Adipocyte lipid storage and adipokine production are modulated by lipoxygenase-derived oxylipins generated from 18-carbon fatty acids

Highlights

• Oxylipins generated from short-chain PUFAs block lipid accumulation by adipocytes.

• The oxylipins operate by modulating the levels of key proteins involved in lipid metabolism.

• The effects of oxylipins on protein levels are not due to changes in gene expression.

Abstract

Generation of oxylipins (oxygenated metabolites of fatty acids) by lipoxygenases may be responsible for the beneficial effects of 20- and 22-carbon n-3 fatty acids on adipose tissue dysfunction in obesity, but the potential actions of oxylipins derived from 18-carbon fatty acids, which are generally at higher levels in the diet, are unknown. We therefore compared the effects of select lipoxygenase-derived oxylipins produced from α-linolenic acid (ALA, C18:3 n-3), linoleic acid (LA, C18:2 n-6), and arachidonic acid (AA, C20:4 n-6) on key adipocyte functions that are altered in obesity. Individual oxylipins were added to the culture medium of differentiating 3T3-L1 preadipocytes for 6 days. Lipid accumulation was subsequently determined by Oil Red O staining, while Western blotting was used to measure levels of proteins associated with lipid metabolism and characteristics of adipocyte functionality. Addition of all oxylipins at 30 nM was sufficient to significantly decrease triglyceride accumulation in lipid droplets, and higher levels completely blocked lipid production. Our results establish that lipoxygenase-derived oxylipins produced from 18-carbon PUFA differentially affect multiple adipocyte processes associated with lipid storage and adipokine production. However, these effects are not due to the oxylipins blocking adipocyte maturation and thus globally suppressing all adipocyte characteristics. Furthermore, these oxylipin species decrease the lipid content of adipocytes regardless from which precursor fatty acid or lipoxygenase they were derived. Consequently, adipocyte characteristics can be altered through the ability of oxylipins to selectively modulate levels of proteins involved in both lipid metabolism and adipokine production.

Introduction

Dietary fat is an important source of energy, however, excess intake is associated with obesity and its associated morbidities, cardiovascular disease (CVD) and type 2 diabetes mellitus (T2DM). On the other hand, fat provides essential fatty acids (FA) that are required for brain development, blood clotting and other critical physiological and cellular processes. Since fat is composed of a variety of different constituent FAs, the composition of dietary fat has become an issue of considerable debate (Schwab and Uusitupa, 2015). Based on a variety of epidemiological and observational studies, there is a consensus for consuming diets low in saturated fatty acids (SFA) and higher in polyunsaturated fatty acids (PUFA). The recommendation to replace SFAs with PUFAs is largely based on data that suggests consumption of PUFAs can reduce the risk of both CVD and T2DM (Mozaffarian et al., 2010, Schwab et al., 2014).

The biological actions of dietary FAs can be mediated a number of different ways, through changes in membrane dynamics and receptor function, as well as activation of various receptors and enzymes that modulate intracellular signaling and gene expression (Mollace et al., 2013). Additionally, the metabolism of FAs, particularly the PUFAs, by several different enzymes can result in the formation of products that act as receptor ligands and exhibit properties that are quite distinct from the precursor FAs. The two main families of lipid mediators that originate from PUFAs are endocannabinoids and oxylipins (Poudyal et al., 2011, Wainwright and Michel, 2013). Oxylipins are oxygenated FA metabolites derived through the actions of cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450, and those that originate from marine long-chain omega-3 FAs (eicosapentaenoic acid, EPA; docosahexaenoic acid, DHA) are of significant interest because of their ability to modulate inflammation. However, information regarding the formation and subsequent actions of oxylipins that are derived from the omega-6 linoleic acid (LA) and omega-3 alpha-linolenic acid (ALA) are almost completely unexplored, especially in the context of obesity.

Under conditions of caloric excess, the adipose tissue depots expand through an increase in both adipocyte number and size in order to enhance storage of fat in the form of triglycerides. Although not all adipocytes become enlarged, hypertrophy of these cells is associated with major changes in gene expression, metabolic properties and the profile of secreted cytokines and adipokines. These altered properties are the hallmarks of adipocyte dysfunction, a condition that is linked to the development of a more pro-inflammatory environment due to a decrease in the production of vasodilatory and anti-inflammatory mediators. While oxylipins are also produced by adipose tissue, and diet can modulate both the amount and species of oxylipin that are synthesized (Balvers et al., 2012), the role of these lipid mediators in this tissue has not been explored in detail.

It has been reported that resolvins and protectins, oxylipins generated from EPA and DHA, inhibit macrophage recruitment and increase adiponectin secretion in adipose explants from obese mice (Borgeson and Godson, 2012). Such observations have generated considerable interest in these novel oxylipins in the context of obesity since alterations in the dietary intake of certain PUFAs could influence adipose tissue functionality in the obese state thus leading to a decrease in comorbidities such as insulin resistance and cardiovascular disease. On the other hand, oxylipins generated from other PUFAs may produce similar or opposite results. However, there is very little data on the effects of oxylipins derived from C18 PUFAs such as LA on specific functions of adipose tissue (Goto et al., 2015), and none with respect to the oxylipins originating from ALA, despite the fact that these 18-carbon-derived oxylipins are present at higher levels in tissues than the more well-characterized oxylipins derived from longer-chain fatty acids such as arachidonic acid (AA) and EPA (Gabbs et al., 2015). Therefore, the effects of select LA and ALA oxylipins on lipid metabolism, specifically lipid storage in lipid droplets, and functional characteristics such as adipokine secretion were examined for the first time with differentiated 3T3-L1 cells, an established cell line derived from mouse that exhibits the metabolic characteristics of mature adipocytes.