Reorganization of the DNA replication landscape during adipogenesis is closely linked with adipogenic gene expression.

The temporal order of DNA replication along the chromosomes is thought to reflect the transcriptional competence of the genome. During differentiation of mouse 3T3-L1 cells into adipocytes, cells undergo one or two rounds of cell division called mitotic clonal expansion (MCE). MCE is an essential step for adipogenesis; however, little is known about the regulation of DNA replication during this period. Here, we performed genome-wide mapping of replication timing (RT) in mouse 3T3-L1 cells before and during MCE, and identified a number of chromosomal regions shifting toward either earlier or later replication through two rounds of replication. These RT changes were confirmed in individual cells by single-cell DNA-replication sequencing. Coordinate changes between a shift toward earlier replication and transcriptional activation of adipogenesis-associated genes were observed. RT changes occurred before the full expression of these genes, indicating that RT reorganization might contribute to the mature adipocyte phenotype. To support this, cells undergoing two rounds of DNA replication during MCE had a higher potential to differentiate into lipid droplet-accumulating adipocytes, compared with cells undergoing a single round of DNA replication and non-replicating cells.

Tipepidine activates AMPK and improves adipose tissue fibrosis and glucose intolerance in high-fat diet-induced obese mice.

Tipepidine (3-[di-2-thienylmethylene]-1-methylpiperidine) (TP) is a non-narcotic antitussive used in Japan. Recently, the potential application of TP in the treatment of neuropsychiatric disorders, such as depression and attention deficit hyperactivity disorder, has been suggested; however, its functions in energy metabolism are unknown. Here, we demonstrate that TP exhibits a metabolism-improving action. The administration of TP reduced high-fat diet-induced body weight gain in mice and lipid accumulation in the liver and increased the weight of epididymal white adipose tissue (eWAT) in diet-induced obese (DIO) mice. Furthermore, TP inhibited obesity-induced fibrosis in the eWAT. We also found that TP induced AMP-activated protein kinase (AMPK) activation in the eWAT of DIO mice and 3T3-L1 cells. TP-induced AMPK activation was abrogated by the transfection of liver kinase B1 siRNA in 3T3-L1 cells. The metabolic effects of TP were almost equivalent to those of metformin, an AMPK activator that is used as a first-line antidiabetic drug. In summary, TP is a potent AMPK activator, suggesting its novel role as an antidiabetic drug owing to its antifibrotic effect on adipose tissues.

Gprc5a is a novel parathyroid hormone-inducible gene and negatively regulates osteoblast proliferation and differentiation.

Teriparatide is a peptide derived from a parathyroid hormone (PTH) and an osteoporosis therapeutic drug with potent bone formation-promoting activity. To identify novel druggable genes that act downstream of PTH signaling and are potentially involved in bone formation, we screened PTH target genes in mouse osteoblast-like MC3T3-E1 cells. Here we show that Gprc5a, encoding an orphan G protein-coupled receptor, is a novel PTH-inducible gene and negatively regulates osteoblast proliferation and differentiation. PTH treatment induced Gprc5a expression in MC3T3-E1 cells, rat osteosarcoma ROS17/2.8 cells, and mouse femurs. Induction of Gprc5a expression by PTH occurred in the absence of protein synthesis and was mediated primarily via the cAMP pathway, suggesting that Gprc5a is a direct target of PTH signaling. Interestingly, Gprc5a expression was induced additively by co-treatment with PTH and 1α, 25-dihydroxyvitamin D3 (calcitriol), or retinoic acid in MC3T3-E1 cells. Reporter analysis of a 1 kb fragment of human GPRC5A promoter revealed that the promoter fragment showed responsiveness to PTH via the cAMP response element, suggesting that GPRC5A is also a PTH-inducible gene in humans. Gprc5a knockdown promoted cell viability and proliferation, as demonstrated by MTT and BrdU assays. Gprc5a knockdown also promoted osteoblast differentiation, as indicated by gene expression analysis and mineralization assay. Mechanistic studies showed that Gprc5a interacted with BMPR1A and suppressed BMP signaling induced by BMP-2 and constitutively active BMP receptors, ALK2 (ACVR1) Q207D and ALK3 (BMPR1A) Q233D. Thus, our results suggest that Gprc5a is a novel gene induced by PTH that acts in an inhibitory manner on both cell proliferation and osteoblast differentiation and is a candidate for drug targets for osteoporosis.

