Macroautophagy/autophagy is a highly regulated process involved in the turnover of cytosolic components, however its pivotal role in maintenance of bone homeostasis remains elusive. In the present study, we investigated the direct role of ATG7 (autophagy related 7) during developmental and remodeling stages in vivo using osteoblast-specific Atg7 conditional knockout (cKO) mice. Atg7 cKO mice exhibited a reduced bone mass at both developmental and adult age. The trabecular bone volume of Atg7 cKO mice was significantly lower than that of controls at 5 months of age. This phenotype was attributed to decreased osteoblast formation and matrix mineralization, accompanied with an increased osteoclast number and the extent of the bone surface covered by osteoclasts as well as an elevated secretion of TNFSF11/RANKL (tumor necrosis factor [ligand] superfamily, member 11), and a decrease in TNFRSF11B/OPG (tumor necrosis factor receptor superfamily, member 11b [osteoprotegerin]). Remarkably, Atg7 deficiency in osteoblasts triggered endoplasmic reticulum (ER) stress, whereas attenuation of ER stress by administration of phenylbutyric acid in vivo abrogated Atg7 ablation-mediated effects on osteoblast differentiation, mineralization capacity and bone formation. Consistently, Atg7 deficiency impeded osteoblast mineralization and promoted apoptosis partially in DDIT3/CHOP (DNA-damage-inducible transcript 3)- and MAPK8/JNK1 (mitogen-activated protein kinase 8)-SMAD1/5/8-dependent manner in vitro, while reconstitution of Atg7 could improve ER stress and restore skeletal balance. In conclusion, our findings provide direct evidences that autophagy plays crucial roles in regulation of bone homeostasis and suggest an innovative therapeutic strategy against skeletal diseases.

The critical regulation of the peripheral circadian gene implicated in osteoarthritis (OA) has been recently recognized; however, the causative role and clinical potential of the peripheral circadian rhythm attributable to such effects remain elusive. The purpose of this study was to elucidate the role of a circadian gene Bmal1 in human cartilage and pathophysiology of osteoarthritis. In our present study, the mRNA and protein levels of circadian rhythm genes, including nicotinamide adenine dinucleotide oxidase (NAD⁺) and sirtuin 1 (Sirt1), in human knee articular cartilage were determined. In OA cartilage, the levels of both Bmal1 and NAD⁺ decreased significantly, which resulted in the inhibition of nicotinamide phosphoribosyltransferase activity and Sirt1 expression. Furthermore, the knockdown of Bmal1 was sufficient to decrease the level of NAD⁺ and aggravate OA-like gene expression changes under the stimulation of IL-1β. The overexpression of Bmal1 relieved the alteration induced by IL-1β, which was consistent with the effect of the inhibition of Rev-Erbα (known as NR1D1, nuclear receptor subfamily 1, group D). On the other hand, the transfection of Sirt1 small interfering RNA not only resulted in a reduction of the protein expression of Bmal1 and a moderate increase of period 2 (per2) and Rev-Erbα but also further exacerbated the survival of cells and the expression of cartilage matrix-degrading enzymes induced by IL-1β. Overexpression of Sirt1 restored the metabolic imbalance of chondrocytes caused by IL-1β. These observations suggest that Bmal1 is a key clock gene to involve in cartilage homeostasis mediated through sirt1 and that manipulating circadian rhythm gene expression implicates an innovative strategy to develop novel therapeutic agents against cartilage diseases.

Sirtuin1 (SIRT1) has recently emerged as a pivotal regulator of glucose metabolism and insulin sensitivity. However, the underlying mechanism has not been fully elucidated. In this study, we investigated the role of SIRT1 in the development of obesity and insulin resistance by generating mice with adipose-specific ablation of Sirt1 (Ad-Sirt1^-/- mice). Ad-Sirt1^-/- mice exhibited increased fat mass, impaired glucose tolerance, attenuated insulin sensitivity, and increased exosomes, whereas the administration of exosomes inhibitor effectively ameliorated the impaired metabolic profile in Ad-Sirt1^-/- mice. Moreover, the increased exosomes were proved to be a result of defective autophagy activity in Ad-Sirt1^-/- mice and restoration of SIRT1 activity efficiently improved metabolic profiles in vitro. Further study demonstrated that Sirt1 deficiency-induced exosomes modulated insulin sensitivity at least partially via the TLR4/NF-κB signaling pathway. Therefore, our findings implicated SIRT1 as a key factor in metabolic regulation, and adipose Sirt1 deficiency could exert an effect on the development of obesity and insulin resistance by promoting exosome release.

