OBJECTIVEType 2 diabetes is a complex disease that is accompanied by elevated levels of nonesterified fatty acids (NEFAs), which contribute to β-cell dysfunction and β-cell loss, referred to as lipotoxicity. Experimental evidence suggests that oxidative stress is involved in lipotoxicity. In this study, we analyzed the molecular mechanisms of reactive oxygen species-mediated lipotoxicity in insulin-producing RINm5F cells and INS-1E cells as well as in primary rat islet cells.RESEARCH DESIGN AND METHODSThe toxicity of saturated NEFAs with different chain lengths upon insulin-producing cells was determined by MTT and propidium iodide (PI) viability assays. Catalase or superoxide dismutase overexpressing cells were used to analyze the nature and the cellular compartment of reactive oxygen species formation. With the new H2O2-sensitive fluorescent protein HyPer H2O2 formation induced by exposure to palmitic acid was determined.RESULTSOnly long-chain (>C14) saturated NEFAs were toxic to insulin-producing cells. Overexpression of catalase in the peroxisomes and in the cytosol, but not in the mitochondria, significantly reduced H2O2 formation and protected the cells against palmitic acid-induced toxicity. With the HyPer protein, H2O2 generation was directly detectable in the peroxisomes of RINm5F and INS-1E insulin-producing cells as well as in primary rat islet cells.CONCLUSIONSThe results demonstrate that H2O2 formation in the peroxisomes rather than in the mitochondria are responsible for NEFA-induced toxicity. Therefore, we propose a new concept of fatty acid-induced β-cell lipotoxicity mediated via reactive oxygen species formation through peroxisomal β- oxidation.

Chronically elevated concentrations of non-esterified fatty acids (NEFAs) in type 2 diabetes may be involved in β-cell dysfunction and apoptosis. It has been shown that long-chain saturated NEFAs exhibit a strong cytotoxic effect upon insulin-producing cells, while short-chain as well as unsaturated NEFAs are well tolerated. Moreover, long-chain unsaturated NEFAs counteract the toxicity of palmitic acid. Reactive oxygen species (ROS) formation and gene expression analyses together with viability assays in different β-cell lines showed that the G-protein-coupled receptors 40 and 120 do not mediate lipotoxicity. This is independent from the role, which these receptors, specifically GPR40, play in the potentiation of glucose-induced insulin secretion by saturated and unsaturated long-chain NEFAs. Long-chain NEFAs are not only metabolized in the mitochondria but also in peroxisomes. In contrast to mitochondrial β-oxidation, the acyl-coenzyme A (CoA) oxidases in the peroxisomes form hydrogen peroxide and not reducing equivalents. As β-cells almost completely lack catalase, they are exceptionally vulnerable to hydrogen peroxide generated in peroxisomes. ROS generation in the respiratory chain is less important because overexpression of catalase and superoxide dismutase in the mitochondria do not provide protection. Thus, peroxisomally generated hydrogen peroxide is the likely ROS that causes pancreatic β-cell dysfunction and ultimately β-cell death. Keywords: β-oxidation, G-protein-coupled receptors, insulin secretion, mitochondria, non-esterified fatty acids, peroxisomes, reactive oxygen species, type 2 diabetes mellitus Date submitted 26 March 2010; date of final acceptance 29 April 2010 IntroductionType 2 diabetes mellitus is a complex metabolic disorder with a dramatically increasing prevalence worldwide [1]. This disorder is characterized by peripheral insulin resistance and pancreatic β-cell dysfunction [2,3], resulting in defective glucose-induced insulin secretion [4][5][6] and ultimately in β-cell loss through apoptosis [7,8]. Hypercaloric Western diets, rich in carbohydrates and saturated fats, are responsible for the manifestation of the metabolic syndrome. This is characterized by dyslipidaemia, hypertension, and obesity, which precede type 2 diabetes manifestation. Accompanying elevated levels of non-esterified fatty acids (NEFAs) [9] can suppress insulin secretion and cause β-cell dysfunction and loss, a phenomenon referred to as lipotoxicity [10,11]. Although lipotoxicity is subject to intensive research and scientific discussion, a conclusive molecular mechanism has not been elucidated. Structural Requirements for LipotoxicityThe effects of NEFAs upon insulin-producing cells are dependent on chain length and degree of saturation [12]. Saturated long-chain NEFAs, such as the physiologically most abundant saturated NEFA palmitic acid, are highly toxic, Correspondence to: Prof. Sigurd Lenzen, Institute of Clinical Biochemistry, Hannover Medical School, 30623 Hannover, Germany. E-mail: lenzen.sigurd@mh-han...

