Curcumin effect on copper transport in HepG2 cells

Funding Information: Acknowledgments: The authors appreciate the support and advice of Uldis Berkis. The authors gratefully acknowledge the financial support from EU 7th Framework program project “Unlocking infectious disease research potential at R¯ıga Stradin,š University” (BALTINFECT) (grant agreement No. 316275). Publisher Copyright: © 2018 by the authors. Licensee MDPI, Basel, Switzerland. Copyright: Copyright 2018 Elsevier B.V., All rights reserved.Background and Objective: In Wilson’s disease, copper metabolism is impaired due to defective copper transporting protein ATP7B, resulting in copper accumulation in liver and brain and causing damage to liver and brain tissues. Published data suggest that one of the possible treatments for Wilson’s disease is curcumin—a compound found in the root of Curcuma longa. In this study, we tested whether curcumin affects copper transport and excretion in HepG2 hepatocytes carrying wildtype ATP7B. Materials and Methods: We examined the impact of 5 µM and 25 µM curcumin on the transport of copper in HepG2 cells incubated with 20 µM and 100 µM CuCl2, as well as copper excretion from cells. First, immunofluorescent staining and co-localization analysis were carried out in HepG2 cells using confocal laser scanning microscope and Nikon NIS Elements software. Second, a concentration of copper extracted into cell culture medium was determined using atomic absorption spectrometry. Results: The analysis of the co-localization between Golgi complex and ATP7B revealed that both 5 µM and 25 µM doses of curcumin improve the ability of liver cells to transport copper to plasma membrane at 20 µM CuCl2, but not at 100 µM CuCl2 concentration. However, atomic absorption spectrometry showed that curcumin rather promotes copper absorption into liver cell line HepG2 than excretion of it. Conclusions: Curcumin accelerates the transport of copper within liver cells, but does not promote copper excretion from HepG2 cells.publishersversionPeer reviewe

Prokaryotic expression, purification and immunogenicity in rabbits of the small antigen of hepatitis delta virus

Funding Information: Expression and purification of HDV antigen was supported by Russian Foundation for Basic Research (grant 16-04-01490a). Evaluation of serum by Western blot and confocal microscopy was supported by Russian Science Foundation (grant 14-14-01021). Experiments in rabbits were supported by the Swedish Institute grants 09272_2013 and 19806_2016. Cross-border collaboration of the partners, exchange of the materials and standard operation procedures used in the study, and dissemination of the data were supported by the EU Twinning project VACTRAIN, contract nr 692293. Publisher Copyright: © 2016 by the authors; licensee MDPI, Basel, Switzerland.Hepatitis delta virus (HDV) is a viroid-like blood-borne human pathogen that accompanies hepatitis B virus infection in 5% patients. HDV has been studied for four decades; however, the knowledge on its life-cycle and pathogenesis is still sparse. The studies are hampered by the absence of the commercially-available HDV-specific antibodies. Here, we describe a set of reproducible methods for the expression in E. coli of His-tagged small antigen of HDV (S-HDAg), its purification, and production of polyclonal anti-S-HDAg antibodies in rabbits. S-HDAg was cloned into a commercial vector guiding expression of the recombinant proteins with the C-terminal His-tag. We optimized S-HDAg protein purification procedure circumventing a low affinity of the His-tagged S-HDAg to the Ni-nitrilotriacetyl agarose (Ni-NTA-agarose) resin. Optimization allowed us to obtain S-HDAg with >90% purity. S-HDAg was used to immunize Shinchilla grey rabbits which received 80 µg of S-HDAg in two subcutaneous primes in the complete, followed by four 40 µg boosts in incomplete Freunds adjuvant. Rabbits were bled two weeks post each boost. Antibody titers determined by indirect ELISA exceeded 107. Anti-S-HDAg antibodies detected the antigen on Western blots in the amounts of up-to 100 pg. They were also successfully used to characterize the expression of S-HDAg in the eukaryotic cells by immunofluorescent staining/confocal microscopy.publishersversionPeer reviewe

