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1.
Growth Horm IGF Res ; 46-47: 24-35, 2019.
Article in English | MEDLINE | ID: mdl-31158782

ABSTRACT

OBJECTIVE: The objective of our study was to examine the direct action of insulin-like growth factor-1(IGF-1) signaling on energy homeostasis in myocytes. DESIGN: We studied the IGF-1 stimulation of mitochondrial uncoupling protein 3 (UCP3) expression in the HEK 293 derived cell line TSA201, murine C2C12 skeletal muscle myoblasts, and rat L6 skeletal myoblasts. We also investigated the direct effect of IGF-1 on the Insulin/IGF-1 receptor (IGF-1R)/phosphatidylinositol 3 (PI3)-Akt/forkhead box O4 (FOXO4) pathway using a combination of a reporter assay, semi-quantitative polymerase chain reaction, western blotting, and animal experiments. RESULTS: We demonstrated that IGF-1 regulates UCP3 expression via phosphorylation of FOXO4, which is a downstream signal transducer of IGF-1. UCP3 expression increased with activated FOXO4 in a dose-dependent manner. We also examined the functional FOXO4 binding site consensus sequences and identified it as the -1922 bp site in the UCP3 promoter region. UCP3 was also found to be concomitantly expressed with IGF-1 during differentiation of C2C12 myoblasts. Our animal experiments showed that high fat diet induced IGF-1 levels which likely influenced UCP3 expression in the skeletal muscle. CONCLUSION: Our findings demonstrate that that IGF-1 directly stimulates UCP3 expression via the IGF-1/IGF-1R/PI3-Akt/FOXO4 pathway.


Subject(s)
Cell Cycle Proteins/metabolism , Forkhead Transcription Factors/metabolism , Gene Expression Regulation/drug effects , Insulin-Like Growth Factor I/pharmacology , Muscle, Skeletal/metabolism , Myoblasts, Skeletal/metabolism , Uncoupling Protein 3/metabolism , Animals , Cell Cycle Proteins/genetics , Cell Differentiation , Forkhead Transcription Factors/genetics , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Muscle, Skeletal/cytology , Muscle, Skeletal/drug effects , Myoblasts, Skeletal/cytology , Myoblasts, Skeletal/drug effects , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Rats , Receptor, IGF Type 1/genetics , Receptor, IGF Type 1/metabolism , Uncoupling Protein 3/genetics
2.
Growth Factors ; 37(5-6): 247-256, 2019 12.
Article in English | MEDLINE | ID: mdl-32156173

ABSTRACT

Mitochondria uncoupling protein2 (UCP2) expressed ubiquitously is a key molecule of energy metabolism. Insulin-like growth factor-1 (IGF-1) is a hormone, a target molecule of growth hormone (GH) signal pathway, which is also known as the drug "mecasermin" for clinical usages. IGF-1 is seemed to be closely related to metabolic diseases, such as adult GH deficiency. However, there has not been reports depicted possible relationship with each other. So, we sought to elucidate the mechanisms by which expression of UCP2 is regulated by IGF-1 via FOXO1. The findings suggested that three sequences in the consensus UCP2 promoter play complementary functional roles in the functional expression of FOXO1. So, we found that FOXO1 is involved in IGF-1-mediated energy metabolism greater than that of direct action of GH via STAT5. Our findings suggested that IGF-1 was involved in energy metabolism by regulating the expression of UCP2 via the PI3K/Akt/FOXO1 pathway.


Subject(s)
Forkhead Box Protein O1/metabolism , Growth Hormone/metabolism , Insulin-Like Growth Factor I/metabolism , Uncoupling Protein 2/metabolism , 3T3-L1 Cells , Adipose Tissue/metabolism , Animals , Energy Metabolism , Gene Expression Regulation , HEK293 Cells , Hep G2 Cells , Humans , Mice , Mitochondria/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Promoter Regions, Genetic , Receptor, IGF Type 1/metabolism , STAT5 Transcription Factor/metabolism , Tumor Suppressor Proteins/metabolism
3.
Growth Horm IGF Res ; 40: 44-54, 2018 06.
Article in English | MEDLINE | ID: mdl-29398371

ABSTRACT

OBJECTIVE: We evaluated the direct action of GH signaling in energy homeostasis in myocytes. DESIGN: We investigated the GH-induced expression of UCP3 in human embryonic kidney 293 cells, human H-EMC-SS chondrosarcoma cells, murine C2C12 skeletal muscle myoblasts, and rat L6 skeletal muscle cells, as well as its direct effect on the GHR/JAK/STAT5 pathway using a combination of a reporter assay, real-time quantitative polymerase chain reaction, and western blotting. RESULTS: We demonstrated that the regulation of energy metabolism by GH involves UCP3 via activated STAT5, a signal transducer downstream of GH. UCP3 expression increased with STAT5 in a dose-dependent manner and was higher than that of UCP2. We confirmed the functional STAT5 binding site consensus sequences at -861 and -507 bp in the UCP3 promoter region. CONCLUSION: The results suggest that GH stimulates UCP3 directly and that UCP2 and that UCP3 participate in the signal transduction pathway that functions downstream of the GHR/JAK/STAT.


