OBJECTIVE: To Investigate the effects of lithocholic acid (LCA) on the balance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). METHODS: Twelve 10-week-old SPF C57BL/6J female mice were randomly divided into an experimental group (undergoing bilateral ovariectomy) and a control group (only removing the same volume of adipose tissue around the ovaries), with 6 mice in each group. The body mass was measured every week after operation. After 4 weeks post-surgery, the weight of mouse uterus was measured, femur specimens of the mice were taken for micro-CT scanning and three-dimensional reconstruction to analyze changes in bone mass. Tibia specimens were taken for HE staining to calculate the number and area of bone marrow adipocytes in the marrow cavity area. ELISA was used to detect the expression of bone turnover markers in the serum. Liver samples were subjected to real-time fluorescence quantitative PCR (RT-qPCR) to detect the expression of key genes related to bile acid metabolism, including cyp7a1, cyp7b1, cyp8b1, and cyp27a1. BMSCs were isolated by centrifugation from 2 C57BL/6J female mice (10-week-old). The third-generation cells were exposed to 0, 1, 10, and 100 μmol/L LCA, following which cell viability was evaluated using the cell counting kit 8 assay. Subsequently, alkaline phosphatase (ALP) staining and oil red O staining were conducted after 7 days of osteogenic and adipogenic induction. RT-qPCR was employed to analyze the expressions of osteogenic-related genes, namely ALP, Runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), as well as adipogenic-related genes including Adiponectin (Adipoq), fatty acid binding protein 4 (FABP4), and peroxisome proliferator-activated receptor γ (PPARγ). RESULTS: Compared with the control group, the body mass of the mice in the experimental group increased, the uterus atrophied, the bone mass decreased, the bone marrow fat expanded, and the bone metabolism showed a high bone turnover state. RT-qPCR showed that the expressions of cyp7a1, cyp8b1, and cyp27a1, which were related to the key enzymes of bile acid metabolism in the liver, decreased significantly ( P<0.05), while the expression of cyp7b1 had no significant difference ( P>0.05). Intervention with LCA at concentrations of 1, 10, and 100 μmol/L did not demonstrate any apparent toxic effects on BMSCs. Furthermore, LCA inhibited the expressions of osteogenic-related genes (ALP, Runx2, and OCN) in a dose-dependent manner, resulting in a reduction in ALP staining positive area. Concurrently, LCA promoted the expressions of adipogenic-related genes (Adipoq, FABP4, and PPARγ), and an increase in oil red O staining positive area. CONCLUSION After menopause, the metabolism of bile acids is altered, and secondary bile acid LCA interferes with the balance of osteogenic and adipogenic differentiation of BMSCs, thereby affecting bone remodelling.
Female ; Mice ; Animals ; Core Binding Factor Alpha 1 Subunit/pharmacology* ; PPAR gamma/metabolism* ; Steroid 12-alpha-Hydroxylase/metabolism* ; Mice, Inbred C57BL ; Cell Differentiation ; Osteogenesis ; Mesenchymal Stem Cells ; Bile Acids and Salts/pharmacology* ; Bone Marrow Cells ; Cells, Cultured ; Azo Compounds

Humans ; PPAR gamma/metabolism* ; Multiple Sclerosis ; Transcription Factors/metabolism* ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha

This study investigated the mechanism of Zexie Decoction(ZXD) in promoting white adipose tissue browning/brown adipose tissue activation based on the GLP-1R/cAMP/PKA/CREB pathway. A hyperlipidemia model was induced by a western diet(WD) in mice, and the mice were divided into a control group, a model group(WD), and low-, medium-, and high-dose ZXD groups. An adipogenesis model was induced in 3T3-L1 cells in vitro, and with forskolin(FSK) used as a positive control, low-, medium-, and high-dose ZXD groups were set up. Immunohistochemistry and immunofluorescence results showed that compared with the WD group, ZXD promoted the expression of UCP1 in white and brown adipose tissues, and also upregulated UCP1, CPT1β, PPARα, and other genes in the cells. Western blot analysis showed a dose-dependent increase in the protein expression of PGC-1α, UCP1, and PPARα with ZXD treatment, indicating that ZXD could promote the white adipose tissue browning/brown adipose tissue activation. Hematoxylin-eosin(HE) staining results showed that after ZXD treatment, white and brown adipocytes were significantly reduced in size, and the mRNA expression of ATGL, HSL, MGL, and PLIN1 was significantly upregulated as compared with the results in the WD group. Oil red O staining and biochemical assays indicated that ZXD improved lipid accumulation and promoted lipolysis. Immunohistochemistry and immunofluorescence staining for p-CREB revealed that ZXD reversed the decreased expression of p-CREB caused by WD. In vitro intervention with ZXD increased the protein expression of CREB, p-CREB, and p-PKA substrate, and increased the mRNA level of CREB. ELISA detected an increase in intracellular cAMP concentration with ZXD treatment. Molecular docking analysis showed that multiple active components in Alismatis Rhizoma and Atractylodis Macrocephalae Rhizoma could form stable hydrogen bond interactions with GLP-1R. In conclusion, ZXD promotes white adipose tissue browning/brown adipose tissue activation both in vivo and in vitro, and its mechanism of action may be related to the GLP-1R/cAMP/PKA/CREB pathway.
Mice ; Animals ; Adipose Tissue, Brown ; Molecular Docking Simulation ; PPAR alpha/metabolism* ; Adipose Tissue, White ; RNA, Messenger/metabolism*

