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1.
Nat Commun ; 5: 4934, 2014 Sep 26.
Article in English | MEDLINE | ID: mdl-25255859

ABSTRACT

Despite growing interest in the relationship between autophagy and systemic metabolism, how global changes in autophagy affect metabolism remains unclear. Here we show that mice with global haploinsufficiency of an essential autophagy gene (Atg7(+/-) mice) do not show metabolic abnormalities but develop diabetes when crossed with ob/ob mice. Atg7(+/-)-ob/ob mice show aggravated insulin resistance with increased lipid content and inflammatory changes, suggesting that autophagy haploinsufficiency impairs the adaptive response to metabolic stress. We further demonstrate that intracellular lipid content and insulin resistance after lipid loading are increased as a result of autophagy insufficiency, and provide evidence for increased inflammasome activation in Atg7(+/-)-ob/ob mice. Imatinib or trehalose improves metabolic parameters of Atg7(+/-)-ob/ob mice and enhances autophagic flux. These results suggest that systemic autophagy insufficiency could be a factor in the progression from obesity to diabetes, and autophagy modulators have therapeutic potential against diabetes associated with obesity and inflammation.


Subject(s)
Autophagy , Diabetes Mellitus/physiopathology , Obesity/physiopathology , Adaptation, Physiological , Animals , Diabetes Mellitus/pathology , Disease Progression , Female , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Obesity/pathology , Stress, Physiological
2.
J Clin Invest ; 124(8): 3311-24, 2014 Aug.
Article in English | MEDLINE | ID: mdl-25036705

ABSTRACT

Islet amyloid accumulation is a hallmark of human type 2 diabetes (T2D). In contrast to human islet amyloid polypeptide (hIAPP), murine islet amyloid polypeptide (mIAPP) does not exhibit amyloidogenic propensity. Because autophagy is important in the clearance of amyloid-like proteins, we studied transgenic mice with ß cell-specific expression of hIAPP to evaluate the contribution of autophagy in T2D-associated accumulation of hIAPP. In mice with ß cell-specific expression of hIAPP, a deficiency in autophagy resulted in development of overt diabetes, which was not observed in mice expressing hIAPP alone or lacking autophagy alone. Furthermore, lack of autophagy in hIAPP-expressing animals resulted in hIAPP oligomer and amyloid accumulation in pancreatic islets, leading to increased death and decreased mass of ß cells. Expression of hIAPP in purified monkey islet cells or a murine ß cell line resulted in pro-hIAPP dimer formation, while dimer formation was absent or reduced dramatically in cells expressing either nonamyloidogenic mIAPP or nonfibrillar mutant hIAPP. In autophagy-deficient cells, accumulation of pro-hIAPP dimers increased markedly, and pro-hIAPP trimers were detected in the detergent-insoluble fraction. Enhancement of autophagy improved the metabolic profile of hIAPP-expressing mice fed a high-fat diet. These results suggest that autophagy promotes clearance of amyloidogenic hIAPP, autophagy deficiency exacerbates pathogenesis of human T2D, and autophagy enhancers have therapeutic potential for islet amyloid accumulation-associated human T2D.


Subject(s)
Autophagy/physiology , Diabetes Mellitus, Type 2/etiology , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/pathology , Islet Amyloid Polypeptide/metabolism , Animals , Apoptosis , Autophagy-Related Protein 7 , Blood Glucose/metabolism , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/pathology , Disease Models, Animal , Female , Humans , Insulin/blood , Insulin-Secreting Cells/drug effects , Islet Amyloid Polypeptide/chemistry , Islet Amyloid Polypeptide/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microtubule-Associated Proteins/deficiency , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Models, Molecular , Protein Multimerization , Protein Structure, Quaternary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Trehalose/pharmacology
3.
Mol Cell Endocrinol ; 367(1-2): 1-10, 2013 Mar 10.
Article in English | MEDLINE | ID: mdl-23246353

ABSTRACT

To shed light on islet cell molecular phenotype in human type 2 diabetes (T2D), we studied the transcriptome of non-diabetic (ND) and T2D islets to then focus on the ubiquitin-proteasome system (UPS), the major protein degradation pathway. We assessed gene expression, amount of ubiquitinated proteins, proteasome activity, and the effects of proteasome inhibition and prolonged exposure to palmitate. Microarray analysis identified more than one thousand genes differently expressed in T2D islets, involved in many structures and functions, with consistent alterations of the UPS. Quantitative RT-PCR demonstrated downregulation of selected UPS genes in T2D islets and beta cell fractions, with greater ubiquitin accumulation and reduced proteasome activity. Chemically induced reduction of proteasome activity was associated with lower glucose-stimulated insulin secretion, which was partly reproduced by palmitate exposure. These results show the presence of many changes in islet transcriptome in T2D islets and underline the importance of the association between UPS alterations and beta cell dysfunction in human T2D.