Subject(s)
Chondrosarcoma/metabolism , Gene Expression Regulation/drug effects , Growth Hormone/pharmacology , Muscle, Skeletal/metabolism , Myoblasts, Skeletal/metabolism , Uncoupling Protein 3/metabolism , Animals , Cells, Cultured , Chondrosarcoma/genetics , Chondrosarcoma/pathology , Humans , Janus Kinase 2/genetics , Janus Kinase 2/metabolism , Mice , Muscle, Skeletal/cytology , Myoblasts, Skeletal/cytology , Rats , STAT5 Transcription Factor/genetics , STAT5 Transcription Factor/metabolism , Signal Transduction , Uncoupling Protein 3/genetics
4.
Growth Horm IGF Res ; 37: 31-39, 2017 12.
Article in English | MEDLINE | ID: mdl-29111497

ABSTRACT

OBJECTIVE: The transition of white adipocytes to beige cells (a phenomenon referred to as browning or beigeing) during obesity has been previously reported. Our study aimed to examine the mechanisms through which obesity induced by a high fat diet (HFD) affects uncoupling protein 1 (UCP1) expression via signal transduction and activator of transcription 5 (STAT5s). DESIGN: Seven-week-old male C57BL/6J mice were fed a normal or HFD for 11weeks. Body weight, white adipose tissue weight, and blood lipid and glucose levels were measured. To unveil the molecular mechanisms of UCP1 expression in adipose tissue, we performed further studying 3T3-L1 cells using qRT-PCR. We also measured UCP1 promoter activity in the TSA201 cell line using a dual luciferase assay. In addition, we analyzed the predicted consensus sequences for STAT5 binding in the UCP1 promoter region. RESULTS: Mice fed an HFD had higher body weight and intra-abdominal adipose tissues weight and a higher expression of UCP1, GH receptor (GHR), STATs, suppressors of cytokine signaling (SOCSs), and cytokine-inducible SH2-containing protein (CISH) compared to control mice. In 3T3-L1 cell studies, GH induced phosphorylation of the STAT5, SOCSs, CISH and UCP1 expressions. UCP1 promoter activity was associated with constitutively active STAT5 in a dose-dependent manner. We confirmed functional STAT5 binding sites at -425, -279, and -178bp of the UCP1 promoter. CONCLUSION: We suggest that endogenous GH induces UCP1 expression in adipose tissue via STAT5.


Subject(s)
Adipose Tissue, White/metabolism , Growth Hormone/metabolism , STAT5 Transcription Factor/genetics , Uncoupling Protein 1/metabolism , 3T3-L1 Cells , Abdominal Fat/metabolism , Adipocytes/metabolism , Animals , Binding Sites , Blood Glucose/metabolism , Body Weight , Calcium Phosphates/metabolism , Diet, High-Fat , HEK293 Cells , Humans , Lipids/blood , Male , Mice , Mice, Inbred C57BL , Mice, Obese , Obesity/metabolism , Phosphorylation , Polymerase Chain Reaction , Promoter Regions, Genetic , RNA, Messenger/metabolism , Uncoupling Protein 1/genetics
5.
J Biomed Sci ; 23(1): 56, 2016 Jul 29.
Article in English | MEDLINE | ID: mdl-27473111

ABSTRACT

BACKGROUND: The impact of vitamin D3 (VD3) on obesity has been reported in the past. Our study was aimed at investigating the possible mechanisms by which VD3 affects obesity induced by a high fat diet. METHODS: Eight-week-old C57BL/6 J male mice were fed a normal- or high-fat diet for 9 weeks and were treated with a gavage of vehicle (corn oil) or cholecalciferol (50 µg/kg, daily). Body weight, white adipose tissue weight, blood lipid and glucose levels were measured. In addition, we investigated the expression of 1,25(OH)2D3 (calcitriol)/VDR-regulated genes involved in energy and lipid metabolism, such as of uncoupling protein 3 (UCP3), by using qRT-PCR in the liver, adipose tissue, skeletal muscle and C2C12, L6, and H-EMC-SS cells. We also measured UCP3 promoter transcription in the same cell lines using a Dual Luciferase Assay. Furthermore, we analyzed the binding site consensus sequences of VDR on the UCP3 promoter. RESULTS: Mice consuming a high-fat diet treated with cholecalciferol had lower body weight and adipose tissue weight and higher expression of UCP3 compared to the other treatment groups. Changes in the expression of genes correlated with calcitriol/VDR. Luciferase activity was dose-dependently associated with calcitriol/VDR levels. We confirmed the functional VDR binding site consensus sequences at -2200, -1561, -634, and +314 bp in the UCP3 promoter region. CONCLUSION: We suggest that VD3/VDR inhibits weight gain by activating UCP3 in the muscles.


