br The lipoxygenase pathway and

The 12/15-lipoxygenase pathway and microvascular complications of diabetes
The 12/15-lipoxygenase pathway in adipose tissue Only recently has adipose tissue been recognized as a highly metabolically active endocrine organ imparting profound local and systemic inflammatory effects. Adipose tissue, either found as white or brown fat, is a complex organ comprised of fat cells (adipocytes) and the stromal vascular compartment containing a mixed population of pre-adipocytes, leukocytes, macrophages, fibroblasts, and endothelial cells. This organ is responsible for the secretion of inflammatory cytokines and numerous adipose-specific hormone-like proteins, called adipokines, that not only affect local adipocyte function, but also systemic bodily functions. There is emerging evidence that the LOX enzymes, expressed in both white and brown fat, are important for proper adipogenesis and ensuing adipocyte function in regulating whole-body energy homeostasis [63]. Study of adipogenesis is possible through the in vitro characterization of the 3T3-L1 fibroblastic pre-adipocyte cell line [64]. The addition of a differentiation cocktail media to these cells promotes full differentiation into a pure population of adipocytes characteristic of accumulated triacylglycerol content in lipid-droplets within eight days [65]. This adipogenesis is dependent on an exogenous supply of free fatty acids to facilitate activation of peroxisome proliferator-activated receptors (PPARs; nuclear receptor proteins that function as transcription factors). PPARγ is a strict requirement for early adipocyte differentiation, and several fatty 74 metabolites of lipoxygenases appear to be necessary for PPARγ activation [63], [66], [67], [68]. Treatment of 3T3-L1 pre-adipocytes with either NDGA (nordihydroguaiaretic acid; a non-specific lipoxygenase inhibitor) or baicalein (a 12-LOX inhibitor) prevents adipogenesis. This phenotype is rescued upon treatment with rosiglitazone (a selective agonist of PPARγ in adipoctyes), consistent with the rise in PPARγ agonists during early adipocyte differentiation (marked by extensive mitotic clonal expansion) and observations that certain lipoxygenase metabolites activate PPARs [69], [70], [71], [72], [73]. In addition, during this period of differentiation, arachidonic acid is necessary for proper glucose uptake and is dependent on LOX activity [74]. These results demonstrate that certain LOXs are responsible for generating the endogenous PPARγ ligands necessary for adipogenesis. This role appears to be specific to the epidermal-derived 12-LOX, as the platelet- and leukocyte-derived 12-LOXs are expressed at very low levels in the pre-adipocytes and early differentiated adipocytes and adipogenic defects were not reported in leukocyte-12-LOX or platelet-12-LOX deficient mice [35], [63], [75], [76], [77]. Additionally, Hallenborg and colleagues recently demonstrated that over-expression of epidermal-12-LOX and its hepoxilin lipid products in 3T3-L1 pre-adipocytes stimulate adipogenesis, whereby epidermal-12-LOX knockdown prevents this differentiation [78]. The hepoxilins also accumulate during early 3T3-L1 differentiation and appear to directly activate PPARγ to promote adipogenesis [78]. It is possible that leukocyte-12-LOX may participate in lipogenesis during late stage adipogenesis since epidermal- and platelet-12-LOX expression are absent and leukocyte-12-LOX expression is maximal by day 8 of 3T3-L1 differentiation, which immediately follows the rise in activity of several key enzymes of fatty acid synthesis [76], [79] (also, our unpublished observations). Leukocyte-12-LOX (12/15-LOX) appears to be a significant player in modulating adipocyte function in vivo in diet-induced mouse models of obesity. Comparison of 12/15-LOX knockout mice with C57BL6/J mice fed either a standard chow or high-fat “Western” type diet (a diet containing 0.2% cholesterol of which 42% calories are from fat, 15.3% calories are from protein, and 42.7% calories are from carbohydrate, primarily sucrose) revealed that 12/15-LOX is the primary enzyme generating the 12(S)-HETE products under obese conditions [35]. This increased 12/15-LOX activity coincides with increased inflammation both systemically and in epididymal adipose tissue [35], [36]. Although both C57BL6/J and 12/15-LOX knockout mice exhibited similar weight gain and increased adiopcyte size when fed the Western diet, fewer incidences of macrophage infiltration and activation were observed in the epididymal adipose fat pads from 12/15-LOX knockout mice when fed the Western diet. Additionally, MCP-1 staining was significantly decreased in adipose tissue from the 12/15-LOX knockout mice. Furthermore, mice were also protected from developing insulin resistance and maintained normal adiponectin (an adipokine that improves insulin sensitivity by increasing energy expenditure and fatty acid oxidation) levels during the high-fat diet [80]. Thus 12/15-LOX activation under diet-induced obese conditions plays a significant role in mediating inflammation via ensuing adipocyte dysfunction. Preliminary data also suggests that the Zucker rat genetic model of obesity and insulin resistance shows higher 12/15-LOX in adipose tissue compared to lean controls (Chakrabarti, Wen, Dobrian, Cole, Ma, Pei, Williams, Bevard, Vandenhoff, Keller, Gu, and Nadler, unpublished observations).