Melatonin Regulates Osteoblast Differentiation through the m6A Reader hnRNPA2B1 under Simulated Microgravity.

Recent studies have confirmed that melatonin and N6-methyladenosine (m6A) modification can influence bone cell differentiation and bone formation. Melatonin can also regulate a variety of biological processes through m6A modification. Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) serves as a reader of m6A modification. In this study, we used the hindlimb unloading model as an animal model of bone loss induced by simulated microgravity and used 2D clinorotation to simulate a microgravity environment for cells on the ground. We found that hnRNPA2B1 was downregulated both in vitro and in vivo during simulated microgravity. Further investigations showed that hnRNPA2B1 could promote osteoblast differentiation and that overexpression of hnRNPA2B1 attenuated the suppression of osteoblast differentiation induced by simulated microgravity. We also discovered that melatonin could promote the expression of hnRNPA2B1 under simulated microgravity. Moreover, we found that promotion of osteoblast differentiation by melatonin was partially dependent on hnRNPA2B1. Therefore, this research revealed, for the first time, the role of the melatonin/hnRNPA2B1 axis in osteoblast differentiation under simulated microgravity. Targeting this axis may be a potential protective strategy against microgravity-induced bone loss and osteoporosis.

Haspin participates in AURKB recruitment to centromeres and contributes to chromosome congression in male mouse meiosis.

Chromosome segregation requires that centromeres properly attach to spindle microtubules. This essential step regulates the accuracy of cell division and must therefore be precisely regulated. One of the main centromeric regulatory signaling pathways is the haspin-H3T3ph-chromosomal passenger complex (CPC) cascade, which is responsible for the recruitment of the CPC to the centromeres. During mitosis, the haspin kinase phosphorylates histone H3 at threonine 3 (H3T3ph), an essential epigenetic mark that recruits the CPC, in which the catalytic component is Aurora B kinase (AURKB). However, the centromeric haspin-H3T3ph-CPC pathway remains largely uncharacterized in mammalian male meiosis. We have analyzed haspin functions by either its chemical inhibition with LDN-192960 in cultured spermatocytes, or the ablation of the Haspin gene in Haspin-/- mice. Our studies suggest that haspin kinase activity is required for proper chromosome congression both during meiotic divisions and for the recruitment of Aurora B and kinesin MCAK (also known as KIF2C) to meiotic centromeres. However, the absence of H3T3ph histone mark does not alter borealin (or CDCA8) and SGO2 centromeric localization. These results add new and relevant information regarding the regulation of the haspin-H3T3ph-CPC pathway and centromere function during meiosis.

MLPH is a novel adipogenic factor controlling redox homeostasis to inhibit lipid peroxidation in adipocytes.

Abnormal adipose tissue formation is associated with metabolic disorders such as obesity, diabetes, and liver and cardiovascular diseases. Thus, identifying the novel factors that control adipogenesis is crucial for understanding these conditions and developing targeted treatments. In this study, we identified the melanosome-related factor MLPH as a novel adipogenic factor. MLPH was induced during the adipogenesis of 3T3-L1 cells and human mesenchymal stem cells. Although MLPH did not affect lipid metabolism, such as lipogenesis or lipolysis, adipogenesis was severely impaired by MLPH depletion. We observed that MLPH prevented excess reactive oxygen species (ROS) accumulation and lipid peroxidation during adipogenesis and in mature adipocytes. In addition, increased MLPH expression was observed under cirrhotic conditions in liver cancer cells and its overexpression also reduced ROS and lipid peroxidation. Our findings demonstrate that MLPH is a novel adipogenic factor that maintains redox homeostasis by preventing lipid peroxidation and ROS accumulation, which could lead to metabolic diseases.

IL-33 regulates adipogenesis via Wnt/ß-catenin/PPAR-γ signaling pathway in preadipocytes.

