Effects of diisononyl phthalate on lipid metabolism in HepG2 cells / 环境与职业医学

Background Exposure to diisononyl phthalate (DINP), an endocrine disruptor associated with metabolic diseases and widely used in plastic products, has been linked to the development of several adverse health outcomes in the liver, including non-alcoholic fatty liver disease (NAFLD). Objective To investigate the effects and the possible molecular mechanisms of DINP exposure on lipid metabolism in human hepatocellular carcinoma cells (HepG2 cells). Methods First, HepG2 cells were treated with DINP at three time spots (24, 48, and 72 h) and eleven doses (0, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 mmol·L−1). Cell viability were detected using cell counting kit 8 (CCK8). Intracellular lipid deposition was determined by oil red O staining and lipid content detection, and triglyceride (TG) and cholesterol (TC) were further detected. Finally, the mRNA expression levels were detected by fluorescence quantitative PCR, including fatty acid synthesis related genes [acetyl-CoA carboxylase alpha (Accα), fatty acid synthase (Fasn), malonyl-CoA decarboxylase (Mlycd), and sterol regulatory element binding protein 1 (Srebp1)] and β-oxidation related genes [peroxisome proliferator activated receptor alpha (Pparα), AMP-activated protein kinase (Ampk), carnitine palmitoyltransferase 1A (Cpt-1a), transcription factor A, mitochondrial (Tfam), nuclear respiratory factor 1 (Nrf1), and peroxisome proliferator-activated receptor gamma and coactivator 1 alpha (Pgc1-α)]. Results Compared with the control group (0 mmol·L−1), the no observed adverse effect levels (NOAEL) of HepG2 cell viability were 0.3, 0.1, and 0.1 mmol·L−1 after 24, 48, and 72 h exposure to DINP, respectively, and the corresponding lowest observed adverse effect levels (LOAEL) were 1, 0.3, and 0.3 mmol·L−1, respectively (P<0.05). After exposure to 30 mmol·L−1 and 100 mmol·L−1 DINP for 24 h, the intracellular lipid content, lipid deposition, TG, and TC levels were increased significantly compared with the control group (P<0.01). Compared with the control group, the mRNA expression levels of genes related to fatty acid synthesis, such as Mlycd, Srebp1, Fasn, and Accα, were down-regulated after the 100 mmol·L−1 DINP exposure for 24 h, while the mRNA expression level of Mlycd was up-regulated in the 30 mmol·L−1 group. The β-oxidation related genes such as Ampk, Pparα, and Tfam were up-regulated significantly after the 100 mmol·L−1 DINP exposure, while Cpt-1a mRNA expression level was down-regulated (P<0.05). Conclusion Exposure to DINP at 30 mmol·L−1 and 100 mmol·L−1 can interfere with fatty acid synthesis and β-oxidation in lipid metabolism of HepG2 cells, resulting in lipid deposition.

Total Saponins of Dioscorea Mitigate Nonalcoholic Steatohepatitis in Mice by Regulating AMPK/SREBP-1c/ACC Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the role and mechanism of total saponins of Dioscorea （TSD） in mitigating nonalcoholic steatohepatitis （NASH） in mice. MethodForty-eight C57BL/6J mice were randomized into a normal group and a modeling group. The mice for modeling were fed with a high-fat and high-cholesterol diet + 20% fructose solution for 16 weeks and randomized into model， atorvastatin （4 mg·kg-1·d-1）， and high-， medium-， and low-dose （200， 60， and 20 mg·kg-1·d-1） TSD groups. The mice were administrated with corresponding doses of drugs by gavage for 8 weeks. The mouse activity， liver index， levels of total cholesterol （TC）， triglycerides （TG）， and free fatty acids （FFAs） in the liver， and levels of TC， TG， aspartate aminotransferase （AST）， alanine aminotransferase （ALT）， γ-glutamyl transferase （GGT）， interleukin-1β （IL-1β）， and tumor necrosis factor-α （TNF-α） in the serum were measured. Hematoxylin-eosin staining， Masson staining， oil red O staining， and transmission electron microscopy were employed to observe the pathological changes， lipid accumulation， and morphological changes of liver ultrastructure. Western blot was employed to determine the protein levels of AMP-activated protein kinase （AMPK）， sterol regulatory element-binding protein-1c （SREBP-1c）， acetyl-CoA carboxylase （ACC）， and phosphorylated ACC （p-ACC） in the liver tissue. ResultCompared with the normal group, the activity of mice in the model group decreased(P<0.05， P<0.01), the levels of TC, TG, FFA and serum TC, TG, ALT, AST, GGT, IL-1β and TNF-α, liver coefficient and liver pathology scores were significantly increased, the expression of p-AMPK/AMPK and p-ACC proteins in liver tissues was significantly reduced, and the expressions of SREBP-1c and ACC proteins were significantly increased (P<0.01). Compared with the model group， atorvastatin increased the mouse activity （P<0.05）， while each dose of TSD caused no significant changed in the mouse activity. The levels of TC, TG, FFA in liver and serum TC, TG, ALT, AST, GGT, IL-1β, TNF-α, liver coefficient and liver pathological score in TSD and atorvastatin groups were significantly decreased, and the expressions of p-AMPK/AMPK and p-ACC in liver tissue were significantly increased. The expressions of SREBP-1c and ACC were significantly decreased (P<0.05,P<0.01). ConclusionTSD may alleviate NASH in mice by regulating the AMPK/SREBP-1c/ACC signaling pathway to reduce lipid synthesis.

