ABSTRACT
Brown seaweed Sargassum confusum (C. Agardh) has been used in traditional Chinese medicine to treat a variety of diseases. The aim of the present study was to evaluate the anti-diabetic effect of oligosaccharides from brown seaweed S. confusum (SCO). The anti-diabetic effect of SCO was evaluated in vivo using high-fat/high-sucrose fed hamsters. Molecular mechanisms of modulating gene expression of specific members of insulin signaling pathways were determined. The components of the intestinal microflora in diabetic animals were also analyzed by high-throughput 16S rRNA gene sequencing. And it was found that SCO had a sequence of sulfated anhydrogalactose and methyl sulfated galactoside units. Fasting blood glucose levels were significantly decreased after SCO administration. Histology showed that SCO could protect the cellular architecture of the liver. SCO could also significantly increase the relative abundance of Lactobacillus and Clostridium XIVa and decrease that of Allobaculum, Bacteroides and Clostridium IV. The active role of SCO in anti-diabetic effect was revealed by its regulation of insulin receptor substrate 1/phosphatidylinositol 3-kinase and c-Jun N-terminal kinase pathways. These results suggested that SCO might be used as a functional material to regulate gut microbiota in obese and diabetic individuals.
Subject(s)
Diabetes Mellitus/drug therapy , Gastrointestinal Microbiome/drug effects , Hypoglycemic Agents/therapeutic use , MAP Kinase Signaling System/drug effects , Oligosaccharides/therapeutic use , Sargassum/chemistry , Animals , Bacteria/genetics , Base Sequence , Blood Glucose/drug effects , Body Weight/drug effects , Diet, Carbohydrate Loading , Diet, High-Fat , Hypoglycemic Agents/isolation & purification , Liver/drug effects , Liver/pathology , Male , Mesocricetus , Oligosaccharides/isolation & purification , Protective Agents/isolation & purification , Protective Agents/therapeutic use , RNA, Ribosomal, 16S/genetics , Seaweed/chemistryABSTRACT
Hepatic stellate cells (HSCs) play a critical role in the pathogenesis and reversal of liver fibrosis. Targeting HSCs is of great significance in the treatment of hepatic fibrosis, and has attracted wide attention of scholars. Here we demonstrated that expression of geranylgeranyldiphosphate synthase (GGPPS) predominantly increased in HSCs in murine fibrotic liver. HSC-specific knockdown of GGPPS using vitamin A-coupled liposome carrying siRNA-ggpps decreased activation of HSCs and alleviated fiber accumulation in vivo. Furthermore, our in vitro studies showed that GGPPS was up-regulated during HSCs activation in TGF-ß1-dependent manner. Inhibition of GGPPS suppressed TGF-ß1 induced F-actin reorganization and HSCs activation in LX-2 cells. Further, we found that GGPPS regulated HSCs activation and liver fibrosis possibly by enhancing RhoA/Rock kinase signaling. So its concluded that GGPPS promotes liver fibrosis by activating HSCs, which may represent a potential target for anti-fibrosis therapies.
ABSTRACT
Research on mevalonate kinase deficiency has revealed that it may lead to the development of renal angiomyolipomas (RAMLs). Thus, it was suspected that geranylgeranyl pyrophosphate synthase (GGPPS), a key enzyme in the mevalonate pathway, may be involved in the development of RAMLs. In the present study, the expression of GGPPS in RAMLs and renal epithelioid angiomyolipomas (REAs) was assessed, and paraffin embedded specimens from 60 patients, including 9 cases with REA and 51 cases with RAML, were examined. Immunoreactivity was evaluated semi-quantitatively according to the intensity of staining and the percentage of positively stained cells. The results indicated that GGPPS was predominantly present in the cytoplasm, and REA tissues exhibited higher expression of GGPPS in the cytoplasm compared with RAML tissues. It was also identified that GGPPS was upregulated in TSC2-null cells, and inhibition of GGPPS could induce apoptosis of TSC2-null cells by autophagy. In conclusion, the increased expression of GGPPS in RAMLs and REAs indicated that mevalonate pathways may be involved in disease progression. GGPPS may serve as a potential therapeutic target and the current results may provide a novel therapeutic strategy for RAML and lymphangioleiomyomatosis.