Interleukin-33 (IL-33), an emerging cytokine within the IL-1 family, assumes a pivotal function in the control of obesity. However, the specific mechanism of its regulation of obesity formation remains unclear. In this study, we found that the expression level of IL-33 increased in visceral adipose tissue in mice fed with a high-fat diet (HFD) compared with that in mice fed with a normal diet (ND). In vitro, we also found the expression level of IL-33 was upregulated during the adipogenesis of 3T3-L1 cells. Functional test results showed that knockdown of IL-33 in 3T3-L1 cells differentiation could promote the accumulation of lipid droplets, the content of triglyceride and the expression of adipogenic-related genes (i.e. PPAR-γ, C/EBPα, FABP4, LPL, Adipoq and CD36). In contrast, overexpression of IL-33 inhibits adipogenic differentiation. Meanwhile, the above tests were repeated after over-differentiation of 3T3-L1 cells induced by oleic acid, and the results showed that IL-33 played a more significant role in the regulation of adipogenesis. To explore the mechanism, transcriptome sequencing was performed and results showed that IL-33 regulated the PPAR signaling pathway in 3T3-L1 cells. Further, Western blot and confocal microscopy showed that the inhibition of IL-33 could promote PPAR-γ expression by inhibiting the Wnt/ß-catenin signal in 3T3-L1 cells. This study demonstrated that IL-33 was an important regulator of preadipocyte differentiation and inhibited adipogenesis by regulating the Wnt/ß-catenin/PPAR-γ signaling pathway, which provided a new insight for further research on IL-33 as a new intervention target for metabolic disorders.

Comparison of two biological systems used for phototoxicity testing: Cellular and tissue.

The OECD has approved two similar methods for testing the phototoxic potency of chemicals. The first method, OECD 432, is based on the cytotoxicity properties of materials to the mouse 3T3 (clone A31) cell line (fibroblasts) after exposure to light. The second method, OECD 498, is based on the same properties but using reconstructed human epidermis - EpiDerm (stratified keratinocytes). The aim of this study was to compare these two methods using statistical tests (specificity, sensitivity, negative predictive value, positive predictive value and accuracy) and non-statistical characteristics (e.g. price and experimental duration, amount of material, level of complications, cell type, irradiation dose). Both tests were performed according to the relevant guidelines using the same 11 control substances. Higher performance values were observed for OECD 432 in both phototoxic and non-phototoxic classifications. The accuracy of OECD 432 was 90.9%, while that of OECD 498 was 72.7%. OECD 432 was also shorter and less expensive. On the other hand, OECD 498 was less complicated, and used human cells with stratum corneum, which better reflects real skin. This method can also be used with oily substances that are poorly soluble in water. However, both methods are important for testing the phototoxic properties of materials, and can be used alone or in a tiered strategy.

The flower of Abelmoschus manihot (L.) medik exerts antioxidant effects by regulating the Nrf2 signalling pathway in scald injury.

Scald is a common skin injury in daily life. It is well known that skin burns are associated with inflammation and oxidative stress. In our previous study, we found that Abelmoschus manihot (L.) medik had excellent therapeutic effects on scald-induced inflammation, but its effect on scald-induced oxidative stress was not reported. In this study, a deep second-degree scald model in mice was established, and the wound healing rate, healing time, malondialdehyde (MDA) and total superoxide dismutase (T-SOD) levels, and nuclear factor erythroid 2-related Factor 2 (Nrf2) expression in wound tissue were measured to evaluate the scald wound healing performance of extraction from A. manihot (L.) medik (EAM). Scalding activity in mice was examined in vivo by hot water-induced finger swelling. The treatment scald activities were also examined in vivo by subjecting mice to thermal water-induced digit swelling. Additionally, the antioxidant effect of EAM on fibroblasts was also used to determine the mechanism in vitro. The results showed that EAM not only decreased the wound healing time but also effectively regulated the levels of oxidising, MDA and T-SOD in wound tissue. Concurrently, EAM suppressed digit swelling and hyperalgesia. Furthermore, EAM had a significant protective effect on NIH-3T3 cells after H2 O2 injury by regulating the Nrf2 signalling pathway against oxidative injury. Therefore, EAM is a promising drug for the treatment of scald-induced inflammation.

ATAD3 is a limiting factor in mitochondrial biogenesis and adipogenesis of white adipocyte-like 3T3-L1 cells.