Objective To investigate the effects of long noncoding RNA H19 on lipid accumulation of macrophages under high fat stress and its mechanism. Methods Human THP-1 cells-derived macrophages were incubated with ox-LDL, and the effects of H19 siRNA intervention on lipid accumulation was observed. The THP-1 cells were divided into control group (conventional culture), ox-LDL group, siRNA negative control (NC siRNA) combined with ox-LDL treatment group, and H19 siRNA combined with ox-LDL treatment group. Oil red O staining was used to determine the lipid accumulation in cells, and cholesterol concentration was analyzed by enzymatic method; ATP assay kit for detecting celluar ATP content; colorimetry was used to detect the levels of oxidative stress indicators and ELISA was used to detect the levels of monocyte chemoattractant protein-1 (MCP-1) in the cell supernatant. Western blot analysis was used to detect the protein expression of ATP binding cassette transporter A1 (ABCA1), peroxisome proliferator-activated receptor α (PPARα), peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and nuclear factor κB p-p65 (NF-κB p-p65). Results Knockdown H19 significantly inhibited intracellular lipid accumulation, decreased total cholesterol (TC) and cholesterol ester (CE) content, and decreased CE/TC ratio. Knockdown H19 significantly alleviated cell damage including an increase in ATP content, a decrease in oxidative stress levels and a decrease in MCP-1 levels, which caused by high-fat stress. H19 siRNA upregulated expression of ABCA1, PPARα and PGC-1α in THP-1 derived macrophages, downregulated NF-κB signal pathway. Conclusion Knockdown H19 upregulates PGC-1α expression in THP-1 cells and downregulates NF-κB pathway, which promotes cholesterol reverse transport, reduces inflammatory reaction and inhibits lipid accumulation.
Humans ; Adenosine Triphosphate ; Cholesterol ; NF-kappa B ; PPAR alpha ; RNA, Long Noncoding/genetics* ; RNA, Small Interfering/genetics* ; THP-1 Cells ; Macrophages/metabolism* ; Lipid Metabolism

The aim of the present study was to investigate the effects of short-term ketogenic diet on the low temperature tolerance of mice and the involvement of peroxisome proliferator-activated receptor α (PPARα). C57BL/6J mice were divided into two groups: normal diet (WT+ND) group and ketogenic diet (WT+KD) group. After being fed with normal or ketogenic diet at room temperature for 2 d, the mice were exposed to 4 °C low temperature for 12 h. The changes in core temperature, blood glucose, blood pressure of mice under low temperature condition were detected, and the protein expression levels of PPARα and mitochondrial uncoupling protein 1 (UCP1) were detected by Western blot. PPARα knockout mice were divided into normal diet (PPARα^-/-+ND) group and ketogenic diet (PPARα^-/-+KD) group. After being fed with the normal or ketogenic diet at room temperature for 2 d, the mice were exposed to 4 °C low temperature for 12 h. The above indicators were also detected. The results showed that, at room temperature, the protein expression levels of PPARα and UCP1 in liver and brown adipose tissue of WT+KD group were significantly up-regulated, compared with those of WT+ND group. Under low temperature condition, compared with WT+ND, the core temperature and blood glucose of WT+KD group were increased, while mean arterial pressure was decreased; The ketogenic diet up-regulated PPARα protein expression in brown adipose tissue, as well as UCP1 protein expression in liver and brown adipose tissue of WT+KD group. Under low temperature condition, compared to WT+ND group, PPARα^-/-+ND group exhibited decreased core temperature and down-regulated PPARα and UCP1 protein expression levels in liver, skeletal muscle, white and brown adipose tissue. Compared to the PPARα^-/-+ND group, the PPARα^-/-+KD group exhibited decreased core temperature and did not show any difference in the protein expression of UCP1 in liver, skeletal muscle, white and brown adipose tissue. These results suggest that the ketogenic diet promotes UCP1 expression by up-regulating PPARα, thus improving low temperature tolerance of mice. Therefore, short-term ketogenic diet can be used as a potential intervention to improve the low temperature tolerance.
Animals ; Mice ; Adipose Tissue, Brown/metabolism* ; PPAR alpha/pharmacology* ; Diet, Ketogenic ; Uncoupling Protein 1/metabolism* ; Blood Glucose/metabolism* ; Temperature ; Mice, Inbred C57BL ; Liver ; Adipose Tissue/metabolism*

