Cholecalciferol inhibits lipid accumulation by regulating early adipogenesis in cultured adipocytes and zebrafish

Highlights

• CCF inhibits adipogenesis by regulating early adipogenic factors.

• CCF inhibits mitotic clonal expansion via cell cycle arrest.

• CCF inhibits serine threonine-specific kinase and mammalian target of rapamycin.

• CCF activates 5'adenosine monophosphate-activated protein kinase α signaling.

• CCF inhibits lipid accumulation in zebrafish.

Abstract

Cholecalciferol (CCF) is a common dietary supplement as a precursor of active vitamin D. In the present study, the effect of CCF on lipid accumulation was investigated in adipocyte cells and zebrafish models. CCF effectively inhibited lipid accumulation in both experimental models; this effect was attributed to the CCF-mediated regulation of early adipogenic factors. CCF down-regulated the expressions of CCAAT-enhancer-binding protein-β (C/EBPβ), C/EBPδ, Krueppel-like factor (KLF) 4, and KLF5, while KLF2, a negative adipogenic regulator, was increased by CCF treatment. CCF inhibited cell cycle progression of adipocytes through down-regulation of cyclin A and cyclinD; p-Rb was suppressed by CCF, but p27 was up-regulated with CCF treatment. This CCF-mediated inhibition of cell cycle progression is highly correlated to the inhibitions of extracellular signal-regulated kinase (ERK), serine threonine-specific kinase (AKT), and mammalian target of rapamycin (mTOR). Furthermore, CCF-induced inactivation of acetyl-CoA carboxylase (ACC), a fatty acid synthetic enzyme, with the activation of AMP-activated protein kinase α (AMPKα) was also observed. Consistent with the observations in adipocytes, CCF effectively inhibited lipid accumulation with the down-regulation of adipogenic factors in zebrafish. The present study indicates that CCF showed anti-adipogenic effect in adipocytes and zebrafish, and its inhibitory effect was involved in the regulation of early adipogenic events including cell cycle arrest and activation of AMPKα signaling.

Introduction

Cholecalciferol (CCF or vitamin D₃) is a precursor of active vitamin D (1,25- hydroxyvitamin D) [1]. CCF is endogenously synthesized from 7-dehydrocholesterol under the epidermal layer of skin via ultraviolet B (UVB) exposure from sunlight. It is then converted to 25-hydroxy CCF by hepatic 25-hydroxylase and is further hydroxylated by renal 1-a-hydroylase to produce 1,25-hydroxyvitamin D, a biologically active form [2]. CCF is also the most commonly-consumed dietary source of vitamin D. Dietary CCF taken up by enterocytes is incorporated into chylomicrons and thereby reaches the liver and kidneys via the blood stream for further hydroxylation [2].

Accumulated studies reveal the existence of strong links between vitamin D and obesity [3], [4]. The study of vitamin D on obesity or adipogenesis was mostly performed using 1,25-hydroxyvitamin D. A recent study reported that 1,25-hydroxyvitamin D levels were lower in obese people, compared to normal weight individuals [4]. Kong and Li showed that the inhibition of adipogenesis by 1,25-hydroxyvitamin D is associated with the blockade of adipogenic factors, such as peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT-enhancer-binding protein-alpha (C/EBPα) [3]. They also suggested that 1,25-hydroxyvitamin D-induced stabilization of cellular VDR is linked to the inhibition of adipogenesis. Besides, the levels of vitamin D have been associated with various diseases such as inflammation and cancer [5], [6].

Excessive adipogenesis results from an imbalance in energy homeostasis, and thereby causes obesity, which is associated with other metabolic complications such as diabetes, atherosclerosis, and hypertension [7]. Accordingly, the proper control of the process of adipogenesis is important for reducing the risk of metabolic diseases. Cultured adipogenic processes occur in post-confluent cells with re-progression of the cell cycle and a dramatic increase in cell number, called mitotic clonal expansion (MCE) [8]. This increase in cell number (hyperplasia) is accompanied by the activation of cell signaling pathways, such as PI3K/AKT and ERK pathways [9]. Other signaling molecules affecting lipid biosynthesis include AMPK and mTOR [10], [11]. Along with these signaling events, various genes necessary to trigger the differentiation are transcriptionally activated; early adipogenic factors, including the consecutively activated C/EBPβ, KLF4, and KLF5, as well as the up-regulated C/EBPα and PPARγ [12].

The synthesis of CCF via exposure to sunlight is needed to satisfy the proper vitamin D requirements of the body. However, since many factors such as sunburn, skin aging, and change of skin color weaken this method of cutaneous synthesis of vitamin D, people have been increasingly interested in dietary sources of vitamin D [13]. CCF is naturally found in many dietary sources, including fish liver oils, oily fish, egg yolk, and wild mushrooms, even if the content varies [14]. Although the effects of 1,25-hydroxyvitamin D on adipogenesis have been studied, there is limited information about the effects of its precursor, CCF. Accordingly, the present study investigated the effect of CCF on adipogenesis in both cultured adipocytes and a zebrafish model, showing that it modulates the early stage of adipogenesis; the CCF-modulated signaling in this process reveals different aspects from findings reported in the previous studies of 1,25-hydroxyvitamin D.