Background/Aims: Elevated levels of non-esterified fatty acids (NEFAs) are under suspicion to mediate β-cell dysfunction and β-cell loss in type 2 diabetes, a phenomenon known as lipotoxicity. Whereas saturated fatty acids show a strong cytotoxic effect upon insulin-producing cells, unsaturated fatty acids are not toxic and can even prevent toxicity. Experimental evidence suggests that oxidative stress mediates lipotoxicity and there is evidence that the subcellular site of ROS formation is the peroxisome. However, the interaction between unsaturated and saturated NEFAs in this process is unclear. Methods: Toxicity of rat insulin-producing cells after NEFA incubation was measured by MTT and caspase assays. NEFA induced H₂O₂ formation was quantified by organelle specific expression of the H₂O₂ specific fluorescence sensor protein HyPer. Results: The saturated NEFA palmitic acid had a significant toxic effect on the viability of rat insulin-producing cells. Unsaturated NEFAs with carbon chain lengths >14 showed, irrespective of the number of double bonds, a pronounced protection against palmitic acid induced toxicity. Palmitic acid induced H₂O₂ formation in the peroxisomes of insulin-producing cells. Oleic acid incubation led to lipid droplet formation, but in contrast to palmitic acid induced neither an ER stress response nor peroxisomal H₂O₂ generation. Furthermore, oleic acid prevented palmitic acid induced H₂O₂ production in the peroxisomes. Conclusion: Thus unsaturated NEFAs prevent deleterious hydrogen peroxide generation during peroxisomal β-oxidation of long-chain saturated NEFAs in rat insulin-producing cells.

Hydrogen peroxide is an important mediator in cell signalling and cell death. Apart from the mitochondrion the peroxisome is the most important cellular site for the generation and scavenging of hydrogen peroxide. Peroxisomes contain various oxidases, e.g. for the metabolism of long-chain fatty acids, polyamines, and for the oxidation of urate, which form hydrogen peroxide. Widely-used chemical probes for the detection of hydrogen peroxide like dichlorofluorescein diacetate (DCFDA) often lack in specificity and the possibility of compartment-specific measurement. To overcome these disadvantages, Belousov et al. developed the novel hydrogen peroxide sensitive fluorescent protein HyPer. In the present study the HyPer protein was fused with the PTS1 tag for a specific hydrogen peroxide detection in peroxisomes. The localization of the HyPer protein in the peroxisomes was confirmed by immunofluorescence and the functionality by fluorescence microscopy and flow cytometry analyses. The presented HyPer-Peroxi fluorescent protein is a valuable tool for studying hydrogen peroxide generation within the peroxisomes.

BACKGROUND AND AIMS Renal fibrosis is a central feature of chronic kidney disease (CKD), and the severity of CKD correlates with the magnitude of renal fibrosis. Despite the causative role attributed to fibrosis in CKD progression, there is still no treatment available that directly targets renal fibrosis. Upon kidney injury, embryonic signalling pathways are activated, promoting the repair and regeneration of injured tissue. Maladaptive repair mechanisms have been associated with the development of renal fibrosis and sustained activation of Wnt/β-catenin signalling contributes to CKD progression. GPR124 (ADGRA2 gene), has been identified as a potential fibrotic mediator through its upregulation in pericytes during pericyte to myofibroblast transition. GPR124 has been identified to function as a co-receptor for Wnt7 mediating canonical Wnt signaling in endothelial cells. We observed elevated GPR124 expression in mouse kidneys after chronic ischaemia reperfusion injury (cIRI). To further explore the role of GPR124 in CKD progression, we have studied its correlation with disease severity in human NURTuRE kidney biopsies [1]. We have also looked at GPR124 therapeutic potential in mouse cIRI. Given its potential disease relevance, we set out to develop inhibitory GPR124 antibodies as novel therapeutics to treat renal fibrosis. METHOD Diagnostic FFPE kidney cortex biopsy samples from 497 NURTuRE patients were subjected to bulk RNA-Seq. Library preparation with Illumina Enrichment Tagmentation technology was followed by sequencing and alignment of paired-end reads to the reference genome GRCh38/ENSEMBL 97 and quantification. Monoclonal Anti-GPR124 antibodies were generated against the extracellular domain of human GPR124. Top candidates were selected by FACS binding and further characterized in a TCF/LEF luciferase assay for the inhibition of Wnt7 signaling. One hu/ms cross-reactive candidate was studied in a 14-day IRI mouse model to assess its anti-fibrotic effect in vivo. Mice were exposed to 30-min unilateral renal ischaemia, followed by 14 days recovery. Kidneys were studied for gene expression by RT-PCR. Animals were divided into sham (n = 5), control IRI (no antibody, n = 12) and treated groups (each n = 12). The antibody was administered before ischemia and during the recovery phase in two doses [50 mg/kg and 150 mg/kg]. RESULTS CONCLUSION NURTuRE provides us with a unique opportunity to assess relevance of a potential target candidate gene in kidney disease patients. The potential of GPR124 as a therapeutic target was highlighted by the upregulation of expression levels along disease severity and biological pathway analysis, where our data supports a potential role in extracellular matrix reorganization. The administration of GPR124 antibodies and inhibition of the GPR124-mediated Wnt signaling in vivo inhibited the development of ischaemia-induced fibrosis in the mouse kidney. The results strongly support a role for GPR124 in human kidney disease progression, through inhibition of fibrosis, and its potential as a future clinical target.