The aim of this study was to investigate the effect of activating transcription factor 3 (ATF3) on the differentiation of intramuscular preadipocytes in goat, and to elucidate its possible action pathway at the molecular level. In this study, the recombinant plasmid of goat pEGFP-N1-ATF3 was constructed, and the intramuscular preadipocytes were transfected with liposomes. The relative expression levels of adipocyte differentiation marker genes were detected by quantitative real-time PCR (qRT-PCR). After transfection of goat intramuscular preadipocytes with the goat pEGFP-N1-ATF3 overexpression vector, it was found that the accumulation of lipid droplets was inhibited, and the adipocyte differentiation markers PPARγ, C/EBPα and SREBP1 were extremely significantly down-regulated (P < 0.01), while C/EBPβ and AP2 were significantly down-regulated (P < 0.05). The ATF3 binding sites were predicted to exist in the promoter regions of PPARγ, C/EBPα and AP2 by the ALGGEN PROMO program. The overexpression of goat ATF3 inhibits the accumulation of lipid droplets in intramuscular preadipocytes, and this effect may be achieved by down-regulating PPARγ, C/EBPα and AP2. These results may facilitate elucidation of the regulatory mechanism of ATF3 in regulating the differentiation of goat intramuscular preadipocytes.
3T3-L1 Cells ; Activating Transcription Factor 3/pharmacology* ; Adipocytes ; Adipogenesis/genetics* ; Animals ; CCAAT-Enhancer-Binding Protein-alpha/pharmacology* ; Cell Differentiation ; Goats ; Mice ; PPAR gamma/metabolism*

Tanshinone Ⅱ_A( Tan Ⅱ_A),the liposoluble constituents of Salvia miltiorrhiza,can not only ameliorate the lipidic metabolism and decrease the concentration of lipid peroxidation,but also resist oxidation damage,scavenge free radicals and control inflammation,with a protective effect on prognosis after liver function impairment. Therefore,the studies on the exact mechanism of Tan Ⅱ_A in protecting the liver can provide important theoretical and experimental basis for the prevention and treatment effect of Tan Ⅱ_A for liver injury. In the present study,the protective effects and mechanism of Tan Ⅱ_A on 4-hydroxynonenal( 4-HNE)-induced liver injury were investigated in vitro. Normal liver tissues NCTC 1469 cells were used to induce hepatocytes oxidative damages by 4-HNE treatment. The protective effect of Tan Ⅱ_A on hepatocytes oxidative damages was detected by release amount of lactate dehydrogenase( LDH) analysis and hoechst staining. The protein expression changes of peroxisome proliferator-activated receptor α( PPARα) and peroxisome proliferator response element( PPRE) were analyzed by Western blot analysis in NCTC 1469 cells before and after Tan Ⅱ_A treatment. The gene expression changes of fatty aldehyde dehydrogenase( FALDH) were analyzed by Real-time polymerase chain reaction( PCR) analysis. The results showed that 4-HNE increased the release amount of LDH,lowered the cell viability of NCTC 1469 cells,and Tan Ⅱ_A reversed 4-HNE-induced hepatocyte damage. Western blot analysis and RT-PCR analysis results showed that 4-HNE decreased the expression of PPARα and FALDH and increased the expression of 4-HNE. However,the expression of PPARα and FALDH were increased significantly and the expression of 4-HNE was decreased obviously after Tan Ⅱ_A treatment. This study confirmed that the curative effect of Tan Ⅱ_A was obvious on hepatocytes damage,and the mechanism may be associated with activating PPARα and FALDH expression as well as scavenging 4-HNE.
Aldehyde Oxidoreductases ; metabolism ; Aldehydes ; Animals ; Cell Line ; Diterpenes, Abietane ; pharmacology ; Hepatocytes ; drug effects ; Lipid Peroxidation ; Mice ; Oxidative Stress ; PPAR alpha ; metabolism

The plasticizer di(2-ethylhexyl) phthalate (DEHP) has been widely used in the manufacture of polyvinyl chloride-containing products such as medical and consumer goods. Humans can easily be exposed to it because DEHP is ubiquitous in the environment. Recent research on the adverse effects of DEHP has focused on reproductive and developmental toxicity in rodents and/or humans. DEHP is a representative of the peroxisome proliferators. Therefore, peroxisome proliferator-activated receptor alpha (PPARα)-dependent pathways are the expected mode of action of several kinds of DEHP-induced toxicities. In this review, we summarize DEHP kinetics and its mechanisms of carcinogenicity and reproductive and developmental toxicity in relation to PPARα. Additionally, we give an overview of the impacts of science policy on exposure sources.
Animals ; Diethylhexyl Phthalate ; toxicity ; Environmental Pollutants ; toxicity ; Haplorhini ; Humans ; Mice ; PPAR alpha ; genetics ; metabolism ; Plasticizers ; toxicity ; Rats