Effects of AR on proliferation and lipid synthesis of myocardial fibroblasts in high glucose environment / 中国药理学通报

Aim To explore the effect of androgen receptor AR on the proliferation and lipid synthesis of cardiac fibroblasts under high-glucose conditions and the possible molecular mechanism.Methods The hearts of neonatal rats were dissected for primary culture of cardiac fibroblasts. Then the growth status of CFs was observed under the inverted microscope, and the identification of CFs was performed by immunofluorescence staining using anti-vimentin. After cell adherence, the cells were divided into blank control group, high glucose model group, negative control group, and overexpressed AR group. The glucose concentration was 33.0 mmol·L-1 except that the blank control group was 5.5 mmol·L-1. After 24 hours of CFs culture, Western blot and RT-qPCR were used to detect the expression of AR, FASN, PCNA, cyclin D1, α-SMA, and collagen . Oil red O and CCK-8 were used to detect the changes in lipid synthesis and cell proliferation ability, respectively.Results Compared with the blank control group, the lipid synthesis and proliferation of CFs in the high glucose model group were enhanced. Western blot and RT-qPCR results showed that the expression of AR decreased, while the expression of fat lipid synthase(FASN), proliferation marker PCNA, cyclin D1 and fibrosis marker α-SMA and collagen increased. After AR overexpressed plasmid was transfected into the CFs treated by high glucose, AR overexpression markedly decreased the expression of FASN, PCNA, cyclin D1, α-SMA and collagen compared with the empty plasmid‐transfected group. Meanwhile, oil red O staining and CCK-8 results showed that the lipid synthesis and proliferation ability of the overexpressed AR group decreased compared with the empty vector group, respectively. Conclusions High glucose promotes the proliferation and lipid synthesis of cardiac fibroblasts. Besides, the mechanism may be related to the regulation of lipid synthesis regulated by AR.

Diosgenin alleviates NAFLD induced by a high-fat diet in rats via mTOR/SREBP-1c/HSP60/MCAD/SCAD signaling pathway / 中国中药杂志

This study aims to observe the effects of diosgenin on the expression of mammalian target of rapamycin(mTOR), sterol regulatory element-binding protein-1c(SREBP-1c), heat shock protein 60(HSP60), medium-chain acyl-CoA dehydrogenase(MCAD), and short-chain acyl-CoA dehydrogenase(SCAD) in the liver tissue of the rat model of non-alcoholic fatty liver disease(NAFLD) and explore the mechanism of diosgenin in alleviating NAFLD. Forty male SD rats were randomized into five groups: a control group, a model group, low-(150 mg·kg~(-1)·d~(-1)) and high-dose(300 mg·kg~(-1)·d~(-1)) diosgenin groups, and a simvastatin(4 mg·kg~(-1)·d~(-1)) group. The rats in the control group were fed with a normal diet, while those in the other four groups were fed with a high-fat diet. After feeding for 8 weeks, the body weight of rats in the high-fat diet groups increased significantly. After that, the rats were administrated with the corresponding dose of diosgenin or simvastatin by gavage every day for 8 weeks. The levels of triglyceride(TG), total cholesterol(TC), alanine transaminase(ALT), and aspartate transaminase(AST) in the serum were determined by the biochemical method. The levels of TG and TC in the liver were measured by the enzyme method. Oil-red O staining was employed to detect the lipid accumulation, and hematoxylin-eosin(HE) staining to detect the pathological changes in the liver tissue. The mRNA and protein levels of mTOR, SREBP-1c, HSP60, MCAD, and SCAD in the liver tissue of rats were determined by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) and Western blot, respectively. Compared with the control group, the model group showed increased body weight, food uptake, liver index, TG, TC, ALT, and AST levels in the serum, TG and TC levels in the liver, lipid deposition in the liver, obvious hepatic steatosis, up-regulated mRNA and protein expression levels of mTOR and SREBP-1c, and down-regulated mRNA and protein expression levels of HSP60, MCAD, and SCAD. Compared with the model group, the rats in each treatment group showed obviously decreased body weight, food uptake, liver index, TG, TC, ALT, and AST levels in the serum, TG and TC levels in the liver, lessened lipid deposition in the liver, ameliorated hepatic steatosis, down-regulated mRNA and protein le-vels of mTOR and SREBP-1c, and up-regulated mRNA and protein levels of HSP60, MCAD, and SCAD. The high-dose diosgenin outperformed the low-dose diosgenin and simvastatin. Diosgenin may prevent and treat NAFLD by inhibiting the expression of mTOR and SREBP-1c and promoting the expression of HSP60, MCAD, and SCAD to reduce lipid synthesis, improving mitochondrial function, and promoting fatty acid β oxidation in the liver.

