Extracellular Elaidate, a Trans Fatty Acid, Tends to be Incorporated into Triglycerides and Incorporated Elaidate is Released by Lipolysis

Intake of elaidate, an industrially produced trans fatty acid, is associated with the development of cardiovascular disease. Recently, we revealed that persistent exposure to elaidate impairs the insulin responsiveness of adipocytes. Moreover, extracellular elaidate is incorporated into phospholipids and triglycerides and exists mainly as triglycerides in adipocytes. Because fatty acids in adipocytes are not only used as an energy source but also released as cytokines to regulate cellular function and whole-body metabolism, we hypothesized that elaidate is released from adipocytes. Here, we examined the intracellular behavior of elaidate to explain that incorporated elaidate exists mainly as triglycerides, and whether it is released from adipocytes. Extracellular elaidate was incorporated into triglycerides rather than phospholipids, and elaidate incorporated into triglycerides did not decrease during the study period. Under lipolytic stimulation, incorporated elaidate—together with other fatty acids—was released from adipocytes. These results imply that adipocytes act as a reservoir of elaidate.


INTRODUCTION
Trans fatty acids (TFAs) are unsaturated fatty acids with non-conjugated trans double bonds. TFAs are produced naturally as well as industrially, and daily dietary intake of industrially produced TFAs is associated with an elevated risk of cardiovascular disease. 1) On this basis, the World Health Organization published a roadmap to food becoming 'trans fat free by 2023. 2) Intake of industrially produced TFAs has been implicated in the development of insulin resistance and type 2 diabetes mellitus (T2DM), [3][4][5] and persistent exposure to a physiological concentration of elaidate, an industrially produced TFA, impairs the insulin responsiveness of cultured adipocytes. 6,7) Moreover, extracellular elaidate is incorporated into phospholipids and triglycerides and exists mainly as triglycerides in adipocytes. 8) Intracellular fatty acids in adipocytes are not only used as an energy source but also released as cytokines to regulate cellular function and whole-body metabolism. 9,10) Therefore, we hypothesized that elaidate incorporated into triglycerides is released from adipocytes and affects the surrounding environment. In this study, we examined the behavior of elaidate in adipocytes to explain that incorporated elaidate exists mainly as triglycerides, and whether it is released from adipocytes following stimulation.
Analysis of Fatty Acid Composition of Triglycerides, Free Fatty Acids, and Phospholipids The amount of fatty acids was analyzed as described by Ishibashi et al., 2016 and 2018. 6 ,8 ) In brief, total lipids were extracted using the Bligh & Dyer method, and triglycerides, free fatty acids, and phospholipids were separated by thin-layer chromatography (Silica Gel 60, Sigma-Aldrich/Merck KGaA) developed using petroleum ether: diethyl ether: acetic acid (82:18:1, v/v/v) and then hydrolyzed and methylated. The fatty acid content of each lipid was quantitated using a GC-MS-QP2010 Ultra (Shimadzu, Kyoto, Japan) in the selected ion-monitoring mode ( m/z 55 for palmitoleate, oleate, and elaidate; m/z 74 for palmitate; m/z 77 for stearate-d35; m/z 298 for stearate; m/z 301 for stearate-d3). The effi ciencies of extraction, isolation, and methylation were normalized to those of an internal standard.

Extracellular Elaidate Tended to be Incorporated into Triglycerides and Remained in Adipocytes
To examine the behavior of elaidate in adipocytes, we focused on the directivity of incorporation of extracellular elaidate into lipid classes and the residual amounts of incorporated elaidate. First, to examine the directivity of incorporation of elaidate, it was added into the culture medium and the amounts of elaidate in the lipid classes were measured. As shown in Fig. 1A , because there was little free elaidate, elaidate was immediately incorporated into triglycerides or phospholipids. Elaidate was incorporated more frequently into triglycerides than into phospholipids. Next, we examined the directivity of stearate incorporation using stearate-d35. In contrast to elaidate, stearate-d35 tended to be incorporated into phospholipids, implying that the directivity of elaidate to triglycerides is not a common property of fatty acids.
Next, to examine the residual amounts of incorporated elaidate, elaidate-incorporated adipocytes were cultured in the absence of elaidate for 6 d ( Fig. 1B ). The fraction of elaidate in phospholipids gradually decreased, but that in triglyceride did not decrease. Moreover, similar findings were obtained using stearate-d3, implying that residuality of elaidate is not specifi c to elaidate. Therefore, extracellular elaidate tends to be incorporated into triglycerides and not naturally released,

Fig. 1. Extracellular Elaidate Tended to be Incorporated into Triglycerides and Remained in Adipocytes
(A) 3T3-L1 adipocytes were exposed to 10 µM elaidate or stearate-d35 for 1, 3, or 6 h. Values are means ± SD (n = 3). Asterisk indicates signifi cant diff erences (* p < 0.05) by Student's t -test. (B) 3T3-L1 adipocytes were persistently exposed to 10 µM elaidate or stearate-d3 before and during diff erentiation for 18 d. The cells were washed with PBS and administered fresh medium lacking elaidate or stearate-d3, which was refreshed every 2 d. The amount of elaidate or stearate-d3 in triglycerides and phospholipids was measured at the indicated times. Values are means ± SD (n = 3). Asterisks indicate signifi cant diff erences (* p < 0.05; versus 0 h) by Student's t -test. resulting in intracellular elaidate existing mainly as triglycerides.

Incorporated Elaidate is Released by Lipolytic Stimulation
Lipolytic stimulation enhances the release of fatty acids from adipocytes. Therefore, we examined whether lipolytic stimulation enhances the release of elaidate from elaidate-incorporated cells. Stimulation of vehicle-exposed cells with isoproterenol increased the amounts of palmitate, palmitoleate, and oleate in the supernatant (Fig. 2). When elaidate-exposed cells were stimulated with isoproterenol, the amount of elaidate in the supernatant increased. These results imply that elaidateincorporated adipocytes supply elaidate under lipolytic conditions, such as starvation.

DISCUSSION
Although dietary intake of elaidate has been evaluated and incorporation of elaidate into cells has been revealed, the amount of elaidate incorporated into cells remains unclear. Here, we report that adipocytes may act as a reservoir of elaidate ingestion. Extracellular elaidate tended to be incorporated into triglycerides in adipocytes, and incorporated elaidate was released by lipolytic stimulation. Moreover, the amount of elaidate in triglycerides did not decrease naturally.
Adipocytes isolated from obese individuals release 2.5-4 µmol free fatty acids per hour from 10 7 cells under basal conditions. 12) Because humans have approximately 4 to 8×10 10 adipocytes, 13) adipose tissue can release 10-20 mmol fatty acids per hour. Given that elaidate accounts for 1.4% of the released fatty acids (Fig. 2), 0.14-0.28 mmol elaidate is released by human adipose tissue, potentially reaching millimolar concentrations in the blood and possibly higher near adipocytes. Macrophages are recruited to adipose tissue under inflammation, 14) and bone-marrow-derived macrophages are activated by 1-100 µM elaidate for 24 h in vitro, 15) implying that elaidate released from adipocytes activates macrophages and enhances inflammation.
Elaidate in triglycerides lacks a specific removal system, resulting in accumulation of elaidate in adipocytes, which may be released during starvation. Therefore, in the development of cardiovascular disease and diabetes, the amount of elaidate accumulated in adipose tissue is as important as its ingestion.