Variants in the FFAR1 Gene Are Associated with Beta Cell Function

The FFAR1 receptor is expressed mainly in pancreatic beta cells and is activated by medium to long chain free fatty acids (FFAs), as well as by thiazolidinediones, resulting in elevated Ca(2+) concentrations and promotion of insulin secretion. These properties suggest that FFAR1 could be a mediator of lipotoxicity and a potential candidate gene for Type 2 diabetes (T2D). We therefore investigated whether variations at the FFAR1 locus are associated with T2D and beta cell function.We re-sequenced the FFAR1 region in 96 subjects (48 healthy and 48 T2D individuals) and found 13 single nucleotide polymorphisms (SNPs) 8 of which were not previously described. Two SNPs located in the upstream region of the FFAR1 gene (rs1978013 and rs1978014) were chosen and genotyped in 1929 patients with T2D and 1405 healthy control subjects. We observed an association of rs1978013 and rs1978014 with insulinogenic index in males (p = 0.024) and females (p = 0.032), respectively. After Bonferroni corrections, no association with T2D was found in the case-control material, however a haplotype consisting of the T-G alleles conferred protection against T2D (p = 0.0010).Variation in the FFAR1 gene may contribute to impaired beta cell function in T2D

Beta Cell Function: from Human Genetics to Animal Models

Beta cell function is an important factor in the development of both Type 1 (T1D) and Type 2 (T2D) diabetes mellitus. T1D is characterized by a primary defect in insulin secretion due to the immune-mediated beta cell destruction, however, the more common T2D beside insulin resistance also include impaired beta cell function as a consequence to abnormal glucose homeostasis. Genetic susceptibility is involved in both types of diabetes. We have studied several genetic and immunological factors affecting beta cell function. First, we tested whether single nucleotide polymorphisms (SNPs) of the human Free Fatty Acid Receptor 1 (FFAR1) are associated with T2D and insulin secretion. Another genetic study focused on FOXP3 association with T1D and the disease-related clinical parameters. The role of microRNAs (miRNAs) on beta cell function was studied in the third project using a novel genetically engineered mouse model. Subsequently, the effect of Alpha 1-Antitrypsin (AAT) on cytokine-induced apoptosis and on insulin secretion was studied in beta cells in vitro. In Study I, we concluded that SNPs rs1978013 and rs1978014 in the upstream region of FFAR1 gene might contribute to impaired beta cell function in T2D. Study II showed that the minor A allele in the FOXP3 rs2232365 SNP might represent a protective factor in T1D pathogenesis and suggest a possible role of FOXP3 in the regulation of autoimmunity against pancreatic beta cells. We have demonstrated for the first time in Study III that targeted disruption of the Dicer1 gene specifically in beta cells leads to progressive impairment of insulin secretion and diabetes development. Our findings of Study IV show that AAT stimulates insulin secretion and protects beta cells against cytokine-induced apoptosis, and these effects of AAT seems to be mediated through the cAMP pathway

α 1-antitrypsin enhances insulin secretion and prevents cytokine-mediated apoptosis in pancreatic β-cells.

α1-antitrypsin (AAT) is a serine protease inhibitor, which recently has been shown to prevent type 1 diabetes (T1D) development, to prolong islet allograft survival and to inhibit β-cell apoptosis in vivo. It has also been reported that T1D patients have significantly lower plasma concentrations of AAT suggesting the potential role of AAT in the pathogenesis of T1D. We have investigated whether plasma-purified AAT can affect β-cell function in vitro. INS-1E cells or primary rat pancreatic islets were used to study the effect of AAT on insulin secretion after glucose, glucagon-like peptide-1 (GLP-1) and forskolin stimulation and on cytokine-mediated apoptosis. The secreted insulin and total cyclic AMP (cAMP) were determined using radioimmunoassay and apoptosis was evaluated by propidium iodide staining followed by FACS analysis. We found that AAT increases insulin secretion in a glucose-dependent manner, potentiates the effect of GLP-1 and forskolin and neutralizes the inhibitory effect of clonidine on insulin secretion. The effect of AAT on insulin secretion was accompanied by an increase in cAMP levels. In addition, AAT protected INS-1E cells from cytokine-induced apoptosis. Our findings show that AAT stimulates insulin secretion and protects β-cells against cytokine-induced apoptosis, and these effects of AAT seem to be mediated through the cAMP pathway. In view of these novel findings we suggest that AAT may represent a novel anti-inflammatory compound to protect β-cells under the immunological attack in T1D but also therapeutic strategy to potentiate insulin secretion in type 2 diabetes (T2D)