Metformin regulates AMPK/SREBP-1 pathway and its clinical application / 中国临床药理学与治疗学

Metformin is one of the commonly used hypoglycemic drugs in clinical practice. In addition to hypoglycemia, there are a variety of medical biological values that have been constantly discovered and attracted much attention. In recent years, studies have shown that metformin through activation of AMPK inhibition of sterols regulating element binding protein 1 (SREBP-1) reduce lipid synthesis, in the treatment of liver steatosis, improve insulin sensitivity, prevention Metformin is one of the commonly used hypoglycemic drugs in clinical practice. In addition to hypoglycemia, there are a variety of medical biological values that have been constantly discovered and attracted much attention. In recent years, studies have shown that metformin through activation of AMPK inhibition of sterols regulating element binding protein 1 (SREBP-1) reduce lipid synthesis, in the treatment of liver steatosis, improve insulin sensitivity, prevention of atherosclerosis and cardiovascular dysfunction, tumor, polycystic ovary syndrome and adjuvant therapy of COVID-19 aspects play a role. Therefore, this article reviews the possible mechanism and clinical application of metformin in regulating glucose and lipid metabolism by inhibiting SREBP-1 through activating AMPK.

Effect and mechanism of muscle injury on liver lipid metabolism in Duchenne muscular dystrophy mice / 中国药科大学学报

@#Duchene muscular dystrophy (DMD) is a serious progressive muscular dystrophy.Reports in recent years about abnormal lipid in DMD patients have increased, yet little attention has been paid to liver lipid.This study aimed to explore the effect of dystrophin gene defect on liver lipid synthesis.7-week-old mdx male mice were used as DMD model.The conditions of liver function, liver lipid accumulation and liver lipid synthesis were determined through liver tissue morphological examination, blood biochemical examination, and detection of hepatic gene and protein expression.The results showed that lipid droplets in liver of mdx mice increased significantly.The contents of total cholesterol and triglyceride in liver, aspartate aminotransferase and alanine aminotransferase in serum increased.The gene and protein expression of hepatic lipid synthesis-related enzymes such as fatty acid synthase, acetyl CoA carboxylase, and sterol regulatory element binding protein 1-c were up-regulated.These results showed accumulation of liver lipid in 7-week-old mdx male mice.

Dihydromyricin inhibited hepatic lipid deposition induced by high-fat diet in obese mice by activating SIRT1-AMPK pathway / 中国药理学通报

Aim To investigate the effect of dihydromyricetin (DHM) on lipid accumulation in liver of obese mice induced by high fat diet and its mechanism. Methods Sixty C57BL/6J mices were randomly divided into six groups (n = 10); (1)ND group; normal diet, (2)ND + L-DHM group; normal diet and treatment with low-dose DHM (125 mg • kg

Central cellular signaling pathways involved with the regulation of lipid metabolism in the liver: a review

The liver is primarilyresponsible for energy homeostasis and the regulation of lipid, carbohydrate and protein metabolism. Lipid metabolism consists of distributing lipids to peripheral tissues or ensuring their return to the liver to be reprocessed. Additionally, cellular metabolism isregulated by several molecules in different signaling pathways. Lipid homeostasis in the liver is mainly regulated by AKT, AMPK, SREBP, PPAR, and JNK. The PI3K/AKT/mTOR signaling pathway results in the biosynthesis of macromolecules and regulates lipogenesis and the expression of lipogenic genes. AMPK is an energy sensor that regulates metabolism and is activated when stored ATP is depleted, and it is responsible for the suppression of several key lipogenic factors in the liver related to cholesterol and fatty acid synthesis. SREBPs control lipogenic geneexpressionandcholesterol metabolism and actin the nutritional regulation of fatty acids and triglycerides. The continued activation of SREBPs is associated with cellular stress, inflammation and ultimately steatosis. PPARs are intrinsically important regulators of lipid metabolism. These genes are essential tovarious metabolic processes, especially lipid and glucose homeostasis, and can play a role in cell differentiation. JNK signaling is related to insulin resistance and its activation results in decreased mitochondrial activity and fat accumulation. Therefore, the study of cell signaling pathways related to lipid metabolism and liver function may help to identify abnormalities and develop strategies to manage and regulate metabolic disorders and resulting complications.

