Cogent evidence indicates a unique role of leptin in the promotion of liver fibrosis. Hepatic stellate cell (HSC) activation is a pivotal step in the process of liver fibrosis. Sterol regulatory element binding protein (SREBP)- 1c, a critical transcription factor for lipid synthesis and adipocyte differentiation, functions as a key transcription factor in inhibition of HSC activation. SREBP- 1c is highly expressed in quiescent HSCs and downregulated upon HSC activation. The aim of this study is to examine the effect of leptin on SREBP- 1c gene expression in HSCs in vitro and in vivo and elucidate the underlying mechanisms. The results of the present study demonstrated that leptin strongly inhibited SREBP- 1c expression in HSCs in vivo and in vitro. In Vivo Profiling of Estrogen Receptor/Specificity Protein-Dependent. In Vivo Profiling of Estrogen Receptor/Specificity Protein-Dependent.Leptin- induced activation of p. MAPK led to the decreases in liver X receptor (LXR)- . Moreover, leptin inhibition of SREBP- 1c expression via p. MAPK increased the expression of alpha. I) collagen in HSCs. Paraspeckle protein 1 alpha isoform (PSPC1, previously known as PSP1) and Splicing factor, proline- and. Diet-Induced Obesity in Male Mice Is Associated with Reduced Fertility and Potentiation of Acrylamide-Induced. A simple diet change can improve almost everything in your life. Eliminating Fatigue with These Five Superfoods! Eliminating Fatigue with These Five Superfoods! Our results might provide new insights into the mechanisms of the unique role of leptin in the development of liver fibrosis and might have potential implications for clarifying the molecular mechanisms underlying liver fibrosis in diseases in which circulating leptin levels are elevated such as nonalcoholic steatohepatitis, type 2 diabetes mellitus and alcoholic cirrhosis. INTRODUCTIONLeptin, the product of the obese gene, is mainly produced by adipocyte (1). It inhibits free fatty acid uptake and de novo fatty acid synthesis, stimulates free fatty acid mobilization and oxidation and reverses adipocyte differentiation (2,3). Accumulating evidence has indicated that leptin plays critical roles in the development of liver fibrosis in vivo and in vitro (4–7). Hepatic stellate cell (HSC) activation is a pivotal step in the development of liver fibrosis. Leptin can directly target HSCs via activation of its receptor (8) and stimulate HSC activation and fibrogenesis (8–1. PSP1 homologs have been identified from other. The constitutively expressed vanillic acid-sensitive olfactory G protein. A closed-loop synthetic gene circuit for the treatment of diet-induced obesity. P38 mitogen-activated protein kinase and liver X receptor. The mice were given free access to water and standard chow diet. Plasmid pdnP38, plas-mid pwtP38, plasmid pSP1, plasmid pSp1-Luc, plasmid pNFY-Luc. Peak Nutrition started off as a couple of guys selling supplements out of their car. Now Peak is a full time nutrition store with 6 St. Louis area locations. Isolation and Identification of a Plasmatocyte-spreading Peptide from the Hemolymph. Quiescent HSCs, once called fat- storing cells, are much like adipocytes. It is proposed that HSC transdifferentiation from quiescent to myofibroblastic cells is analogous to adipocyte to preadipocyte (fibroblast) transdifferentiation (1. Pre-work out; Protein; Super Stacks; Weight Gain. Forgotten your password? Home > Pre-work out Pre-work out. USN DIET FUEL ULTRALEAN 3. The major transcription factors for adipocyte differentiation include the CCAAT/enhanced binding protein family (C/EBP), peroxisome proliferator–activated receptor (PPAR)- . SREBP- 1 belongs to the SREBP family members and exists as two isoforms (SREBP- 1a and SREBP- 1c). SREBP- 1c (also known as adipocyte determination and differentiation- dependent factor 1) is a key transcription factor for the promotion of lipid synthesis and adipocyte differentiation (1. The ratio of SREBP- 1a to SREBP- 1c m. RNA is 1: 9 in liver, and SREBP- 1c is the predominant transcript in liver (1. A gain of function manipulation for transcription factors such as PPAR. Interestingly, the process of HSC activation is coupled with the sequential upregulation of leptin and downregulation of SREBP- 1c (1. Therefore, it is of interest to examine the relationship between leptin and the gene expression of SREBP- 1c and elucidate the underlying mechanisms in HSCs, which is still largely unknown. The aim of this study is to examine the role of leptin in SREBP- 1c expression in HSCs in vitro and in vivo and reveal the underlying mechanisms. MATERIALS AND METHODSMaterials Leptin was purchased from Pro. Spec- Tany Techno. Gene (Rehovot, Israel) and used to treat cells at 1. L for 2. 4 h (if no specific indication). SB2. 03. 58. 0 (a p. Thioacetamide (TAA), isobutylmethylxanthine, dexamethasone and insulin were all purchased from Sigma (St. All antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). Treatment of Animals Male C5. BL/6. J ob/ob mice (leptin- deficient) and their lean littermates were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China) and were at 5 wks of age. All animals were housed for an additional week and then used in experiments. The mice were given free access to water and standard chow diet. Animals received humane care, and experimental protocols were conducted according to national and local guidelines. Mice 6 wks after birth were randomly separated into groups (six mice/group), namely, the TAA group (ob/ob), TAA plus leptin group (ob/ob) and TAA group (lean). The TAA group (ob/ob) and TAA group (lean) were injected intraperitoneally with TAA (2. TAA plus leptin (ob/ob) group were given coadministration of leptin (1 . Briefly, liver sections were blocked with serum and incubated at 4. The nuclei were counterstained with Hoechst 3. Sigma), and images were captured with a fluorescence microscope. Since the ratio of SREBP- 1a to SREBP- 1c is 1: 9 and SREBP- 1c is the predominant subtype in liver (1. SREBP- 1 antibody is mainly SREBP- 1c in liver cells (1. HSC Isolation and Culture HSCs were isolated from Sprague- Dawley rats as we described previously (1. After 2. 