12-Lipoxygenase Products Reduce Insulin Secretion and β-Cell Viability in Human Islets

Abstract

Context: Inflammation is increasingly recognized as an important contributing factor in diabetes mellitus. Lipoxygenases (LOs) produce active lipids that promote inflammatory damage by catalyzing the oxidation of linoleic and arachidonic acid, and LO is expressed in rodent and human islets. Little is known about the differential effect of the various hydroxyeicosatetraenoic acids (HETEs) that result from LO activity in human islets. Design: We compared the effect of stable compounds derived from LOs: 12(S)-HETE, 15HETE, 12HPETE, and 12RHETE. Interventions: At both 1 and 100 nm, insulin secretion was consistently reduced by 12(S)-HETE and 12HPETE. 12(S)-HETE also reduced viability activity by 32% at 1 nm. Insulin and reduced viability were partially reversed by treatment with lisofylline, a small-molecule antiinflammatory compound that protects mitochondrial function. 12(S)-HETE also increased cell death rate of human islets by 50% at 100 nm. To investigate mechanisms of 12-LO-mediated islet inhibition, we examined the p38-MAPK and JNK stress-activated pathways. Results: Treatment of 12(S)-HETE significantly increased phosphorylated p38-MAPK (pp38) protein activity in human islets. We further explored the in vivo role of 12-LO in regulating p38-MAPK using mouse models. Knockdown of 12-LO by injecting 12-LO siRNA into C57BL/6 mice decreased pp38 protein levels in mouse islets. The addition of proinflammatory cytokines increased pp38 levels in normal mouse islets but not in siRNA-treated islets. Conclusions: These data suggest that 12(S)-HETE reduces insulin secretion and increases cell death in human islets. The 12-LO pathway is present in human islets, and expression is up-regulated by inflammatory cytokines. Reduction of 12-LO activity could thus provide a new therapeutic approach to protect human -cells from inflammatory injury. Genotype and Tissue-Specific Effects on Alternative Splicing of the Transcription Factor 7-Like 2 Gene in Humans Ashis K. Mondal, Swapan K. Das, Giulia Baldini, Winston S. Chu, Neeraj K. Sharma, Oksana G. Hackney, Jianhua Zhao, Struan F. A. Grant, and Steven C. Elbein (J Clin Endocrinol Metab, 10.1210/jc.2009-2064) ABSTRACT Context: Noncoding single-nucleotide polymorphisms (SNPs) within the TCF7L2 gene are confirmed risk factors for type 2 diabetes, but the mechanism by which they increase risk is unknown. Objective: We hypothesized that associated SNPs alter TCF7L2 splicing and that splice forms have altered biological roles. Design: Splice forms and 5 and 3 untranslated regions were characterized in sc adipose, muscle, liver, HepG2 cells, pancreas, and islet. Isoform-specific transcript levels were quantified in sc adipose. Alternative splice forms were characterized in HepG2 liver cells under glucose and insulin conditions and in SGBS cells with differentiation. Major isoforms were characterized by transfection. Setting: The study was conducted at an ambulatory general clinical research center. Patients: Patients included 78 healthy, nondiabetic study subjects characterized for insulin sensitivity and secretion. Results: We identified 32 alternatively spliced transcripts and multiple-length 3 untranslated region transcripts in adipose, muscle, islet, and pancreas. Alternative exons 3a, 12, 13, and 13a were observed in all tissues, whereas exon 13b was islet specific. Transcripts retaining exons 13 and 13a but not total TCF7L2 transcripts were significantly correlated with both obesity measures (P 0.01) and rs7903146 genotype (P 0.026) in sc adipose. Insulin (5–10 nM) suppressed all TCF7L2 isoforms in SGBS cells but T R A N S L A T I O N A L H I G H L I G H T S F R O M J C E MContext: Noncoding single-nucleotide polymorphisms (SNPs) within the TCF7L2 gene are confirmed risk factors for type 2 diabetes, but the mechanism by which they increase risk is unknown. Objective: We hypothesized that associated SNPs alter TCF7L2 splicing and that splice forms have altered biological roles. Design: Splice forms and 5 and 3 untranslated regions were characterized in sc adipose, muscle, liver, HepG2 cells, pancreas, and islet. Isoform-specific transcript levels were quantified in sc adipose. Alternative splice forms were characterized in HepG2 liver cells under glucose and insulin conditions and in SGBS cells with differentiation. Major isoforms were characterized by transfection. Setting: The study was conducted at an ambulatory general clinical research center. Patients: Patients included 78 healthy, nondiabetic study subjects characterized for insulin sensitivity and secretion. Results: We identified 32 alternatively spliced transcripts and multiple-length 3 untranslated region transcripts in adipose, muscle, islet, and pancreas. Alternative exons 3a, 12, 13, and 13a were observed in all tissues, whereas exon 13b was islet specific. Transcripts retaining exons 13 and 13a but not total TCF7L2 transcripts were significantly correlated with both obesity measures (P 0.01) and rs7903146 genotype (P 0.026) in sc adipose. Insulin (5–10 nM) suppressed all TCF7L2 isoforms in SGBS cells but T R A N S L A T I O N A L H I G H L I G H T S F R O M J C E M