Our results are consistent with the central importance of selective uptake and alkylating activity in the mechanism of streptozotocin diabetogenicity. Alkylation of DNA leads to necrosis of pancreatic beta cells and thus to a state of insulin-dependent diabetes mellitus, well-known as streptozotocin diabetes in experimental diabetes research.

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...

This Type I diabetic rat develops a spontaneous insulin-dependent autoimmune diabetes through beta cell apoptosis. It could prove to be a valuable new animal model for clarifying the mechanisms involved in the development of autoimmune diabetes.

Regulation of glucose-induced insulin secretion is crucially dependent on glucokinase function in pancreatic beta-cells. Glucokinase mRNA expression was metabolically regulated allowing continuous translation into enzyme protein. Glucokinase enzyme activity in the beta-cell was exclusively regulated by glucose. Using a selective permeabilization technique, different intracellular activity states of the glucokinase enzyme in bioengineered glucokinase-overexpressing RINm5F tissue culture cells were observed. These results could be confirmed in analogous experiments with dispersed islet cells. A diffusible glucokinase fraction with high enzyme activity could be distinguished from an intracellularly bound fraction with low activity. Glucose induced a significant long-term increase of the active glucokinase fraction. This effect was accomplished through the release of glucokinase enzyme protein from an intracellular binding site of protein character. The inhibitory function of this protein factor was abolished through proteolytic digestion or heat inactivation. Northern blot analyses revealed that this binding protein was not identical to the well-known liver glucokinase regulatory protein. This hitherto unknown new protein factor may have the function of a glucokinase regulatory protein in the pancreatic beta-cell, which may regulate glucokinase enzyme activity in a glucose-dependent manner.