While high fat diet models of insulin resistance are

While high-fat diet models of insulin resistance are acceptable approaches for the study of pre-diabetes in humans, they are not suitable for modeling progression towards established type2 diabetes. The pattern of activation of the lipoxygenase pathway in pre-clinical models of type2 diabetes has not been reported. In mice there are two LO isoforms, leukocyte 12/15 LO and platelet 12LO that primarily generate 12(S)-HETE, the major lipid mediator in the pathway that exerts pro-inflammatory actions [[1], [16]]. Both isoforms can exert pro-inflammatory and anti-inflammatory actions via their respective metabolites and were shown to have complex roles in regulation of inflammation and immune responses in the pathology of metabolic diseases [[1], [17], [18]]. C57BLKS/J-db/db mice express the gene encoding the truncated and inactive leptin receptor [19]. This animal model is characterized by hyperphagia, obesity, hyperglycemia, and elevated plasma insulin and in several respects resembles human type 2 diabetes with peripheral insulin resistance [[20], [21], [22]]. The model is characterized by progressive β cell destruction and onset of type 2 diabetes from ∼10–12 weeks of age [[20], [22]]. The db/db mice display anti-β cell immunity [[23], [24]]. Also, inflammation is present systemically as well as in pancreatic islets of db/db mice [[25], [26]], along with increased numbers of islet-associated macrophages [25]. This model may be ideally suited to test the effect of 12LO inhibition in adipose tissue and islet inflammation in prevention studies, targeting early stages of type2 diabetes. Due to accelerated progression towards type 2 diabetes and islet loss, this model could be also valuable for interventional studies targeted to more advanced stages of the disease, possibly involving immune components. Selective and highly potent inhibitors of human 12 and 15-LO were recently reported [[27], [28], [29], [30]]. Both inhibitors have high selectivity, do not have antioxidant properties and irreversibly inhibit enzymatic activity [[30], [31]]. The 12/15 lipoxygenase inhibitors are also potent against the rodent lipoxygenase isoforms and were used successfully in vivo in a model of stroke in mice29. Understanding changes in 12LO KC7F2 and activity in the early stages of obesity and diabetes is important in order to establish the optimal intervention window to test such novel isoform-specific 12LO inhibitors. Therefore, the objective of this study was to characterize the 12- and 15- lipoxygenases pathways and to find a suitable interventional window for testing selective lipoxygenase inhibitors and their effects on insulin resistance and development of type 2 diabetes.
Materials and methods
Results
Discussion Pre-diabetes and type2 diabetes associated with obese state are characterized by inflammation in AT and pancreatic islets [[2], [36], [37]]. Cross-talk between AT and islets resulting in propagation of inflammation has been substantiated by several recent studies [[2], [37]]. Therefore, targeting both tissues to reduce production of inflammatory mediators and to improve functional responses may be an appropriate goal for future therapies aiming to reduce the onset and progression of insulin resistance and diabetes. Lipoxygenases are enzymes expressed in key tissues responsible for metabolic homeostasis, including pancreatic islets [38] and AT [[34], [39]] in humans. Several proof of concept studies showed that either global or tissue-specific germline deletion of 12/15-LO in rodents prevents development of insulin resistance and improves islet function in rodent models of diet-induced obesity and insulin resistance [[4], [6], [35], [40]] suggesting that 12-lipoxygenases are significant contributors to inflammation in obesity and insulin resistance. Also, in vitro studies using human islets showed that ALOX 12 (the human counterpart isoform of mouse platelet 12-LO) is involved in islet inflammation via the prime metabolite 12(S)-HETE and that selective ALOX 12 inhibitors alleviate islet inflammation in vitro [[11], [41], [42]]. We also showed that in human visceral fat of obese subjects with type2 diabetes there is an increased production of 12(S)-HETE that is largely originating from SVF cells, likely macrophages and vascular endothelial cells [[34], [39]]. Collectively these data suggest that 12-lipoxygenases are attractive therapeutic targets to limit inflammation in obesity and diabetes. However, despite the wealth of proof of concept data, in vivo pre-clinical studies using selective lipoxygenase inhibitors in obesity and type 2 diabetes are missing. One barrier to such studies was the limitation of adequate parenteral formulation due to lack of aqueous solubility for the inhibitors that now seems to be overcome in a recently identified new class of selective inhibitors [43]. Another translational barrier is the species-specific mechanism of action and pattern of expression of the mammalian lipoxygenases [1]. While platelet 12-LO (ALOX12) is the only enzyme that produces 12(S)-HETEs in humans, mice have two enzymes that contribute to 12(S)-HETE production: platelet 12-LO and leukocyte 12/15-LO, which is the counterpart isoform to human ALOX15, but unlike the latter produces predominantly 12(S)-HETEs. Compounds that inhibit both the human and mouse isoforms of 12LO are now available for preclinical studies. However, there are few rodent models that mimic human type 2 diabetes. Finally, there are limited rodent models that mimic full onset type2 diabetes in human disease. One such model is the C57BLKS/J-db/db mouse, that shows obesity and accelerated decline in islet function, hyperinsulinemia and severe hyperglycemia with fully onset of type2 diabetes by 12 weeks of age [[20], [21], [22]]. In this study we used the db/db mouse model to characterize longitudinal changes in expression and activity of the two LO isoforms to determine the suitability of this model for testing selective LO inhibitors prior to fully onset of type 2 diabetes.