P-239

Human islet isolation alters zinc transporter gene expression

C.J. Drogemuller¹, D.M. Mohanasundaram¹, C. Mee¹, C. Murgia², P.D. Zalewski¹, W. Hawthorne³, T. Loudovaris⁴, G.R. Russ¹, P.T.H. Coates^1
1 South Australian and Northern Territory Islet Program, Royal Adelaide Hospital, CNARTS, Adelaide, Australia; ² INRAN, Rome, Italy; ³ Centre for Transplant and Renal Research, Westmead Millenium Institute, Sydney, Australia; ⁴ Tom Mandel Islet Program, Melbourne, Australia

Objectives: To examine the effect of islet isolation on zinc transporter gene expression. Zinc transporter genes control movement of zinc from the extracellular matrix to the cytoplasm and intracellular vesicles of islet cells and are known to play an important role in insulin synthesis, secretion and storage.

Methods: Human islets from ten donors were separated by the Ricordi method. RNA from native pancreas, purified acinar and purified islets was isolated using Qiagen® RNA kits, prior to Q-PCR analysis using gene specific Taqman® primers. Gene expression analysis was performed using the ??Ct method.

Results: Relative expression levels of 24 zinc transporter genes, 2 glucose transporters (GLUT1 and GLUT2), glucokinase(GcK), GLP1R and the islet hormone genes insulin(INS), somatostatin(SST) and glucagon(GCG) were determined. GcK, GCG, GLUT1, SST, ZIP5,8&14 and ZnT8 displayed highly variable expression profiles in isolated islets, with ZnT8 and ZIP5 the most variable within the two zinc transporter families. ZIP14 was the most abundantly expressed zinc transporter in islets and was highly variable across the ten isolations. Little or no gene expression of ZIP2,4,12 or ZnT3,4,10 was detected. The expression level of ZIP1 and GLUT1 was elevated in isolated islets and purified acinar tissue compared to native pancreas. Zip5 and ZnT2 were the most abundant zinc transporters in purified acinar tissue, 3 fold higher when compared to their expression level in islets.

Conclusions: Human islets expressed a range of zinc transporter genes with ZnT8 and ZIP14 the most abundant transporters in each family. Their level of gene expression was also highly variable across the different human islet preparations. The zinc transporter ZIP1 and glucose transporter GLUT1 had elevated gene expression in both isolated islets and purified acinar tissue. These changes in gene expression profiles following islet isolation could potentially perturb normal glucose and insulin regulation and lead to impaired islet function.

P-239

Human islet isolation alters zinc transporter gene expression

C.J. Drogemuller¹, D.M. Mohanasundaram¹, C. Mee¹, C. Murgia², P.D. Zalewski¹, W. Hawthorne³, T. Loudovaris⁴, G.R. Russ¹, P.T.H. Coates^1
1 South Australian and Northern Territory Islet Program, Royal Adelaide Hospital, CNARTS, Adelaide, Australia; ² INRAN, Rome, Italy; ³ Centre for Transplant and Renal Research, Westmead Millenium Institute, Sydney, Australia; ⁴ Tom Mandel Islet Program, Melbourne, Australia

Objectives: To examine the effect of islet isolation on zinc transporter gene expression. Zinc transporter genes control movement of zinc from the extracellular matrix to the cytoplasm and intracellular vesicles of islet cells and are known to play an important role in insulin synthesis, secretion and storage.

Methods: Human islets from ten donors were separated by the Ricordi method. RNA from native pancreas, purified acinar and purified islets was isolated using Qiagen® RNA kits, prior to Q-PCR analysis using gene specific Taqman® primers. Gene expression analysis was performed using the ??Ct method.

Results: Relative expression levels of 24 zinc transporter genes, 2 glucose transporters (GLUT1 and GLUT2), glucokinase(GcK), GLP1R and the islet hormone genes insulin(INS), somatostatin(SST) and glucagon(GCG) were determined. GcK, GCG, GLUT1, SST, ZIP5,8&14 and ZnT8 displayed highly variable expression profiles in isolated islets, with ZnT8 and ZIP5 the most variable within the two zinc transporter families. ZIP14 was the most abundantly expressed zinc transporter in islets and was highly variable across the ten isolations. Little or no gene expression of ZIP2,4,12 or ZnT3,4,10 was detected. The expression level of ZIP1 and GLUT1 was elevated in isolated islets and purified acinar tissue compared to native pancreas. Zip5 and ZnT2 were the most abundant zinc transporters in purified acinar tissue, 3 fold higher when compared to their expression level in islets.

Conclusions: Human islets expressed a range of zinc transporter genes with ZnT8 and ZIP14 the most abundant transporters in each family. Their level of gene expression was also highly variable across the different human islet preparations. The zinc transporter ZIP1 and glucose transporter GLUT1 had elevated gene expression in both isolated islets and purified acinar tissue. These changes in gene expression profiles following islet isolation could potentially perturb normal glucose and insulin regulation and lead to impaired islet function.