This study shows that MSC-derived adipocytes exhibited decreased activity of the arachidonic acid metabolic pathway that yields EETs, that is, epoxygenase levels. The inability of MSC-derived adipocytes to sustain normal levels of EETs may be the result of the increased levels of sEH coupled with decreased expression of P450 epoxygenases. We further report that expression of CYP2J2 was significantly decreased in adipocytes. Further, we demonstrate that MSCs stay in a pluripotent condition. We have previously shown that MSCs are pleiotrophic cells that can differentiate to other lineage such as osteoblasts as a result of crosstalk by specific signaling pathways, including HO-1/-2 expression [7
In this report, we show that epoxygenase product activities, EETs, are effective in suppression of adipogenesis. 8,9-EET is more effective in suppression of adipogenesis compared to 5,6- and 11,12-EET but equally effective as 14,15-EET. Further, inhibition of sEH potentiated the EET-mediated decrease of adipogenesis. Adipocyte stem cells in culture treated with AUDA, an inhibitor of sEH, caused an increase in effectiveness of both 11,12- and 14,15-EET in suppression of adipocyte stem cell differentiation. The antiadipogenic effect of an EET agonist, when taken together with the inhibition of sEH, highlights the therapeutic potential of EET in the management of cardiovascular disease [35
]. An association of sEH gene polymorphism with insulin resistance has been reported, implying that sEH plays an important role in the pathogenesis of insulin resistance [37
]. EET agonist decreased O2−
production and prevented the rapid degradation of EET and subsequent activation of pAKT [32
]. Thus, EETs appear capable of reprogramming adipocyte stem cells, resulting in expression of a new phenotype that contains adipocytes of reduced cell size, that are associated with an increase in adiponectin and a decrease in inflammatory cytokines. In this report, we also demonstrate in vitro that the EET agonist 12-(3-hexylureido)dodec-8(Z)-enoic acid is very effective in suppression of adipogenesis and that suppression occurs in a dose-dependent manner.
Several reports show that oxidative stress and O2−
reduce EET levels [33
]. EETs are rapidly degraded by O2−
to DHET [40
], and EETs are also inactivated by sEH to DHETre [33
]. Since EET agonists induce HO-1 and vice versa, HO-1 induction increases EET levels [22
]. HO-1-mediated induction of EETs may be related to the ability of HO-1 to decrease oxidative stress and O2−
. Previously, it was shown that overexpression of HO-1 attenuated and AngII mediated oxidative stress [41
] and vascular injury and dysfunction in hypertensive rats [42
]. The mechanism by which HO-1 attenuated ROS involves an increase in extracellular superoxide dismutase (EC-SOD) and the restoration of mitochondrial function [43
]. In addition, a lack of HO-2 creates a setting that promotes oxidative-stress-related disturbances, including increases in O2−
and decreases in EC-SOD [44
]. The fact that HO-1 and −2 serve an antioxidative function and preserve EET levels suggests that the activation of this system in adipose tissue in obesity, a condition of high oxidative stress, represents an adaptive mechanism that confers MSCs resistance against oxidative stress and inhibits adipogenesis.
The role of EETs in decreasing oxidative stress via an increase in HO-1-mediated adiponectin and pAMPK is in agreement with the report that EETs increase ERK 1/2 MAP kinase.
EETs have been shown to mediate MAP kinase activation and ERK 1/2 MAP kinase phosphorylation [32
]. To date, the effect of EET on adipocyte MAP kinase has not been investigated. In addition, the crosstalk between AMPK–AKT and activation of AMPK is essential for the cellular processes that are controlled by the energy state of the cell. AMPK is activated by a decrease in ATP and rise in cellular AMP [45
], which leads to the phosphorylation of eNOS and key enzymes that subsequently inhibit the synthesis of cholesterol and increase glucose uptake. Activation of AMPK in skeletal muscle leads to an enhancement of glucose transport mediated by the translocation of GLUT4 to the membrane and this appears to be additive to the stimulation in response to insulin [45
]. In addition, pharmacological activation of AMPK by AICAR in obese Zucker rats improves glucose tolerance and reduces systolic blood pressure [47
This study provides direct evidence that the EET-agonist-mediated inhibition of adipogenesis is accompanied by the decrease of FAS, PPARγ
, and C/EBPα in MSC-derived adipocyte stem cells. Additionally, these perturbations occur in sequence, commencing with increased levels of HO-1 expression and decreased lipid accumulation. The lipid-lowering effect of the EET agonist was completely blocked by pharmacological suppression of HO activity. In addition, PPARγ
and C/EBPα are known to increase adipogenesis [48
]. The ability of the EET agonist to stimulate pAKT and decrease FAS, PPARγ
, and C/EBPα supports this hypothesis and that EETs have a negative effect on adipogenesis. These effects can be duplicated by inducers of HO-1 such as CoPP and/or L-4F [7
], suggesting that HO-1 plays a key role in lipid metabolism. These novel observations underscore the importance of EETs in regulation of MSCs to adipocyte lineages.
Since pharmacological activation of AKT and AMPK in obese Zucker rats improves obesity and glucose tolerance and reduces systolic blood pressure [49
], our finding of the effect of the EET-HO-1 module on AKT activation in adipocytes may be crucial to increase glucose uptake, lipid homeostasis, and inhibition of PPARγ
FAS mRNA levels were shown to be increased dramatically during 3T3-L1 adipocyte differentiation [50
]. In our experiments, expression of FAS, PPARγ
, and C/EBPα increased during adipogenesis; however, FAS, PPARγ
, and C/EBPα expression decreased after EET agonist treatment. The action of EET agonist treatment as manifest by increased levels of HO-1 and pAKT is associated in an improvement in glucose uptake. Further, EET agonist effectively restored expression of adiponectin, which was accompanied with a significant increase in cellular glucose uptake. In agreement with our results, adiponectin-deficient cells showed marked downregulation of GLUT4, and adipose triglyceride lipase [51
]. As seen in , inhibition of pAKT by LY294002 increased adipogenesis. In agreement with this, LY294002 was shown to inhibit GLUT4 translocation [52
]. This suggests that EET agonist treatment may increases translocation of GLUT4.