ABSTRACT
Postprandial insulin desensitization plays a critical role in maintaining whole-body glucose homeostasis by avoiding the excessive absorption of blood glucose; however, the detailed mechanisms that underlie how the major player, skeletal muscle, desensitizes insulin action remain to be elucidated. Herein, we report that early growth response gene-1 ( Egr-1) is activated by insulin in skeletal muscle and provides feedback inhibition that regulates insulin sensitivity after a meal. The inhibition of the transcriptional activity of Egr-1 enhanced the phosphorylation of the insulin receptor (InsR) and Akt, thus increasing glucose uptake in L6 myotubes after insulin stimulation, whereas overexpression of Egr-1 decreased insulin sensitivity. Furthermore, deletion of Egr-1 in the skeletal muscle improved systemic insulin sensitivity and glucose tolerance, which resulted in lower blood glucose levels after refeeding. Mechanistic analysis demonstrated that EGR-1 inhibited InsR phosphorylation and glucose uptake in skeletal muscle by binding to the proximal promoter region of protein tyrosine phosphatase-1B (PTP1B) and directly activating transcription. PTP1B knockdown largely restored insulin sensitivity and enhanced glucose uptake, even under conditions of EGR-1 overexpression. Our results indicate that EGR-1/PTP1B signaling negatively regulates postprandial insulin sensitivity and suggest a potential therapeutic target for the prevention and treatment of excessive glucose absorption.-Wu, J., Tao, W.-W., Chong, D.-Y., Lai, S.-S., Wang, C., Liu, Q., Zhang, T.-Y., Xue, B., Li, C.-J. Early growth response-1 negative feedback regulates skeletal muscle postprandial insulin sensitivity via activating Ptp1b transcription.
Subject(s)
Early Growth Response Protein 1/metabolism , Insulin/metabolism , Muscle, Skeletal/metabolism , Postprandial Period/physiology , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , Animals , Blood Glucose , Cells, Cultured , Glucose/metabolism , Homeostasis/physiology , Insulin Resistance/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle Fibers, Skeletal/metabolism , Phosphorylation/physiology , Rats , Receptor, Insulin/metabolism , Signal Transduction/physiology , Transcription, Genetic/physiologyABSTRACT
BACKGROUND & AIMS: Liver injury triggers a highly organized and ordered liver regeneration (LR) process. Once regeneration is complete, a stop signal ensures that the regenerated liver is an appropriate functional size. The inhibitors and stop signals that regulate LR are unknown, and only limited information is available about these mechanisms. METHODS: A 70% partial hepatectomy (PH) was performed in hepatocyte-specific PP2Acα-deleted (PP2Acα(-/-)) and control (PP2Acα(+/+)) mice. LR was estimated by liver weight, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and cell proliferation, and the related cellular signals were analyzed. RESULTS: We found that the catalytic subunit of PP2A was markedly upregulated during the late stage of LR. PP2Acα(-/-) mice showed prolonged LR termination, an increased liver size compared to the original mass and lower levels of serum ALT and AST compared with control mice. In these mice, cyclin D1 protein levels, but not mRNA levels, were increased. Mechanistically, AKT activated by the loss of PP2Acα inhibited glycogen synthase kinase 3ß (GSK3ß) activity, which led to the accumulation of cyclin D1 protein and accelerated hepatocyte proliferation at the termination stage. Treatment with the PI3K inhibitor wortmannin at the termination stage was sufficient to inhibit cyclin D1 accumulation and hepatocyte proliferation. CONCLUSIONS: PP2Acα plays an essential role in the proper termination of LR via the AKT/GSK3ß/Cyclin D1 pathway. Our findings enrich the understanding of the molecular mechanism that controls the termination of LR and provides a potential therapeutic target for treating liver injury.
Subject(s)
Cyclin D1/metabolism , Glycogen Synthase Kinase 3 beta/metabolism , Hepatocytes/metabolism , Liver Regeneration/physiology , Protein Phosphatase 2/metabolism , Animals , Apoptosis/physiology , Cell Proliferation/physiology , Mice , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/physiologyABSTRACT
The mammalian clock system is composed of a master clock and peripheral clocks. At the molecular level, the rhythm-generating mechanism is controlled by a molecular clock composed of positive and negative feedback loops. However, the underlying mechanisms for molecular clock regulation that affect circadian clock function remain unclear. Here, we show that Egr1 (early growth response 1), an early growth response gene, is expressed in mouse liver in a circadian manner. Consistently, Egr1 is transactivated by the CLOCK/BMAL1 heterodimer through a conserved E-box response element. In hepatocytes, EGR1 regulates the transcription of several core clock genes, including Bmal1, Per1, Per2, Rev-erbα and Rev-erbß, and the rhythm amplitude of their expression is dependent on EGR1's transcriptional function. Further mechanistic studies indicated that EGR1 binds to the proximal region of the Per1 promoter to activate its transcription directly. When the peripheral clock is altered by light or feeding behavior transposition in Egr1-deficient mice, the expression phase of hepatic clock genes shifts normally, but the amplitude is also altered. Our data reveal a critical role for EGR1 in the regulation of hepatic clock circuitry, which may contribute to the rhythm stability of peripheral clock oscillators.
Subject(s)
Early Growth Response Protein 1/metabolism , Liver/metabolism , Period Circadian Proteins/metabolism , ARNTL Transcription Factors/deficiency , ARNTL Transcription Factors/genetics , ARNTL Transcription Factors/metabolism , Animals , CLOCK Proteins/chemistry , CLOCK Proteins/genetics , CLOCK Proteins/metabolism , Chromatin Immunoprecipitation , Early Growth Response Protein 1/antagonists & inhibitors , Early Growth Response Protein 1/genetics , Feedback, Physiological , Mice , Mice, Knockout , Period Circadian Proteins/antagonists & inhibitors , Period Circadian Proteins/genetics , Promoter Regions, Genetic , Protein Binding , RNA Interference , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Response Elements , Transcriptional ActivationABSTRACT
GGPPS catalyses the expression of GGPP, a key protein in the mevalonate metabolic pathway. HMG-CoA reductase inhibitor statins can induce liver injury by inhibiting GGPP. However, the function of GGPPS in liver injury has not yet been revealed. In this study, we found that GGPPS increased in liver injury and that GGPPS deletion augmented liver injury and fibrosis. GGPPS inhibition induced hepatocyte apoptosis, inflammation and TGF-ß1 secretion, which activated hepatic stellate cells. Our findings imply that GGPPS deletion induces hepatocyte apoptosis, which makes the liver vulnerable to hepatotoxicity.
Subject(s)
Carbon Tetrachloride Poisoning/enzymology , Farnesyltranstransferase/metabolism , Hepatocytes/enzymology , Liver Cirrhosis/enzymology , Liver/enzymology , Animals , Carbon Tetrachloride Poisoning/genetics , Carbon Tetrachloride Poisoning/pathology , Farnesyltranstransferase/genetics , Gene Deletion , Hepatocytes/pathology , Liver/pathology , Liver Cirrhosis/chemically induced , Liver Cirrhosis/genetics , Liver Cirrhosis/pathology , Mice , Mice, Knockout , Polyisoprenyl Phosphates/biosynthesis , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/metabolismABSTRACT
The sequential secretion of insulin and glucagon delicately maintains glucose homeostasis by inhibiting or enhancing hepatic gluconeogenesis during postprandial or fasting states, respectively. Increased glucagon/insulin ratio is believed to be a major cause of the hyperglycemia seen in type 2 diabetes. Herein, we reveal that the early growth response gene-1 (Egr-1) can be transiently activated by glucagon in hepatocytes, which mediates glucagon-regulated gluconeogenesis by increasing the expression of gluconeogenesis genes. Blockage of Egr-1 function in the liver of mice led to lower fasting blood glucose, better pyruvate tolerance, and higher hepatic glycogen content. The mechanism analysis demonstrated that Egr-1 can directly bind to the promoter of C/EBPa and regulate the expression of gluconeogenesis genes in the later phase of glucagon stimulation. The transient increase of Egr-1 by glucagon kept the glucose homeostasis after fasting for longer periods of time, whereas constitutive Egr-1 elevation found in the liver of db/db mice and high serum glucagon level overactivated the C/EBPa/gluconeogenesis pathway and resulted in hyperglycemia. Blockage of Egr-1 activation in prediabetic db/db mice was able to delay the progression of diabetes. Our results suggest that dysregulation of Egr-1/C/EBPa on glucagon stimulation may provide an alternative mechanistic explanation for type 2 diabetes.
Subject(s)
CCAAT-Enhancer-Binding Proteins/metabolism , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 2/metabolism , Early Growth Response Protein 1/metabolism , Gluconeogenesis , Liver/metabolism , Animals , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 2/pathology , Glucagon/metabolism , Glucose/metabolism , Liver/pathology , Male , MiceABSTRACT
Mumps commonly affects children 5-9 yr of age, and can lead to permanent adult sterility in certain cases. However, the etiology of this long-term effect remains unclear. Mumps infection results in progressive degeneration of the seminiferous epithelium and, occasionally, Sertoli cell-only syndrome. Thus, the remaining Sertoli cells may be critical to spermatogenesis recovery after orchitis healing. Here, we report that the protein farnesylation/geranylgeranylation balance is critical for patients' fertility. The expression of geranylgeranyl diphosphate synthase 1 (GGPPS) was decreased due to elevated promoter methylation in the testes of infertile patients with mumps infection history. When we deleted GGPPS in mouse Sertoli cells, these cells remained intact, whereas the adjacent spermatogonia significantly decreased after the fifth postnatal day. The proinflammatory MAPK and NF-κB signaling pathways were constitutively activated in GGPPS(-/-) Sertoli cells due to the enhanced farnesylation of H-Ras. GGPPS(-/-) Sertoli cells secreted an array of cytokines to stimulate spermatogonia apoptosis, and chemokines to induce macrophage invasion into the seminiferous tubules. Invaded macrophages further blocked spermatogonia development, resulting in a long-term effect through to adulthood. Notably, this defect could be rescued by GGPP administration in EMCV-challenged mice. Our results suggest a novel mechanism by which mumps infection during childhood results in adult sterility.
Subject(s)
Infertility, Male/etiology , Infertility, Male/metabolism , Mumps/complications , Protein Prenylation , Sertoli Cells/metabolism , Adult , Animals , Apoptosis/genetics , Child , Cytokines/biosynthesis , DNA Methylation , Farnesyltranstransferase/genetics , Gene Deletion , Gene Expression Profiling , Gene Expression Regulation , Humans , Inflammation Mediators/metabolism , Macrophages/immunology , Macrophages/pathology , Male , Mice , Mice, Knockout , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Promoter Regions, Genetic , Protein Prenylation/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Seminiferous Tubules/pathology , Spermatogonia/metabolism , Spermatogonia/pathology , Testis/metabolism , Testis/pathology , Young AdultABSTRACT
The receptor tyrosine kinase (RTK) Ror2 plays important roles in developmental morphogenesis and mediates the filopodia formation in Wnt5a-induced cell migration. However, the function of Ror2 in noncanonical Wnt signaling resulting in cancer metastasis is largely unknown. Here, we show that Ror2 expression is higher in the highly metastatic murine B16-BL6 melanoma cells than in the low metastatic variant B16 cells. Overexpression of Ror2 increases the metastasis ability of B16 cells, and knockdown of Ror2 reduces the migration ability of B16-BL6 cells. Furthermore, the inhibition of Src kinase activity is critical for the Ror2-mediated cell migration upon Wnt5a treatment. The C-terminus of Ror2, which is deleted in brachydactyly type B (BDB), is essential for the mutual interaction with the SH1 domain of Src. Intriguingly, the Neurotrophin receptor-interacting MAGE homologue (NRAGE), which, as we previously reported, can remodel the cellular skeleton and inhibit cell-cell adhesion and metastasis of melanoma and pancreatic cancer, sharply blocks the interaction between Src and Ror2 and inhibits Ror2-mediated B16 cell migration by decreasing the activity of Src and focal adhesion kinase (FAK). Our data show that Ror2 is a potential factor in the tumorigenesis and metastasis in a Src-dependent manner that is negatively regulated by NRAGE.
Subject(s)
Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Neoplasm Proteins/metabolism , Receptor Tyrosine Kinase-like Orphan Receptors/metabolism , src-Family Kinases/metabolism , Adenoviridae , Animals , Blotting, Western , Cell Line, Tumor , Cell Movement/genetics , Focal Adhesion Kinase 1/metabolism , Gene Knockdown Techniques , Humans , Immunoprecipitation , Male , Melanoma, Experimental/genetics , Mice , Mice, Inbred C57BL , Neoplasm Metastasis , Receptor Tyrosine Kinase-like Orphan Receptors/biosynthesis , Receptor Tyrosine Kinase-like Orphan Receptors/genetics , Signal TransductionABSTRACT
Cigarette smoke activates the extracellular signal-regulated kinase (ERK) 1/2 mitogen activated-protein kinase pathway, which, in turn, is responsible for early growth response gene-1 (EGR-1) activation. Here we provide evidence that EGR-1 activation can also reactivate ERK 1/2 mitogen activated-protein kinase through a positive feedback loop through its target gene (geranylgeranyl diphosphate synthase) GGPPS. For the first time, the GGPPS gene is identified as a target of EGR-1, as EGR-1 can directly bind to the predicted consensus-binding site in the GGPPS promoter and regulate its transcription. Long-term observations show that there are two ERK 1/2 phosphorylation peaks after cigarette smoke extract stimulation in human lung epithelial Beas-2B cells. The first peak (at 10 minutes) is responsible for EGR-1 accumulation, and the second (at 4 hours) is diminished after the disruption of EGR-1 transcriptional activity. EGR-1 overexpression enhances Ras prenylation and membrane association in a GGPPS-dependent manner, and it augments ERK 1/2 activation. Likewise, a great reduction of the second peak of ERK 1/2 phosphorylation is observed during long-term cigarette smoke extract stimulation in cells where GGPPS is disrupted. Thus, we have uncovered an intricate positive feedback loop in which ERK 1/2-activated EGR-1 promotes ERK 1/2 reactivation through promoting GGPPS transcription, which might affect cigarette smoke-related lung pathological processes.