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  • In pancreatic islets we found an

    2024-05-06

    In pancreatic islets we found an increase in protein expression of leukocyte 12/15-LO that paralleled the metabolic decline characterized by severe hyperglycemia and reduced islet numbers. Similar increase in 12/15-LO expression by Western blot was found in the control mice, which have a normal metabolic phenotype, suggesting that lipoxygenase expression alone may not be sufficient to support pathological changes noted in db/db mice. However, immunostaining showed progressive increases in 12/15 LO protein in islets of db/db mice. Db/db mice are known to display exacerbation of inflammation and immune activation in various tissues [44]. Given such pro-inflammatory phenotype, activation of 12/15LO pathway could be a significant contributor to exacerbation of inflammation and metabolic decline. Therefore, inhibition of lipoxygenase pathway activation in db/db mice may have beneficial effects for lowering local inflammation and the delay of islet functional demise. In fact, a recent paper showed that baicalein, a pan-lipoxygenase inhibitor [45], improves liver inflammation in db/db diabetic mice [46], providing proof of concept for beneficial metabolic effects of lipoxygenase pathway inhibition in this mouse model. Platelet 12-LO expression did not significantly change between weeks 5 and 10. Interestingly, 12(S)-HETE production in the islets was 10-fold higher than 15(S)-HETE. The large increase in 12(S)-HETE in the absence of a significant change in 12LO protein expression may be explained by the complex interplay between substrate availability and how metabolites themselves can influence enzyme activity. Furthermore, it is known that the substrate affinity of 12/15-LO for ω3 fatty acids is allosterically regulated by the lipid products generated in the reaction [[47], [48]]. For example human 15-LO-1 has a preference to metabolize linoleic over arachidonic ci-1033 and this is reversed in the presence of excess 12-HETE in the media [47]. These types of studies were not performed using mouse enzyme isoforms but is conceivable that murine 12-LOs may be subject to equally complex regulation. The types and relative amounts of lipid metabolites generated in a tissue that expresses different 12/15LO isoforms are therefore dependent not only on 12/15LO expression but also on substrate availability, phospholipase A2 activity, local metabolite concentration or oxygen partial pressure. One limitation of our lipidomics analysis is the absence of the final products formed via ω3 pathway (protectins, resolvins, maresins). However, our study reports the levels of 14(S)-HDoHA and 17(S)-HDoHA that are accepted as surrogate markers for production of downstream metabolites via the ω3 pathway [49]. Nevertheless, this study highlights the importance of measuring substrate availability and lipid products generated in a tissue, in parallel with molecular measurements of 12/15LO mRNA or protein expression, as a preferred read-out to explain biological effects. Indeed, our data on protein expression and metabolite abundance did not always show proportional changes in db/db and control mice (Figs. 2A and 4A). This can be explained by potential differences in glutathione peroxidase or phospholipase A2 activity between the db/db and heterozygote mice. Glutathione peroxidases, responsible for conversion of H(p)ETEs to HETEs, are decreased in obesity and insulin resistance [[50], [51]] and such changes may explain differences in metabolite production when similar protein expression is found in db/db and control mice. We predict that inhibition of either of the lipoxygenase isoforms may reduce formation of 12(S)-HETE, the most abundant eicosanoid measured in the islets of db/db mice that is known to contribute to islet functional decline via different mechanisms including oxidative and ER stress and production of pro-inflammatory cytokines [[40], [41], [52]]. Activation of the lipoxygenase pathway in AT in human obesity and diabetes was recently reported [34]. While a wide range of eicosanoid metabolites derived from both ω3 and ω6 substrates were found [53], a predominance of pro-inflammatory metabolites was reported in human subjects with obesity and type 2 diabetes [34]. In this study we report for the first time the eicosanoid production in AT and the adipose SVF and adipocytes in a mouse model of type 2 diabetes. Interestingly, we found a wide range of eicosanoids, however they were detectable only in the db/db pre-diabetic mice and not in heterozygous controls, with the exception of small amounts of 12(S)-HETE. This may be explained by the much larger amounts of PUFAs that were found in db/db mice likely due to hyperphagia and excessive accumulation of dietary fatty acids in adipose tissue of db/db mice. Of the two lipoxygenase isoforms, 12/15-LO was expressed between weeks 5–10 in SVF with a tendency for a steady decline. However, platelet 12-LO was highly expressed at week 10 in db/db mice, indicating that this isoform is the one highly expressed in SVF and accountable for the majority of 12(S)-HETE production, in parallel with metabolic decline. In this case the pattern of enzyme isoform expression was recapitulated by the eicosanoid produced at 8 and 10 weeks of diet. Interestingly both the pro- and anti-inflammatory mediators were produced in balanced amounts at both time points. This pattern may be explained by the predominance of the DHA and EPA ω3 fatty acids over the ω6 AA in the SVF of db/db mice. Overall, the two lipoxygenase isoforms expressed in adipocytes, both of which increase in db/db-/- mice with age, may be more meaningful contributors to adipose tissue inflammation. The selective increase in HETEs in db/db but not control mice over time suggests that they may have pathogenic roles contributing to metabolic decline, since AT HETEs were shown to be causative for onset and progression of insulin resistance in obesity [[35], [54], [55]]. Our data suggests that total AT 12(S)-HETE and 15(S)-HETE production may be an adequate surrogate measurement for lipoxygenase inhibition in AT.