ATAD3 is a vital ATPase of the inner mitochondrial membrane of pluri-cellular eukaryotes, with largely unknown functions but early required for organism development as necessary for mitochondrial biogenesis. ATAD3 knock-down in C. elegans inhibits at first the development of adipocyte-like intestinal tissue so we used mouse adipocyte model 3T3-L1 cells to analyze ATAD3 functions during adipogenesis and lipogenesis in a mammalian model. ATAD3 function was studied by stable and transient modulation of ATAD3 expression in adipogenesis- induced 3T3-L1 cells using Knock-Down and overexpression strategies, exploring different steps of adipocyte differentiation and lipogenesis. We show that (i) an increase in ATAD3 is preceding differentiation-induced mitochondrial biogenesis; (ii) downregulation of ATAD3 inhibits adipogenesis, lipogenesis, and impedes overexpression of many mitochondrial proteins; (iii) ATAD3 re-expression rescues the phenotype of ATAD3 KD, and (iv) differentiation and lipogenesis are accelerated by ATAD3 overexpression, but inhibited by expression of a dominant-negative mutant. We further show that the ATAD3 KD phenotype is not due to altered insulin signal but involves a limitation of mitochondrial biogenesis linked to Drp1. These results demonstrate that ATAD3 is limiting for in vitro mitochondrial biogenesis and adipogenesis/lipogenesis and therefore that ATAD3 mutation/over- or under-expression could be involved in adipogenic and lipogenic pathologies.

Osteoking prevents bone loss and enhances osteoblastic bone formation by modulating the AGEs/IGF-1/ß-catenin/OPG pathway in type 2 diabetic db/db mice.

Type 2 diabetic osteoporosis (T2DOP) is a skeletal metabolic syndrome characterized by impaired bone remodeling due to type 2 diabetes mellitus, and there are drawbacks in the present treatment. Osteoking (OK) is widely used for treating fractures and femoral head necrosis. However, OK is seldom reported in the field of T2DOP, and its role and mechanism of action need to be elucidated. Consequently, this study investigated whether OK improves bone remodeling and the mechanisms of diabetes-induced injury. We used db/db mice as a T2DOP model and stimulated MC3T3-E1 cells (osteoblast cell line) with high glucose (HG, 50 mM) and advanced glycation end products (AGEs, 100 µg/mL), respectively. The effect of OK on T2DOP was assessed using a combined 3-point mechanical bending test, hematoxylin and eosin staining, and enzyme-linked immunosorbent assay. The effect of OK on enhancing MC3T3-E1 cell differentiation and mineralization under HG and AGEs conditions was assessed by an alkaline phosphatase activity assay and alizarin red S staining. The AGEs/insulin-like growth factor-1(IGF-1)/ß-catenin/osteoprotegerin (OPG) pathway-associated protein levels were assayed by western blot analysis and immunohistochemical staining. We found that OK reduced hyperglycemia, attenuated bone damage, repaired bone remodeling, increased tibial and femoral IGF-1, ß-catenin, and OPG expression, and decreased receptor activator of nuclear kappa B ligand and receptor activator of nuclear kappa B expression in db/db mice. Moreover, OK promoted the differentiation and mineralization of MC3T3-E1 cells under HG and AGEs conditions, respectively, and regulated the levels of AGEs/IGF-1/ß-catenin/OPG pathway-associated proteins. In conclusion, our results suggest that OK may lower blood glucose, alleviate bone damage, and attenuate T2DOP, in part through activation of the AGEs/IGF-1/ß-catenin/OPG pathway.

New stilbenes from Cajanus cajan inhibit adipogenesis in 3T3-L1 adipocytes through down-regulation of PPARγ.

Two new stilbenes, denominated Cajanotone B (CAB) and Cajanotone C (CAC), were isolated from the leaves of Cajanus cajan. In this study, the structures of CAB and CAC were unambiguously elucidated by a combination of various spectral methods. Both compounds significantly inhibited the adipogenesis in 3T3-L1 adipocytes by reducing the lipid accumulation, triglyceride content and FFA secretion. CAB and CAC also substantially inhibit the mRNA expression of HSL, ATGL, C/EBPα and PPARγ as deciphered based by RT-PCR assay. Down-regulation of PPAR is believed to be the primary mechanism underlying which CAB and CAC inhibited adipogenic differentiation because the lipid-promoting activity of PPAR agonists can be counteracted by these compounds. The molecular interaction between CAB/CAC and PPARγ was revealed with the help of molecular docking. Taken together, CAB and CAC could serve as new lead compounds with the potential to speed up the development of novel lipid-lowering and weight-control therapies.