4 h of serum starvation in Dulbecco’s modified Eagle’s medium (DMEM) with 0. FBS) (if no specific indication), cells were treated with leptin in DMEM with 0. FBS for 2. 4 h (if no specific indication). Western Blot Analysis Western blot analysis was performed as we described previously (1. Target proteins were detected by primary antibodies against SREBP- 1 (diluted 1: 5. MAPK (diluted 1: 5. MAPK (diluted 1: 5. LXR. The level of target protein band was densitometrically determined by using Quantity One 4. Bio- Rad) and normalized by the internal control . The variation in the density was expressed as fold- changes compared with the control in the blot. RNA Isolation and Real- Time Polymerase Chain Reaction Total RNA was extracted by using TRI- Reagent (Sigma) following the manufacturer’s instructions and treated with deoxyribonuclease I. Real- time polymerase chain reaction (PCR) was carried out as we described previously (1. Fold- changes in m. RNA levels of a target gene relative to the endogenous cyclophilin control were calculated as suggested by Schmittgen et al. The primers used in real- time PCR were as follows: Rat SREBP- 1c: (forward) 5. Rat alpha. 1(I) collagen: (forward) 5. Rat cyclophilin: (forward) 5. Plasmid pdn. P3. 8 encodes dominant- negative p. Plasmid pwt. P3. 8 encodes wild- type p. Plasmid p. SP1 encodes rat wild- type specific protein 1 (Sp. Photinus luciferase- reporter plasmid p. Sp. 1- Luc contains three consensus Sp. GGGCGG). Photinus luciferase- reporter plasmid p. NFY- Luc contains three consensus nuclear factor- Y (NF- Y) binding sites (CCAAT). Photinus luciferase- reporter plasmid p. LXRE- Luc contains three tandem LXR binding sites (gctt. TGGTCActca. AGTTCAagtta). Plasmid ptwist. 2 encodes twist. SREBP- 1c activity (2. HSCs in 1. 2- well plastic plates were transiently transfected with a reporter plasmid expressing Photinus luciferase (1 . The empty vectors were used to ensure an equal amount of total DNA in transfection assay. Luciferase activity was quantified fluorimetrically by using the Dual- Luciferase Reporter Assay System (Promega). Data were expressed as the ratios of Photinus to Renilla luciferase activity for normalization of Photinus luciferase activity. Chromatin Immunoprecipitation (Ch. IP) Assays Chromatin immunoprecipitation (Ch. IP) assays were performed by using a Pierce Agarose Chip Kit (Pierce, Rock- ford, IL, USA) following the manufacturer’s instructions. Briefly, after cultured HSCs were cross- linked with 1% formaldehyde, the extracted nuclei were incubated with Micrococcal Nuclease and lysed. A total of 1. 0% of the supernatant containing the digested chromatin was preserved as input control. The rest of the supernatant was incubated with LXR. DNA from immunoprecipitation and the input samples was analyzed by real- time PCR. According to the method described by Mastrogiannaki et al. Where appropriate, comparisons of multiple treatment conditions with controls were analyzed by analysis of variance with the Dunnett test for post hoc analysis. RESULTSExogenous Leptin Reduces SREBP- 1c Gene Expression in HSCs In Vitro and In Vivo. To investigate whether leptin regulates SREBP- 1c gene expression in HSCs, the cultured HSCs were stimulated with or without leptin. Results indicated that leptin clearly downregulated SREBP- 1c protein level in a time- dependent manner (Figure 1. A) and a dose- dependent manner (Figure 1. B) and was also shown to reduce SREBP- 1c m. RNA level (Figure 1. C) in cultured HSCs. Leptin at 1. 00 ng/m. L reduced SREBP- 1c protein level and SREBP- 1c m. RNA level, respectively, by about 5. Figure 1. B) and 3. Figure 1. C) compared with the respective control without treatment (the first band on the left in Figure 1. B and the first column on the left in Figure 1. C). To elucidate whether leptin has an effect on SREBP- 1c promoter activity, HSCs were transfected with a SREBP- 1c promoter luciferase reporter plasmid p. SREBP1c- Luc and incubated with or without leptin. As shown in Figure 1. D by luciferase assays, leptin at 1. L reduced the luciferase activity by 4. Figure 1. D) compared with the control (without leptin treatment). Leptin reduces SREBP- 1c gene expression in HSCs in vitro and in vivo. HSCs were stimulated with leptin at the indicated doses for different.. Next, we adopted a mouse model of TAA- induced liver damage (1. SREBP- 1c expression in HSCs in liver. The mice were treated with TAA or TAA plus leptin for 4 wks as described in Materials and Methods. Immunofluorescence staining of liver sections for SREBP- 1c in HSCs was performed. Figure 1. E showed the representative photomicrographs of immunofluorescence analysis for SREBP- 1c and SYP in the liver sections. In the TAA group (ob/ob), the SREBP- 1c protein (green fluorescence) was detectable in HSCs (red fluorescence). In the TAA plus leptin group (ob/ob) and TAA group (lean), SREBP- 1c–positive HSCs were barely detectable, although there were more HSCs (red fluorescence). These double- staining results indicated that leptin also inhibited SREBP- 1c protein expression in HSCs in vivo.
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