Subject(s)
Diabetes Mellitus, Type 2/genetics , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/pathology , Oligonucleotide Array Sequence Analysis , Proteasome Endopeptidase Complex/metabolism , Transcriptome/genetics , Ubiquitin/metabolism , Aged , Animals , Cell Line , Diabetes Mellitus, Type 2/pathology , Diabetes Mellitus, Type 2/physiopathology , Female , Gene Expression Profiling , Gene Expression Regulation/drug effects , Humans , Immunohistochemistry , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/drug effects , Male , Middle Aged , Palmitates/pharmacology , Proteasome Endopeptidase Complex/genetics , Proteasome Inhibitors/pharmacology , Rats , Reverse Transcriptase Polymerase Chain Reaction , Ubiquitin/genetics , Ubiquitinated Proteins/genetics , Ubiquitinated Proteins/metabolism
4.
Diabetes Res Clin Pract ; 91(1): 72-9, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21093089

ABSTRACT

We studied the effect of a novel dipeptidyl peptidase IV (DPP IV) inhibitor, DA-1229, on blood glucose profile and pancreatic ß-cell mass in established diabetes after streptozotocin (STZ) treatment. Mice that developed diabetes after administration of STZ 100mg/kg were treated with DA-1229 for 13 weeks. DA-1229 significantly reduced plasma DPP IV activity, and enhanced glucagon-like peptide 1 (GLP-1) levels. In STZ-treated mice fed DA-1229 (STZ-DA), blood glucose levels were significantly lower than those in diabetic mice fed normal chow (STZ-NC). Basal and glucose-stimulated insulin secretion and glucose tolerance assessed by intraperitoneal glucose tolerance test were significantly improved by DA-1229 administration. Volume density of ß-cell was significantly increased in STZ-DA mice compared to STZ-NC mice, suggesting that DA-1229-mediated amelioration of established diabetes was due to beneficial effect of DA-1229 on ß-cell mass. The number of replicating ß-cells and that of scattered small ß-cell unit representing ß-cell neogenesis were significantly increased in STZ-DA mice compared to STZ-NC mice, explaining increased ß-cell mass by DA-1229. The expression of PDX-1, a downstream mediator of GLP-1 action, was increased in islets of STZ-DA mice compared to STZ-NC mice. These results suggest a therapeutic potential of DA-1229 in diabetes, particularly that associated with decreased ß-cell mass.


Subject(s)
Cell Differentiation/drug effects , Cell Proliferation/drug effects , Diabetes Mellitus, Experimental/drug therapy , Dipeptidyl-Peptidase IV Inhibitors/pharmacology , Dipeptidyl-Peptidase IV Inhibitors/therapeutic use , Insulin-Secreting Cells/drug effects , Piperazines/pharmacology , Piperazines/therapeutic use , Animals , Blood Glucose/analysis , Cell Count , Cell Size/drug effects , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Experimental/physiopathology , Dipeptidyl Peptidase 4/blood , Glucagon-Like Peptide 1/blood , Glucose Intolerance/drug therapy , Homeodomain Proteins/metabolism , Insulin/blood , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/pathology , Insulin-Secreting Cells/physiology , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Islets of Langerhans/pathology , Islets of Langerhans/physiopathology , Male , Mice , Mice, Inbred C57BL , Regeneration/drug effects , Streptozocin/toxicity , Trans-Activators/metabolism
5.
Oncol Rep ; 23(3): 779-86, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20127020

ABSTRACT

Mitochondrial DNA (mtDNA) mutations are frequently found in a variety of tumors. However, the role of mtDNA mutations in tumor behavior is poorly understood. We explored the effects of mtDNA mutations on tumor phenotype employing mtDNA-depleted SK-Hep1 rho0 hepatoma cells. Expression of hypoxia inducible factor (HIF)-2alpha mRNA was markedly increased in rho0 cells compared to control cells. Protein level of HIF-2alpha was increased in SK-Hep1 rho0 cells compared to control cells in hypoxic but not in normoxic conditions, suggesting that mitochondrial dysfunction increases angiogenic potential of tumor cells. Expression of HIF-2alpha was increased at the RNA level after treatment of SK-Hep1 hepatoma cells with ethidium bromide (EtBr) or inhibitors of mitochondrial complexes. HIF reporter activity and the expression of vascular endothelial growth factor (VEGF), an angiogenic key molecule induced by HIF, were increased in SK-Hep1 rho0 cells compared to their normal counterparts. Tube formation assay and chick chorioallantoic membrane (CAM) assay showed that conditioned medium (CM) from mtDNA-depleted SK-Hep1 rho0 cells increased formation of tube-like structures and new blood vessels relative to that from control cells. In SK-Hep1 rho0 cells, expression of genes related to invasion such as urokinase-type plasminogen activator (uPA) or matrix metalloproteases (MMPs) was also upregulated compared to control cells, suggesting that mitochondrial dysfunction could also increase invasive potential of tumor cells. These results strongly suggest that HIF-2alpha mRNA expression is increased in tumor cells with mtDNA mutations or deletions, which contributes to the angiogenic and invasive potential of tumor cells.


Subject(s)
Carcinoma, Hepatocellular/pathology , DNA, Mitochondrial/genetics , Liver Neoplasms/pathology , Neovascularization, Pathologic/etiology , Basic Helix-Loop-Helix Transcription Factors/genetics , Carcinoma, Hepatocellular/genetics , Cell Line, Tumor , Extracellular Matrix Proteins/metabolism , Gene Deletion , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Liver Neoplasms/genetics , Mutation , Neoplasm Invasiveness , RNA, Messenger/analysis , Vascular Endothelial Growth Factor A/genetics
6.
Mol Cell Endocrinol ; 321(2): 131-7, 2010 Jun 10.
Article in English | MEDLINE | ID: mdl-20176078

ABSTRACT

c-Jun N-terminal kinase (JNK) is activated by cellular stress and plays critical roles in diverse types of cell death. However, role of JNK in beta-cell injury is obscure. We investigated the role for JNK in streptozotocin (STZ)-induced beta-cell death. STZ induced JNK activation in insulinoma or islet cells. JNK inhibitors attenuated insulinoma or islet cell death by STZ. STZ-induced JNK activation was decreased by PARP inhibitors, suggesting that JNK activation is downstream of PARP-1. Phosphatase inhibitors induced activation of JNK and abrogated the suppression of STZ-induced JNK activation by PARP inhibitors, suggesting that the inhibition of phosphatases is involved in the activation of JNK by STZ. STZ induced production of reactive oxygen species (ROS) as potential inhibitors of phosphatases, which was suppressed by PARP inhibitors. PARP-1 siRNA attenuated insulinoma cell death and JNK activation after STZ treatment, which was reversed by MKP (MAP kinase phosphatase)-1 siRNA. These results suggest that JNK is activated by STZ downstream of PARP-1 through inactivation of phosphatases such as MKP, which plays important roles in STZ-induced beta-cell death.


Subject(s)
Antibiotics, Antineoplastic/pharmacology , Cell Death/drug effects , Enzyme Activation/drug effects , Insulin-Secreting Cells/drug effects , JNK Mitogen-Activated Protein Kinases/metabolism , Streptozocin/pharmacology , Animals , Blotting, Western , Enzyme Inhibitors/pharmacology , Mice , Mice, Transgenic , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerase Inhibitors , Poly(ADP-ribose) Polymerases/metabolism , Reactive Oxygen Species/metabolism , Reactive Oxygen Species/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction
7.
Oncol Rep ; 18(5): 1311-4, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17914590

ABSTRACT

Metastasis-associated protein 1 (MTA1) is highly upregulated in cancer cells with metastatic potential; however, the molecular mechanism by which MTA1 increases the metastatic potential of cancer cells is far from clear. We characterized the functional consequences of MTA1 overexpression on p53-induced apoptosis of cancer cells. MTA1 was associated with p53 in a co-immunoprecipitation assay. MTA1 also had deacetylation activity on p53 in human non-small cell lung cancer cells H1299 and human hepatoma cells SK-Hep1. MTA1 attenuated the transactivation and p21 induction by p53. Moreover, MTA1 expression decreased p53-mediated apoptosis. These results indicate that MTA1 inhibits p53-induced apoptosis by deacetylation of p53, which might be related to the increased metastatic potential of cancer cells with high MTA1 expression.


Subject(s)
Apoptosis , Gene Expression Regulation, Neoplastic , Histone Deacetylases/pharmacology , Repressor Proteins/pharmacology , Tumor Suppressor Protein p53/metabolism , Acetylation , Blotting, Western , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Humans , Immunoprecipitation , Kidney/metabolism , Kidney/pathology , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Luciferases/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Trans-Activators , Transcription, Genetic , Tumor Suppressor Protein p53/genetics
8.
Oncol Rep ; 16(4): 929-35, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16969516

ABSTRACT

Metastasis-associated protein 1 (MTA1) is highly upregulated in cancer cells with metastatic potential; however, the molecular mechanism by which MTA1 increases the metastatic potential of cancer cells is unknown. We characterized the functional consequences of MTA1 overexpression in cancer cells with an emphasis on its potential role as a deacetylator of hypoxia-inducible factor-1alpha (HIF-1alpha). MTA1 increased the expression of HIF-1alpha protein, but did not increase the expression of its mRNA. Glutathione S-transferase pull-down and coimmunoprecipitation assays demonstrated direct interaction of MTA1 with HIF-1alpha both in vitro and in vivo. Immunoprecipitation and acetylation assays also showed that MTA1 has deacetylation activity on HIF-1alpha in vivo. Moreover, MTA1 increased the transcriptional activity of HIF-1alpha and enhanced the expression of vascular endothelial growth factor, a target molecule of HIF-1alpha. Conditioned medium collected from MTA1 transfectants also increased angiogenesis in vitro and in vivo, probably through enhanced HIF-1alpha stabilization. These results indicate that MTA1 enhances angiogenesis by stabilization of the HIF-1alpha protein, which is closely related to the increased metastatic potential of cancer cells with high MTA1 expression.


Subject(s)
Histone Deacetylases/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Neovascularization, Pathologic , Repressor Proteins/metabolism , Cell Line, Tumor , Collagen/chemistry , Culture Media, Conditioned/metabolism , Culture Media, Conditioned/pharmacology , Drug Combinations , Glutathione Transferase/metabolism , Humans , Immunoprecipitation , Laminin/chemistry , Neoplasm Metastasis , Proteoglycans/chemistry , Trans-Activators , Transcription, Genetic , Transcriptional Activation
9.
Biochem Biophys Res Commun ; 325(4): 1399-405, 2004 Dec 24.
Article in English | MEDLINE | ID: mdl-15555582

ABSTRACT

We have reported that mitochondrial DNA-depleted rho(0) cells are resistant to cell death. Because aged cells have frequent mitochondrial DNA mutations, the resistance of rho(0) cells against cell death might be related to the apoptosis resistance of aged cells and frequent development of cancers in aged individuals. We studied if rho(0) cells have features simulating aged cells. SK-Hep1 hepatoma rho(0) cells showed typical morphology associated with aging such as increased size and elongated appearance. They had increased senescence-associated beta-Gal activity, lipofuscin pigment, and plasminogen activator inhibitor-1 expression. Consistent with their decreased proliferation, the expression of mitotic cyclins was decreased and that of cdk inhibitors was increased. Rb hypophosphorylation and decreased telomerase activity were also noted. Features simulating aged cells were also observed in MDA-MB-435 rho(0) cells. These results support the mitochondrial theory of aging, and suggest that rho(0) cells could serve as an in vitro model for aged cells.


Subject(s)
Breast Neoplasms/pathology , Breast Neoplasms/physiopathology , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/physiopathology , Cellular Senescence/genetics , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Apoptosis/genetics , Cell Line, Tumor , Cell Size , Cell Survival , Humans , Liver Neoplasms/pathology , Liver Neoplasms/physiopathology
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