Bacoside-A repressed the differentiation and lipid accumulation of 3T3-L1 preadipocytes by modulating the expression of adipogenic genes.

Obesity is one of the more complicated diseases, it can induce numerous life-threatening diseases mainly diabetes mellitus, cardiovascular disease, hypertension, and certain cancers. In this study, we assessed the efficacy of bacoside-A (a dammarane-type triterpenoid saponin derived from the plant Bacopa monniera Linn.) on the adipogenesis of 3T3-L1 preadipocytes. Results of this study illustrated that bacoside-A decreased the differentiation of 3T3-L1 cell, as evidenced by diminution of lipid droplets, which contains triglycerides and other lipids. During the differentiation process, transcription factors, which are mainly participating in adipogenesis such us CCAAT/enhancer-binding protein α (C/EBPα) and C/EBPß, peroxisome proliferator-activated receptor-γ (PPARγ), and sterol regulatory element-binding protein-1c (SREBP-1c), expressions were significantly suppressed by bacoside-A. In addition, bacoside-A showed a potent reduction in genes precise to adipocytes such as lipoprotein lipase (LPL), fatty acid synthase (FAS), adipocyte fatty acid-binding protein (FABP4), and leptin expressions. Further, bacoside-A stimulated the phosphorylation of acetyl CoA carboxylase (ACC) and AMP-activated protein kinase (AMPK). These results demonstrated that bacoside-A has anti-adipogenic effects by regulating the transcription factors involved in adipocyte differentiation. Therefore, bacoside-A might be considered as a potent therapeutic agent for alleviating obesity and hyperlipidemia.

Accumulation and toxicity of biologically produced gold nanoparticles in different types of specialized mammalian cells.

The biologically produced gold nanoparticles (AuNPs) are novel carriers with promising use in targeted tumor therapy. Still, there are no studies regarding the efficacy of nanoparticle internalization by cancer and noncancer cells. In this study, AuNPs were produced by Fusarium oxysporum and analyzed by spectrophotometry, transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDS), and Zetasizer. Obtained AuNPs were about 15 nm in size with a zeta potential of -35.8 mV. The AuNPs were added to cancer cells (4T1), noncancer cells (NIH/3T3), and macrophages (RAW264.7). The viability decreased in 4T1 (77 ± 3.74%) in contrast to NIH/3T3 and RAW264.7 cells (89 ± 4.9% and 90 ± 3.5%, respectively). The 4T1 cancer cells also showed the highest uptake and accumulation of Au (∼80% of AuNPs was internalized) as determined by graphite furnace atomic absorption spectroscopy. The lowest amount of AuNPs was internalized by the NIH/3T3 cells (∼30%). The NIH/3T3 cells exhibited prominent reorganization of F-actin filaments as examined by confocal microscopy. In RAW264.7, we analyzed the release of proinflammatory cytokines by flow cytometry and we found the AuNP interaction triggered transient secretion of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). In summary, we proved the biologically produced AuNPs entered all the tested cell types and triggered cell-specific responses. High AuNP uptake by tumor cells was related to decreased cell viability, while low nanoparticle uptake by fibroblasts triggered F-actin reorganization without remarkable toxicity. Thus, the biologically produced AuNPs hold promising potential as cancer drug carriers and likely require proper surface functionalization to shield phagocytizing cells.

Effect and mechanism of recombinant human fibroblast growth factor 18 on osteoporosis in OVX mice.

OBJECTIVES: This study aimed to investigate the effect and the mechanism of recombinant human fibroblast growth factor 18 (rhFGF18) on postmenopausal osteoporosis. METHODS: The effect of rhFGF18 on the proliferation and apoptosis of osteoblasts and the mechanism underlying such an effect was evaluated using an oxidative stress model of the MC3T3-E1 cell line. Furthermore, ovariectomy was performed on ICR mice to imitate estrogen-deficiency postmenopausal osteoporosis. Bone metabolism and bone morphological parameters in the ovariectomized (OVX) mice were evaluated. RESULTS: The results obtained from the cell model showed that FGF18 promoted MC3T3-E1 cell proliferation by activating the extracellular signal-regulated kinase (ERK) and p38 instead of c-Jun N-terminal kinase (JNK). FGF18 also prevented cells from damage inflicted by oxidative stress via inhibition of apoptosis. After FGF18 administration, the expression level of anti-apoptotic protein Bcl-2 in the mice was upregulated, whereas those of the pro-apoptotic proteins Bax and caspase-3 were downregulated. Administering FGF18 also improved bone metabolism and bone morphological parameters in OVX mice. CONCLUSIONS: FGF18 could effectively prevent bone loss in OVX mice by enhancing osteoblastogenesis and protecting osteoblasts from oxidative stress-induced apoptosis.