The NAD⁺-dependent deacetylase sirtuin-1 (SIRT1) has emerged as an important regulator of chondrogenesis and cartilage homeostasis, processes that are important for physiological skeletal growth and that are dysregulated in osteoarthritis. However, the functional role and underlying mechanism by which SIRT1 regulates chondrogenesis remain unclear. Using cultured rat metatarsal bones and chondrocytes isolated from rat metatarsal rudiments, here we studied the effects of the SIRT1 inhibitor EX527 or of SIRT1 siRNA on chondrocyte proliferation, hypertrophy, and apoptosis. We show that EX527 or SIRT1 siRNA inhibits chondrocyte proliferation and hypertrophy and induces apoptosis. We also observed that SIRT1 inhibition mainly induces the PERK–eIF-2α–CHOP axis of the endoplasmic reticulum (ER) stress response in growth-plate chondrocytes. Of note, EX527- or SIRT1 siRNA–mediated inhibition of metatarsal growth and growth-plate chondrogenesis were partly neutralized by phenylbutyric acid, a chemical chaperone that attenuates ER stress. Moreover, EX527-mediated impairment of chondrocyte function (i.e. of chondrocyte proliferation, hypertrophy, and apoptosis) was partly reversed in CHOP^−/− cells. We also present evidence that SIRT1 physically interacts with and deacetylates PERK. Collectively, our findings indicate that SIRT1 deacetylates PERK and attenuates the PERK–eIF-2α–CHOP axis of the unfolded protein response pathway and thereby promotes growth-plate chondrogenesis and longitudinal bone growth.

The osteoblast-specific secreted molecule osteocalcin behaves as a hormone-regulating glucose and lipid metabolism, but the role of osteocalcin in cardiovascular disease (CVD) is not fully understood. In the present study, we investigated the effect of osteocalcin on autophagy and endoplasmic reticulum (ER) stress secondary to diet-induced obesity in the vascular tissue of mice and in vascular cell models and clarified the intracellular events responsible for osteocalcin-mediated effects. The evidences showed that intermittent injections of osteocalcin in mice fed the high-fat diet were associated with a reduced body weight gain, decreased blood glucose and improved insulin sensitivity compared with mice fed the high-fat diet receiving vehicle. Simultaneously, the administration of osteocalcin not only attenuated autophagy and ER stress but also rescued impaired insulin signaling in vascular tissues of mice fed a high-fat diet. Consistent with these results in vivo, the addition of osteocalcin reversed autophagy and ER stress and restored defective insulin sensitivity in vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) in the presence of tunicamycin or in knockout XBP-1 (a transcription factor which mediates ER stress response) cells or in Atg7−/− cells. The protective effects of osteocalcin were nullified by suppression of Akt, mammalian target of rapamycin (mTOR) or nuclear factor kappa B (NFκB), suggesting that osteocalcin inhibits autophagy, ER stress and improves insulin signaling in the vascular tissue and cells under insulin resistance in a NFκB-dependent manner, which may be a promising therapeutic strategies of cardiovascular dysfunction secondary to obesity.

Dehydroepiandrosterone (DHEA) is an endogenous steroid that is metabolized to androgens and/or estrogens in the human prostate. DHEA levels decline with age, and use of DHEA supplements to retard the aging process is of unproved effectiveness and safety. In this study, rat ventral prostatic epithelial cells were used to determine whether DHEA-modulated proliferation and prostate-specific antigen (PSA listed as KLKB1 in the MGI Database) production were mediated via the androgen receptor (AR) and its potential mechanism. We demonstrated that proliferation of prostatic epithelial cells and increase of PSA expression induced by DHEA were neutralized by Casodex or Ar siRNA, two specific AR blockers. DHEA stimulated Nfkb DNA binding activity, with this effect being blunted by Casodex or Ar siRNA. Moreover, the inhibition of the phosphatidylinositol 3-kinase (PI3K)/AKT nullified the effects of DHEA on NFKB activation. These findings suggested that DHEA stimulated normal prostatic epithelial cell proliferation, and AR is involved in DHEA-induced PSA expression in normal prostatic epithelial cells. This stimulation effect induced by DHEA is mediated by the activation of NFKB via PI3K/AKT pathway.