ABSTRACT
Frataxin (FXN) is a mitochondrial protein involved in iron metabolism and in the modulation of reactive oxygen and/or nitrogen species production. No information is currently available as for the role of frataxin in isolated human pancreatic islets. We studied islets from pancreases of multi-organ donors with (T2DM) and without (Ctrl) Type 2 diabetes mellitus. In these islets, we determined FXN gene and protein expression by qualitative and quantitative Real-Time RT-PCR, nitrotyrosine concentration, and insulin release in response to glucose stimulation (SI). FXN gene and protein were expressed in human islets, though the level of expression was much lower in T2DM islets. The latter also had lower insulin release and higher concentration of nitrotyrosine. A positive correlation was apparent between SI and FXN gene expression, while a negative correlation was found between nitrotyrosine islet concentration and FXN expression. Transfection of Ctrl islets with siRNA FXN caused reduction of FXN expression, increase of nitrotyrosine concentration, and reduction of insulin release. In conclusion, in human pancreatic islets FXN contributes to regulation of oxidative stress and insulin release in response to glucose. In islets from T2DM patients FXN expression is reduced while oxidative stress is increased and insulin release in response to glucose impaired.
Subject(s)
Diabetes Mellitus, Type 2/metabolism , Iron-Binding Proteins/metabolism , Islets of Langerhans/metabolism , Islets of Langerhans/pathology , Tissue Donors , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/pathology , Female , Gene Expression Regulation , Humans , Iron-Binding Proteins/genetics , Male , Middle Aged , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Regression Analysis , Reverse Transcriptase Polymerase Chain Reaction , Tyrosine/analogs & derivatives , FrataxinABSTRACT
BACKGROUND AND AIMS: Islet transplantation is an attractive approach to treat type 1 diabetic patients. However, suboptimal islet engraftment still represents an unsolved problem. It has been shown that human islets release monocyte chemoattractant protein-1 (MCP-1), one of the most powerful macrophage chemokines, which may impair the fate of the transplant. The aim of this study was to evaluate the presence and role of MCP-1 in isolated human islets, including genotyping for a common polymorphism. METHODS: Pancreatic islets were isolated by enzymatic digestion and gradient purification from 41 nondiabetic multiorgan donors. We measured MCP-1 mRNA expression by quantitative real- time reverse-transcriptase polymerization chain reaction, analyzed the MCP-1 single nucleotide polymorphism, -2518 G/A (SNP, rs 1024611) and evaluated glucose-stimulated insulin release (IR; microU/islet/min). RESULTS: MCP-1 mRNA expression was found in all studied batches of islets. Overall, IR was significantly higher at 16.7 mmol/L than 3.3 mmol/L glucose. We observed a significant negative correlation between MCP-1 mRNA expression and stimulation index (SI). We found that MCP-1 mRNA expression was significantly higher in CC and CT compared with TT genotype groups. Finally, SI was significant lower in the CC with respect to the TT genotype group. CONCLUSIONS: These data show that MCP-1 gene expression regulated by the -2518 G/A polymorphism, is correlated with glucose-stimulated insulin release. The study of MCP-1 expression and genotype on isolated islets before transplantation may be useful to understand the inflammatory response after infusion of human islets into patients with type 1 diabetes mellitus.
Subject(s)
Chemokine CCL2/genetics , Islets of Langerhans/physiology , Polymorphism, Single Nucleotide , Adenine/analysis , Diabetes Mellitus, Type 1/surgery , Gene Expression Regulation , Glucose/pharmacology , Guanine/analysis , Humans , Inflammation/etiology , Inflammation/genetics , Insulin/metabolism , Insulin Secretion , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Islets of Langerhans Transplantation/adverse effects , Polymorphism, Genetic , RNA, Messenger/genetics , Regulatory Sequences, Nucleic Acid/genetics , Reverse Transcriptase Polymerase Chain Reaction , Tissue DonorsABSTRACT
BACKGROUND AND AIMS: Pancreatic islet transplantation has become one of the potential treatments for type 1 diabetes. We evaluated functional and viability parameters of isolated islets in relation to donors clinical characteristics and preparation variables. METHODS: Islets were isolated from 70 nondiabetic multiorgan donors of overall age of 62.5 +/- 15.9 years. There were 41 men and 29 women. Their mean body mass index (BMI) was 25.62 +/- 3.09 kg/m(2). We evaluated the islet number (IEQ/g pancreatic tissue) insulin release (IR; microU/islet/min) in response to 3.3 (g) or 16.7 (G) mmol/L glucose; calcium flux concentration (CFC); and islet cell viability. RESULTS: IEQ was 5249 +/- 1505, with 73.7 +/- 14.96% viable islet cells. IR was 0.03 +/- 0.01 at g and 0.11 +/- 0.06 at G (stimulation index [S] = 3.24 +/- 1.96). CFC was 1.95 +/- 1.03 DeltaRFU. We observed positive correlations between viable cells and IR at g (R(2) = 0.260; P = .013), IR at G (R(2) = 0.165; P = .013), and CFC (R(2) = 0.175; P = .047). A positive correlation was documented between BMI and g (R(2) = 0.245; P = .016) and negative correlations between age with SI (R(2) = 0.188; P = .052) and cold ischemia time with IEQ (R(2) = 0.865; P = .0061). CONCLUSIONS: These results showed that quality control of isolated human pancreatic islets allowed assessment of beta-cell function and survival before transplantation, revealing several important variables.
