All-trans retinoic acid induces lipophagy by reducing Rubicon in Hepa1c1c7 cells.

All-trans retinoic acid (atRA), a metabolite of vitamin A, reduces hepatic lipid accumulation in liver steatosis model animals. Lipophagy, a new lipolysis pathway, degrades a lipid droplet (LD) via autophagy in adipose tissue and the liver. We recently found that atRA induces lipophagy in adipocytes. However, it remains unclear whether atRA induces lipophagy in hepatocytes. In this study, we investigated the effects of atRA on lipophagy in Hepa1c1c7 cells and the liver of mice fed a high-fat diet (HFD). First, we confirmed that atRA induced autophagy in Hepa1c1c7 cells by Western blotting and the GFP-LC3-mCherry probe. Next, we evaluated the lipolysis in fatty Hepa1c1c7 cells treated with the knockdown of Atg5, an essential gene in autophagy induction. Atg5-knockdown partly suppressed the atRA-induced lipolysis in fatty Hepa1c1c7 cells. We also found that atRA reduced the protein, but not mRNA, expression of Rubicon, a negative regulator of autophagy, in Hepa1c1c7 cells and the liver of HFD-fed mice. Rubicon-knockdown partly inhibited the atRA-induced lipolysis in fatty Hepa1c1c7 cells. In addition, atRA reduced hepatic Rubicon expression in young mice, but the effect of atRA on it diminished in aged mice. Finally, we investigated the mechanism underlying reduced Rubicon protein expression by atRA in hepatocytes. A protein synthesis inhibitor, but not proteasome or lysosomal inhibitors, significantly blocked the reduction of Rubicon protein expression by atRA in Hepa1c1c7 cells. These results suggest that atRA may promote lipophagy in fatty hepatocytes by reducing hepatic Rubicon expression via inhibiting protein synthesis.

25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) induces ectopic calcification.

Vascular calcification is an important pathogenesis related to cardiovascular disease and high mortality rate in chronic kidney disease (CKD) patients. It has been well-known that hyper-phosphatemia induces osteochondrogenic transition of vascular smooth muscle cells (VSMCs) resulting ectopic calcification in aortic media, cardiac valve, and kidney. However, the detailed mechanism of the ectopic calcification has been not clarified yet. Here, we found that the co-localization of CYP27B1 with the calcified lesions of aorta and arteries in kidney of klotho mutant (kl/kl) mice, and then investigated the role of CYP27B1 in the mineralization of the VSMCs. Under high phosphate condition, overexpression of CYP27B1 induced calcification and osteocalcin mRNA expression in the VSMCs. Inversely, siRNA-CYP27B1 inhibited high phosphate-induced calcification of the VSMCs. We also found that the accumulated CYP27B1 protein was glycosylated in the kidney of kl/kl mice. Therefore, overexpression of CYP27B1-N310A and CYP27B1-T439A, which are a mutation for N-linked glycosylation site (N310A) and a mutation for O-linked glycosylation site (T439A) in CYP27B1, decreased calcium deposition and expression of RUNX2 induced by high phosphate medium in VSMCs compared with wild-type CYP27B1. These results suggest that extra-renal expression of glycosylated CYP27B1 would be required for ectopic calcification of VSMCs under hyperphosphatemia.

Identification of Dietary Phytochemicals Capable of Enhancing the Autophagy Flux in HeLa and Caco-2 Human Cell Lines.

Autophagy is a major degradation system for intracellular macromolecules. Its decline with age or obesity is related to the onset and development of various intractable diseases. Although dietary phytochemicals are expected to enhance autophagy for preventive medicine, few studies have addressed their effects on the autophagy flux, which is the focus of the current study. Herein, 67 dietary phytochemicals were screened using a green fluorescent protein (GFP)-microtubule-associated protein light chain 3 (LC3)-red fluorescent protein (RFP)-LC3ΔG probe for the quantitative assessment of autophagic degradation. Among them, isorhamnetin, chrysoeriol, 2,2',4'-trihydroxychalcone, and zerumbone enhanced the autophagy flux in HeLa cells. Meanwhile, analysis of the structure-activity relationships indicated that the 3'-methoxy-4'-hydroxy group on the B-ring in the flavone skeleton and an ortho-phenolic group on the chalcone B-ring were crucial for phytochemicals activities. These active compounds were also effective in colon carcinoma Caco-2 cells, and some of them increased the expression of p62 protein, a typical substrate of autophagic proteolysis, indicating that phytochemicals impact p62 levels in autophagy-dependent and/or -independent manners. In addition, these compounds were characterized by distinct modes of action. While isorhamnetin and chrysoeriol enhanced autophagy in an mTOR signaling-dependent manner, the actions of 2,2',4'-trihydroxychalcone and zerumbone were independent of mTOR signaling. Hence, these dietary phytochemicals may prove effective as potential preventive or therapeutic strategies for lifestyle-related diseases.