Suppression of adipocyte differentiation by low‐intensity pulsed ultrasound via inhibition of insulin signaling and promotion of CCN family protein 2

Abstract

Adipocyte differentiation is regulated by several transcription factors such as the CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferatoractivated receptor-γ (PPARγ). Here, we demonstrate that low-intensity pulsed ultrasound (LIPUS) suppressed differentiation into mature adipocytes via multiple signaling pathways. When C3H10T1/2, a mesenchymal stem cell line, was treated with LIPUS (3.0 MHz, 60 mW/cm 2) for 20 minutes once a day for 4 days during adipogenesis, and both the number of lipid droplets and the gene expression of PPARγ and C/EBPα were significantly decreased. Furthermore, LIPUS treatment decreased the phosphorylation of the insulin receptor and also that of Akt and ERK1/2, which are located downstream of this receptor. Next, we showed that LIPUS suppressed the gene expression of angiotensinogen (AGT), which is an adipokine produced by mature adipocytes, as well as that of angiotensinconverting enzyme 1 (ACE1) and angiotensin receptor type 1 (AT 1 R) during adipogenesis of pre-adipogenic 3T3-L1 cells. Next, the translocation of Yesassociated protein (YAP) into the nucleus of 3T3-L1 cells was promoted by LIPUS, leading to upregulation of CCN family protein 2 (CCN2), a cellular communication network factor. Moreover, forced expression of CCN2 in 3T3-L1 cells decreased PPARγ gene expression, but it did not increase alkaline phosphatase and osterix gene expression. Finally, gene silencing of CCN2 in C3H10T1/2 cells diminished the effect of LIPUS on the gene expression of PPARγ and C/EBPα. These findings suggest that LIPUS suppressed adipogenesis through inhibition of insulin signaling and decreased PPARγ expression via increased CCN2 production, resulting in a possible decrease of mature adipocytes.

Roles of CCN2 as a mechano-sensing regulator of chondrocyte differentiation

“…Collectively, cortical actin organization via Rac1 activation promotes the translocation of SOX9 into the nucleus; and as a result, CCN2 expression is up-regulated. On the other hand, although differentiating adipocytes undergo cortical actin organization as well, CCN2 expression is remarkably down-regulated in them [65] . Additionally, LIPUS treatment decreases cortical actin and increases CCN2 expression, repressing their adipogenic differentiation [65] .…”

“…On the other hand, although differentiating adipocytes undergo cortical actin organization as well, CCN2 expression is remarkably down-regulated in them [65] . Additionally, LIPUS treatment decreases cortical actin and increases CCN2 expression, repressing their adipogenic differentiation [65] . As it is reported that peroxisome proliferator-activated receptor (PPAR)γ, which is an essential transcription factor for adipogenesis, inhibits CCN2 expression [76] , these findings suggest that the PPARγ decreased by LIPUS treatment increases CCN2 expression in adipocytes.…”

“…It is worthy of notice that YAP and its paralog, TAZ are transcriptional coactivator of the TEAD family regulating CCN2 expression as described in Section 2 . As YAP/TAZ is translocated into the nucleus or stays in the cytoplasm according to the tension of the actin cytoskeleton, the expression of CCN2, which is one of the target genes, is increased by the formation of F-actin stress fibers when the cells are subjected to tension [42] , [65] . Therefore, the induction of CCN2 by activated RhoA is due to the retention of YAP in the nucleus by actin polymerization, at least in fibroblasts and mesenchymal stem cells.…”

Review on experimental study and clinical application of low- intensity pulsed ultrasound in inflammation

“…For example, chronic inflammation has been demonstrated to be a key player in the development of diabetes and its complications (116)(117)(118). The cells involved in these diseases, including adipocytes, have been shown to respond under the stimuli of LIPUS (119,120). It is possible that the anti-inflammatory effects of LIPUS could play an important role in the treatment of diabetes, and future research should be conducted.…”

Effect of Angiotensin II on Chondrocyte Degeneration and Protection via Differential Usage of Angiotensin II Receptors

“…To isolate total RNA from HCS-2/8 cells, RCS cells, and xiphoid process-derived chondrocytes, we used ISOGEN reagent (Nippon Gene, Tokyo, Japan). First-strand cDNA was synthesized from 1 µg of total RNA by a primerScript TM RT reagent kit (Takara Shuzo, Tokyo, Japan), and subsequent quantitative PCR analysis was performed with the SYBR ® Green Real-time PCR Master Mix (Toyobo, Tokyo, Japan) using the StepOne Plus real-time PCR machine (Applied Biosystems, Carlsbad, CA, USA) as described previously [32]. The specific primer sequences and the GenBank accession numbers of the target genes are shown in Table 2.…”