Although elevated serum low-density lipoprotein-cholesterol (LDL-C) is without any doubts accepted as an important risk factor for cardiovascular disease (CVD), the role of elevated triglycerides (TGs)-rich lipoproteins as an independent risk factor has until recently been quite controversial. Recent data strongly suggest that elevated TG-rich lipoproteins are an independent risk factor for CVD and that therapeutic targeting of them could possibly provide further benefit in reducing CVD morbidity, events and mortality, apart from LDL-C lowering. Today elevated TGs are treated with lifestyle interventions, and with fibrates which could be combined with omega-3 fatty acids. There are also some new drugs. Volanesorsen, is an antisense oligonucleotid that inhibits the production of the Apo C-III which is crucial in regulating TGs metabolism because it inhibits lipoprotein lipase (LPL) and hepatic lipase activity but also hepatic uptake of TGs-rich particles. Evinacumab is a monoclonal antibody against angiopoietin-like protein 3 (ANGPTL3) and it seems that it can substantially lower elevated TGs levels because ANGPTL3 also regulates TGs metabolism. Pemafibrate is a selective peroxisome proliferator-activated receptor alpha modulator which also decreases TGs, and improves other lipid parameters. It seems that it also has some other possible antiatherogenic effects. Alipogene tiparvovec is a nonreplicating adeno-associated viral vector that delivers copies of the LPL gene to muscle tissue which accelerates the clearance of TG-rich lipoproteins thus decreasing extremely high TGs levels. Pradigastat is a novel diacylglycerol acyltransferase 1 inhibitor which substantially reduces extremely high TGs levels and appears to be promising in treatment of the rare familial chylomicronemia syndrome.
Apolipoprotein C-III ; Cardiovascular Diseases ; Diacylglycerol O-Acyltransferase ; Fatty Acids, Omega-3 ; Fibric Acids ; Hyperlipoproteinemia Type I ; Life Style ; Lipase ; Lipoprotein Lipase ; Lipoproteins ; Metabolism ; Mortality ; PPAR alpha ; Risk Factors ; Triglycerides

OBJECTIVETo investigate the roles of microRNA-21 (miR-21) in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) with high-fat diet-induced insulin resistance (IR) and diabetes mellitus (DM) mice model.

METHODSEight-week-old C57BL/6 mice were allocated into control group, IR group, and DM group. Body mass was recorded. Intraperitoneal glucose tolerance test was performed to determine any abnormal glucose metabolism. The liver pathological changes were detected by biopsy. Changes in free blood glucose, free serum insulin, blood fat and tumor necrosis factor &Alpha; level were measured. Differences in miR-21 expression and peroxidase proliferator-activated receptor subtypes (PPAR-Γ and PPAR-&Alpha;) and adipocyte fatty acid binding protein (aP2) in the liver were detected both at the mRNA and protein levels.

RESULTSAfter one 8-week high-fat diet, the body mass, free serum insulin, and homeostasis model IR index significantly increased in the IR group (P<0.01, P<0.05, compared with control group), while the free blood glucose increased and the free serum insulin decreased in DM group (P<0.05). Free serum insulin level were significantly increased in IR group (P<0.05). Serum tumor necrosis factor-&Alpha; levels exhibited an upward trend in control group, IR group, and DM group (P<0.05, P<0.01). With exacerbation in NAFLD, liver miR-21 expression level went further down in both IR and DM groups (P<0.05). The downregulated miR-21 expression level showed negative correlation with upregulated PPAR-&Alpha;, &Alpha;P2, and PPAR-Γ genetic expression (r=-0.696, r=-0.664, and r=-0.766, respectively; P<0.05) in IR group and with upregulated PPAR-&Alpha; and PPAR-Γ genetic expression in DM group (r=-0.676 and r=-0.550, respectively; P<0.05). In terms of the changes in protein expression level,only on the protein expressions of aP2 and PPAR-Γ in IR group showed significant change (P<0.05, P<0.01, compared with control group).

CONCLUSIONSThe miR-21 expression is downregulated in both IR and DM-induced NAFLD mice. It may be involved in the pathogenesis of NAFLD by regulating the expressions of PPAR subtypes.

Animals ; Blood Glucose ; analysis ; Diabetes Mellitus, Experimental ; physiopathology ; Diet, High-Fat ; Disease Models, Animal ; Insulin ; blood ; Insulin Resistance ; Mice ; Mice, Inbred C57BL ; MicroRNAs ; metabolism ; Non-alcoholic Fatty Liver Disease ; physiopathology ; PPAR alpha ; metabolism ; PPAR gamma ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism