Insulin-like growth factor-1 directly mediates expression of mitochondrial uncoupling protein 3 via forkhead box O4.

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.

IGF-1 regulate the expression of uncoupling protein 2 via FOXO1.

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.

GH directly stimulates UCP3 expression.

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.

Transcriptional activation of the wild-type and mutant vitamin D receptors by vitamin D3 analogs.

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.

Growth hormone regulates the expression of UCP2 in myocytes.

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.