The role of endoplasmic reticulum stress in the formation of fatty liver in high-fat diet fed mice / 中国医师杂志

Objective To explore the influence of endoplasmic reticulum stress on fatty liver in mice feeding with high-fat diet.Methods The 8-week-old male C57BL/6J mice were randomly divided into two groups:high-fat diet group (with 60％ calories by high saturated fatty acid) and control group (with chow diet),both groups had been fed for 16 weeks.H&E-staining and Sudan Ⅳ-staining reflected lipid deposition in liver.The levels of 78-kDa glucose-regulated protein (GRP78),protein kinase R-like endoplasmic reticulum kinase (PERK),phosphorylated α subunit of eukaryotic initiation factor 2 (p-eIF1 α),C/EBP homologous protein (CHOP),steroid regulated element binding proteins 1 (SREBP-1),and fatty acid synthetase (FAS) protein were determined by Western blot to reflect the endoplasmic reticulum stress and lipid synthesis.Results In liver of high fat diet (HFD) group,H&E staining showed that the cytoplasm of hepatocytes were filled with vacuoles,Sudan Ⅳ staining also displayed that many different sizes of red lipid drops exist in hepatocytes.Compared to the liver of control group,high-fat diet induced endoplasmic reticulum stress and elevated lipid synthesis,as evidenced by increases in the level of peroxisome proliferator-activated receptor alpha (PPARα) mRNA expression,and the protein levels of GRP78,PERK,phosphorylated eIF2α,CHOP were also significantly increased.In primary normal hepatocytes incubated with exogenous oleic acid intervention for 24-72 hours,the expression of GRP78,PERK,phosphorylated eIF2α,CHOP protein levels,and the expression of SREBP-1 and FAS protein were significantly increased in dose-dependent manner.Conclusions Feeding with high-fat diet led to accumulation of lipid deposition in liver and fatty liver,the underlying mechanisms might be related to induction of endoplasmic reticulum stress.

Construction of a new alcoholic liver disease mouse model / 上海交通大学学报（医学版）

Objective·To establish a reliable alcoholic liver disease mouse model (ALDNM) that mimics the drinking pattern of alcoholic liver disease (ALD) patients.Methods·Using the self-designed feeding tubes and liquid diet,ALDNM model was developed through chronic feeding combined with acute gavage of ethanol based on Lieber-DeCarli model and Gao-Binge model.C57BL/6 mice were administered with control liquid diet for adaptation for first 5 d,and then divided into pair-fed group and ethanol-fed group (10 mice each group).Ethanol-fed mice were fed with the liquid diet in which ethanol accounts for 30％ of total energy,while the pair-fed mice were fed with the control diet for 10 d.At the 16th day,ethanol-fed mice and pair-fed mice were respectively gavaged a single dose of 31.5％ ethanol or isocaloric maltose dextrin,and euthanized 9 h later.Sera and livers were collected.The general physiological condition,hepatic tissue pathological changes and serum indexes between Lieber-DeCarli models and ALDNM models were compared.The liver lipids of ALDNM mice were determined by Oil red O (ORO) staining and hepatic triacylglyceride (TAG) test.Meanwhile,the mRNA levels of interleukin-6 (IL-6),tumor necrosis factor α (TNF-α),fatty acid synthase (Fas),long chain fatty acid elongase 6 (Elovl6) and stearyl-CoA desaturase (Scdl) were detected by real-time PCR in ALDNM models.Western blotting was used to detect the changes of phosphorylated signal transduction and transcriptional activator (p-STAT3) in the livers.Results·Lieber-DeCarli model mice were generally in poor condition,and there was no significant change in serum glutamic-pyruvic transaminase (GPT) and glutamic-oxaloacetic transaminase (GOT) compared to pair-fed group.However,in ALDNM models,H-E staining showed that the hepatocytes of ethanol-fed mice were extremely swollen with round volume,increased cytoplasm and filled with large amounts of fat vacuoles.ORO staining analyses showed obvious microsteatosis in the liver cells from all ethanol-fed mice.The hepatosomatic index,liver TAG content,serum GPT and GOT of ALDNM models were significantly higher than those in the pair-fed group,while the serum HDL significantly decreased compared to the pair-fed group.Moreover,the expression levels of both lipid synthesis pathways and inflammatory signaling pathways related genes in livers significantly increased in the ethanol-fed mice of ALDNM model.Conclusion·ALDNM model was successfully constructed.This model is cost-and time-efficient.Moreover,ALDNM model mimics the drinking pattern and pathogenesis of ALD patients with the advantages of stable food intake,good repeatability,and obvious liver damage.