Subject(s)
Calcitriol/pharmacology , Cholecalciferol/pharmacology , Gene Expression Regulation/drug effects , Muscle Proteins/biosynthesis , Muscle, Skeletal/metabolism , Obesity/metabolism , Receptors, Calcitriol/metabolism , Uncoupling Protein 3/biosynthesis , Animals , Dietary Fats/adverse effects , Dietary Fats/pharmacology , Male , Mice , Muscle Proteins/genetics , Muscle, Skeletal/pathology , Obesity/chemically induced , Obesity/genetics , Obesity/pathology , Receptors, Calcitriol/genetics , Uncoupling Protein 3/genetics
6.
J Mol Endocrinol ; 57(1): 23-32, 2016 07.
Article in English | MEDLINE | ID: mdl-27154546

ABSTRACT

The active form of vitamin D3 (1α,25(OH)2D3, also known as calcitriol) controls the expression of target genes via the vitamin D receptor (VDR). Vitamin D-dependent rickets type II (VDDRII) is a congenital disease caused by inactivating mutations in the VDR The condition is treated with high doses of calcitriol, but the therapeutic effects of other synthetic VD3 analogs have not yet been investigated. In the present study, we analyzed the transcriptional activity of seven different VD3 analogs with VDRs carrying ligand-binding domain mutations identified in VDDRII patients. Wild-type VDR (WT-VDR) and seven mutant VDRs were expressed in TSA201 human embryonic kidney cells, HepG2 human liver cancer cells, and MC3T3-E1 mouse calvaria cells, and their transcriptional activation with VD3 analogs were analyzed by performing transient expression assays, western blotting, and quantitative real-time PCR. The results demonstrated that falecalcitriol stimulated significantly higher transcriptional activation of the WT-VDR and some mutant VDRs than did calcitriol. Calcitriol showed almost no transcriptional activation of the VDR with the I268T mutation identified in a severe case of VDDRII, whereas falecalcitriol caused a dose-dependent increase in the activation of this mutant VDR. Our findings demonstrate that falecalcitriol has a VDR activation profile distinct from that of calcitriol and may exhibit therapeutic effects even on difficult-to-treat VDDRII cases resistant to calcitriol. It is also possible that VDDRII patients responding to high doses of calcitriol could be appropriately treated with low doses of falecalcitriol.


Subject(s)
Cholecalciferol/pharmacology , Gene Expression Regulation/drug effects , Mutation , Receptors, Calcitriol/genetics , Transcription, Genetic , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/genetics , Animals , Calcitriol/analogs & derivatives , Calcitriol/pharmacology , Cell Line , Cholecalciferol/analogs & derivatives , Humans , Mice , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Calcitriol/metabolism
7.
Growth Horm IGF Res ; 29: 57-62, 2016 08.
Article in English | MEDLINE | ID: mdl-27150070

ABSTRACT

OBJECTIVE: To determine if and how growth hormone (GH) signaling is involved in energy metabolism. DESIGN: We used human embryonic kidney TSA201 cells, human H-EMC-SS chondrosarcoma cells, rat L6 skeletal muscle cells, and murine C2C12 skeletal muscle myoblasts to investigate GH-induced expression of uncoupling protein2 (UCP2) to the GHR/JAK/STAT5 pathway by a combination of a reporter assay, electrophoretic mobility shift assay (EMSA), real-time quantitative PCR, Western blotting. RESULTS: We demonstrated that the regulation energy metabolism, which was hypothesized to be directly acted on by GH, involves UCP2 via activated STAT5B, a signal transducer downstream of GH. We also showed that the sequence at the -586 'TTCnGA' may function as a novel putative consensus sequence of STAT5s. CONCLUSION: The results suggest that GH regulates energy metabolism directly in myocytes and that UCP2 participates in the signal transduction pathway that functions downstream of the GHR/JAK/STAT.


Subject(s)
Chondrosarcoma/metabolism , Energy Metabolism/genetics , Growth Hormone/metabolism , Janus Kinase 2/metabolism , Muscle Fibers, Skeletal/metabolism , STAT5 Transcription Factor/metabolism , Uncoupling Protein 2/genetics , Animals , Blotting, Western , Cell Line, Tumor , Chondrosarcoma/genetics , Electrophoretic Mobility Shift Assay , Gene Expression Regulation , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , Janus Kinases/metabolism , Rats , Real-Time Polymerase Chain Reaction , Receptors, Somatotropin/metabolism , Signal Transduction
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