Biointerfaces with ultrathin patterns for directional control of cell migration.

In the context of wound healing and tissue regeneration, precise control of cell migration direction is deemed crucial. To address this challenge, polydimethylsiloxane (PDMS) platforms with patterned 10 nm thick TiOx in arrowhead shape were designed and fabricated. Remarkably, without tall sidewall constraints, MC3T3-E1 cells seeded on these platforms were constrained to migrate along the tips of the arrowheads, as the cells were guided by the asymmetrical arrowhead tips which provided large contact areas. To the best of our knowledge, this is the first study demonstrating the use of thin TiOx arrowhead pattern in combination with a cell-repellent PDMS surface to provide guided cell migration unidirectionally without tall sidewall constraints. Additionally, high-resolution fluorescence imaging revealed that the asymmetrical distribution of focal adhesions, triggered by the patterned TiOx arrowheads with arm lengths of 10, 20, and 35 µm, promoted cell adhesion and protrusion along the arrowhead tip direction, resulting in unidirectional cell migration. These findings have important implications for the design of biointerfaces with ultrathin patterns to precisely control cell migration. Furthermore, microelectrodes were integrated with the patterned TiOx arrowheads to enable dynamic monitoring of cell migration using impedance measurement. This microfluidic device integrated with thin layer of guiding pattern and microelectrodes allows simultaneous control of directional cell migration and characterization of the cell movement of individual MC3T3-E1 cells, offering great potential for the development of biosensors for single-cell monitoring.

AEBP1 restores osteoblastic differentiation under dexamethasone treatment by activating PI3K/AKT signalling.

Adipocyte enhancer-binding protein 1 (AEBP1) is closely implicated in osteoblastic differentiation and bone fracture; this research aimed to investigate the effect of AEBP1 on restoring osteoblastic differentiation under dexamethasone (Dex) treatment, and its interaction with the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. Pre-osteoblastic MC3T3-E1 cells were cultured in osteogenic medium and treated by Dex to mimic steroid-induced osteonecrosis cellular model. They were then further transfected with control or AEBP1-overexpressed lentiviral vectors. Finally, cells were treated with the PI3K inhibitor LY294002, with or without AEBP1-overexpressed lentiviral vectors. AEBP1 expression showed a downward trend in MC3T3-E1 cells under Dex treatment in a dose-dependent manner. AEBP1-overexpressed lentiviral vectors increased relative cell viability, alkaline phosphatase (ALP) staining, Alizarin red staining and osteoblastic differentiation markers including osteocalcin (OCN), osteopontin (OPN), collagen type I alpha 1 (COL1A1), runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP2), but decreased cell apoptosis rate in MC3T3-E1 cells under Dex treatment; besides, AEBP1-overexpressed lentiviral vectors positively regulated p-PI3K and p-AKT expressions. Furthermore, LY294002 treatment decreased relative cell viability, Alizarin red staining, osteoblastic differentiation markers including OCN, OPN, RUNX2 and BMP, increased cell apoptosis rate and did not affect ALP staining in MC3T3-E1 cells under Dex treatment; meanwhile, LY294002 treatment weakened the effect of AEBP1 overexpression vectors on the above cell functions. AEBP1 restores osteoblastic differentiation under Dex treatment by activating the PI3K/AKT pathway.

Effect of 4 hydroxy fatty acids on lipid accumulation in the 3T3-L1 cells: a comparative study.

