkage of single nucleotide polymorphisms in ZnT8 gene with type 2 diabetes mellitus （T2DM） has been reported in worldwide studies. The at-risk allele of ZnT8 is associated with reduced insulin secretion stimulated by intravenous glucose tolerance test （IVGTT）, impaired conversion of proinsulin to insulin, and fasting hyperglycemia.Besides of being a genetic marker for T2DM, ZnT8 was also found to be one of the autoantigens of type 1 diabetes mellitus （T1DM）. The association between the risk allele of ZnT8 and T1DM was also reported, though some studies show contrary results. Previous studies strongly indicated that ZnT8 might be a key player in mediating insulin secretion, and therefore might serve as a potential therapeutic drug target for diabetes treatment. In addition to pancreas, the adipose tissue is also a major site of ZnT-8 synthesis. Leptin, a hormone secreted by the adipocytes plays a role in mediating satiety and energy expenditure. The circulating leptin concentration closely correlates with the amount of body fat mass. In adipose tissue, leptin expression is increased in the presence of zinc whereas leptin expression and secretion decreases in response to zinc depletion in adipocytesGlucagon-like peptide-1-（7-36）-amide （GLP-1）, a hormone secreted by endocrine L cells of the intestinal tract, has unique insulinotropic and growth factor–like signal transduction properties, which make it a new therapeutic agent for treatment of T2DM. Recently, Magnusson N et al showed that ZnT-8 is a potential target for pharmacological manipulation of GLP-1. However GLP-1 affects ZnT-8 expression in vivo is still unclear. Native GLP-1, being rapidly degraded by dipeptidyl peptidase-IV, has a short half-life of a few minutes. Exendin-4, an analogue of GLP-1, is a stable GLP-1 analogue which can lower levels of blood glucose as a consequence of its ability to activate the GLP-1 receptor. However, the regulation of ZnT8 expression in pancreas and the adipose tissue in T2DM remains to be established. The role of ZnT8 in T2DM pathogenesis is still to be explored.ObjectiveWe aim to explore the role of ZnT8 in T2DM and the possible mechanism. We use molecular cloning technology to construct ZnT8-C prokaryotic expression vector. ZnT8-C proreins are expressed in Escherichia coli （DE3） and then purified. We get ZnT8 polyclonal antibody through immuning New Zealand rabbits with ZnT8-C proteins and purify the antibody. In order to explore the role of ZnT8 in T2DM, we select db/db mice as the animal model. We test the expression of ZnT8 mRNA and protein in db/db mice. Furthermore we test the expression of ZnT8 mRNA and protein in db/db mice administrated with Exendin-4. Methods1. The cDNA sequence ZnT8 in the GenBank （GenBank NO:NM173851.2） were used to design gene-specific primers contained BamHI and NotI sites for PCR amplification of the cDNA encoding the carboxyl terminal of ZnT8（ZnT8-C）. Then, the PCR products were cloned into the plasmid pET32a, and verified by DNA sequence analysis, respectively. The verified recombinant vector was further transformed into Escherichia coli （DE3）pLysS strain to express the recombinant proteins after IPTG induction. The expressed recombinant proteins were assayed by SDS-PAGE and were purified by Ni-NTA agarose affinity, then were examined by Western blotting.We immunized rabbit with purified fusion protein emulsified with an equal volume of Freund’s immunoadjuvant. Then, we collected the immune sera of rabbits. We separated the sera from the blood. The antibody was purified by ammonium sulfate fractionation. Next, we used the indirect sandwich enzyme immunoussay to detecte the valence of ZnT8 polyclonal antibody. Then we detected the purity and activity of the antibody with Western blotting and immunohistochemisty.2. We selected the db/db mice as the animal model of T2DM. We extracted the total RNA and protein from the pancreas and adipose tissue of db/db mice. We detected the ZnT8 mRNA expression in the pancreas and adipose tissue of db/db mice by real-time PCR technology and we detected the ZnT8 protein expression by Western blotting. The db/db mice administrated with Exendin-4 for 14 days. Then the total RNA and protein were extracted from the pancreas and adipose tissue of the mice. The ZnT8 mRNA expressions in the pancreas and adipose tissue were detected by real-time PCR technology and the ZnT8 protein expressions were detected by Western blotting.Results1. （1） The cDNA of the carboxyl terminal of ZnT8 （ZnT8, 268-369aa） was successfully amplified. The sequence matched completly with the sequence of ZnT8-C in GeneBank. （2） The pET32a-ZnT8-C was successfully constructed. The recombinant ZnT8-C （molecular weight 30kDa）, was successfully expressed in a soluble form in Escherichia coli BL21DE3plysS after optimization of expression conditions. After Ni-NTA agarose affinity purification and Western blotting verification, the interest proteins were obtained. （3）The titer of ZnT8 polyclonal antibody achived 1:3200. The ployconal antibody of ZnT8 had good immuning activity. （4） We found, in the pancreas of db/db mice, ZnT-8 protein level was lowered accompanied by the decrease of ZnT-8 mRNA. ZnT-8 mRNA level and protein level was significantly reduced in epididymal and visceral fat of db/db mice. Treatment with Exendin-4 upregulated ZnT-8 gene expression in the pancreas of db/db mice, but did not affect this gene expression in adipose tissue.Conclusions: （1） The ployconal antibody of ZnT8 that we prepared had good immuning activity. （2） ZnT-8 expression in the pancreas and adipose tissue is down-regulated in db/db mice. The reduced ZnT-8 production in pancreas may advance the defects in insulin secretion in diabetes, which could be rescued, at least partially, by the administration of Exendin-4. |
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