In the present study, we demonstrate that ApoM expression is regulated by DHT in HepG2 cells in a dose-dependent manner and that inhibition of ApoM expression by DHT is mediated by PKC. Moreover, DHT did not affect ApoAI expression and we demonstrated further that inhibition of PI3K did not influence DHT-mediated apoM expression.
Androgens administration lowers HDL-C levels in both genders, but androgens modulate cholesterol metabolism in poorly understood ways. Most studies indicate that administration of natural or synthetic androgens produces a plethora of effects, some of which appear negative, such as reduction of HDL-C levels, and others positive, such as increased lean mass and reduced visceral fat, lower total cholesterol, and improved sensitivity to insulin. To understand the role of endogenous and therapeutic androgens in CVD, it will be necessary to identify the mechanisms responsible for the reduction in HDL-C. One hypothesis considers reduced synthesis of ApoAI, ApoM, or HDL. The second hypothesis considers that there is decreased donation of cholesterol from peripheral tissues to HDL particles, and the third proposes that there is increased clearance of HDL-C. The fourth, a more complex hypothesis, is that androgens lead to HDL remodeling[15, 18], cholesterol redistribution, or changes in lipoprotein catabolism.
ApoAI and apoM are constituents of HDL. In some studies, ApoAI levels are reduced after androgen treatment, suggesting decreased synthesis or increased catabolism of HDL[15, 16]. ApoM was first identified as a component of human postprandial lipoproteins in 1999. It was estimated that ~5% of HDL particles contain ApoM. ApoM content in the healthy human plasma pool was 0.94 mM. This roughly corresponds to 1/50th of the mean molar concentration of apoA-I in plasma. However, the physiopathological functions of ApoM are not fully elucidated. Studies of the regulation of ApoM expression may reveal the clinical importance of ApoM.
The AR is highly expressed in adipocytes and regulates their function by a variety of mechanisms, including local transcriptional regulation of lipases and increased levels of adrenergic receptors as well as inhibition of adipogenesis. The AR is also expressed in liver, a primary site of lipoprotein regulation, in which it could conceivably alter the expression of genes controlling HDL metabolism. ApoM is mainly expressed in the hepatocytes. Here, we administered DHT to examine its effect on ApoM secretion and ApoM mRNA levels in HepG2 cells. The present study supports the view that DHT affects lipoprotein production by demonstrating that DHT significantly decreased ApoM mRNA levels in hepG2 cells and the secretion of ApoM secretion into the medium, whereas there was no effect on the ApoAI mRNA levels. These findings suggest that there are different mechanisms for regulating ApoAI and ApoM expression in this particular cell line. To analyze the effect of androgens on ApoM expression and secretion in vivo, we administrated DHT to the ovariectomized C57BL/6 J mice. Plasma levels of ApoM and liver ApoM mRNA of DHT-treated mice were measured and compared with those of vehicle-treated mice. It demonstrated that DHT reduced the levels of plasma ApoM and liver ApoM mRNA in DHT-treated mice. The present findings, therefore, might partially indicate a mechanism underlying the reduction of plasma HDL cholesterol during administration of DHT in vivo.
Sphingolipids are a large family of glycolipids and phospholipids that share a common sphingoid base backbone. These once called ‘structural’ lipids are now well-established signaling molecules that play multiple roles in a vast number of cellular processes. A growing body of literature has demonstrated the reciprocal interaction between bioactive sphingolipids and steroid hormones. Sphingolipids serve as second messengers in steroidogenic regulatory pathways[23, 24], and meanwhile steroid hormones regulates the metabolism of sphingolipids[25–27]. Plasma sphingosine-1-phosphate (S1P), which maintains vascular integrity, is associated with HDL (∼65%) and albumin (∼35%)[28, 29]. HDL induced vasorelaxation as well as barrier-promoting and prosurvival actions on the endothelium have been attributed to S1P signaling[30–32]. ApoM is a lipocalin that resides mainly in the plasma HDL fraction. The retained hydrophobic NH2-terminal signal peptide anchors ApoM in the phospholipid layer of the lipoprotein and prevents filtration of the ∼ 22-kDa protein in the kidney. Studies in ApoM gene-modified mice suggest that apoM has antiatherogenic effects, possibly related in part to ApoM’s ability to increase cholesterol efflux from macrophage foam cells, to increased preβ-HDL formation, and to antioxidative effects[8, 20, 34]. ApoM is a carrier of S1P in HDL and the HDL-associated ApoM–S1P complex mediates vasoprotective actions on the endothelium. This signaling axis may be critical in normal vascular homeostasis and perturbed in vascular diseases. Whether DHT affected HDL-associated function via regulation of ApoM and ApoM–S1P signaling axis is still to be elucidated.
It is well known that androgens exert both transcriptional and non-transcriptional actions[36–38]. The transcriptional actions of androgens are mediated through the classic androgen receptor. The ligand-bound classic androgen receptor mainly functions as a transcription factor modulating the expression of androgen-receptor target genes. In contrast, non-transcriptional actions of androgens include increasing the concentration of intracellular calcium, and activation of protein tyrosine kinase, such as Src(c-Src), extracellular signal-regulated kinase-1/2 (ERK-1/2), and phosphatidylinositol 3-kinase (PI3K)[39–44]. In our present study, we found that flutamide, a classical androgen receptor blocker, did not modify DHT-mediated apoM secretion. Although these data may suggest that the action of DHT on ApoM secretion is non-transcriptional, the differentiation between non-transcriptional vs. transcriptional effects is much more complex and cannot been firmly concluded from the present study.
We also investigated the intracellular signaling mechanisms by which DHT mediates ApoM secretion by hepG2 cells. Our present study shows that PMA, a PKC agonist, increased ApoM secretion. Staurosporin, a PKC superfamily inhibitor, abolished the DHT-mediated decrease in ApoM secretion. The intracellular signaling mechanisms by which DHT act through PKC to affect apoM secretion remains unknown. It is reported that ApoM gene expression is affected by nuclear receptors such as hepatocyte nuclear factor-1a (HNF-1a), hepatocyte nuclear factor-4a (HNF-4a), liver receptor homolog-1 (LRH-1), and liver X receptor (LXR).
Leptin is the first identified endocrine product of adipose tissue and was found to regulate vascular function through local and central mechanisms. There is some evidence supporting the effects of leptin on the cardiovascular system and Type 2 diabetes mellitus (T2DM). It was shown that a high leptin level predicts subsequent development of T2DM. Plasma leptin levels positively correlated with TG, Lp (a), Apo-A1, glucose, BMI, insulin resistance (HOMA-IR), SBP and DBP levels and negatively with HDL-C levels in T2DM patients[50, 51]. Studies suggest that both leptin and leptin-receptor are essential for ApoM expression in vitro and vivo[9, 52]. In the present study we demonstrated that DHT down-regulated the expression and the secretion of ApoM. Whether DHT-affected ApoM expression is mediated by specific nuclear receptors or leptin remains to be investigated.
It has been previously reported that ApoM expression is regulated by PI3-kinase in HepG2-cells. In the present study, we used the PI3-K antagonist (wortmannin) to study DHT-treated HepG2 cells. We found that wortmannin could not abolish DHT-mediated inhibition of ApoM expression, which indicates that PI3-K might not be involved in the DHT-induced inhibition of ApoM expression. Our present results indicate that PKC is involved in DHT-mediated ApoM secretion. However, the participation of PKC family members whose identities remain to be determined.