Notwithstanding the several investigations of the hydroxy fatty acids (hFAs)' physiological functions, studies focusing on their anti-obesity effects are limited. This study investigated the anti-obesity effects of 4 hFAs-10-hydroxy stearic acid (10-hSA), 12-hydroxy stearic acid (12-hSA), 9,12-hydroxy stearic acid (9,12-dhSA), and 12-hydroxy oleic acid (12-hOA)-on the 3T3-L1 cells. All hFAs suppressed lipid accumulation, with 10-hSA and 12-hOA exhibiting the strongest suppression, followed by 12-hSA and 9,12-hSA. This trend was similar to that observed for the glycerol-3-phosphate dehydrogenase (GPDH) activity level. Contrastingly, only 9,12-dhSA suppressed cell viability. The mRNA levels of HK1 and Aldoa were markedly suppressed by 10-hSA and 12-hSA compared to the control. Additionally, mRNA expression of Gyk was considerably suppressed by 12-hSA. Thus, all hFAs suppressed lipid accumulation by suppressing GPDH activity, although their molecular mechanisms were different. These findings will aid the application of hFAs in the food and medical industries.

Evaluation of the anti-androgenic and cytotoxic effects of benzophenone-3 in male Sprague-Dawley rats.

Benzophenone-3 (BP-3, 2-hydroxy-4-methoxybenzophenone, oxybenzone) is one of the most widely used types of benzophenone organic sunscreen. However, this compound is a potentially harmful toxicant. The aim of this study was 2-fold to: (1) utilize a Hershberger bioassay in vivo in castrated male Sprague-Dawley rats to investigate the anti-androgenic activities of BP-3, and (2) use in vitro a methyl tetrazolium assay to compare the toxicity between Leydig cells (TM3 cells) and mouse fibroblast (NIH-3T3) cell lines. In the Hershberger assay, rats were divided into 6 groups (each of n = 7): a vehicle control, negative control, positive control, PB-3 low (40 mg/kg), BP-3 intermediate (200 mg/kg), and BP-3 high (1000 mg/kg)-dose. The weight of the ventral prostate was significantly decreased at BP-3 doses of 200 or 1,000 mg/kg/day. In addition, the levator anibulbocavernosus muscle weights were also significantly reduced at BP-3 doses of 40, 200, or 1,000 mg/kg/day. In the MTT assay, the viability of NIH-3T3 mouse fibroblast cells was within the normal range. However, the TM3 mouse testis Leydig cell viability was significantly lowered in a concentration-dependent manner. Therefore, data indicate that BP-3 might exert in vivo anti-androgenic and in vitro cytotoxic effects in cells associated with the male reproductive system compared to normal non-reproductive cells.Abbreviation: BP-3: benzophenone-3; CG: Cowper's gland; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; GP: glans penis; LABC: levator anibulbocavernosus muscle; MTT: methyl tetrazolium; NC: negative control; PC: positive control; SV: seminal vesicle; TP: testosterone propionate; VC: vehicle control; VP: ventral prostate.

Effects of Low-Intensity Pulsed Ultrasound on the Regulation of Free Fatty Acid Release in 3T3-L1 Cells.

OBJECTIVES: To investigate the effects of low-intensity pulsed ultrasound (LIPUS) on the proliferation, differentiation, and tumor necrosis factor-α (TNF-α)-induced lipolysis of 3T3-L1 cells, and to explore the feasibility of regulating the release of free fatty acids (FFA) to prevent lipotoxicity. METHODS: Different intensities (30, 60, 90, and 120 mW/cm2) of LIPUS were applied to 3T3-L1 preadipocytes for different durations (5, 10, 15, 20, 25, and 30 minutes). Appropriate parameters for subsequent experiments were selected by assessing cell viability. The effect of LIPUS on the proliferation and differentiation of 3T3-L1 cells was evaluated by microscope observation, flow cytometry, and lipid content determination. After treated with LIPUS and TNF-α (50 ng/mL), the degree of lipolysis was assessed by measuring the extracellular FFA content. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression of relevant genes. RESULTS: Different parameters of LIPUS significantly enhance the viability of 3T3-L1 cells (P < .05), with 20 minutes and 30 mW/cm2 as the most suitable settings. After LIPUS treatment, 3T3-L1 cell proliferation accelerated, apoptosis rate and G1 phase cell proportion decreased, the content of lipid droplets and TG was increased in differentiated cells, while FFA release decreased (P < .05). The expression of PCNA, PPARγ, C/EBPα, Perilipin A mRNA increased, and the expression of TNF-α, ATGL, HSL mRNA decreased (P < .05). CONCLUSIONS: LIPUS could promote the proliferation and differentiation of 3T3-L1 cells and inhibit TNF-α-induced lipolysis, indicating its potential as a therapy for mitigating lipotoxicity caused by decompensated adipocytes.