Cigarette Smoke Extract Modulates Functions of Peroxisome Proliferator-Activated Receptors.

Cigarette smoke extract (CSE) contains many toxicants and may derange the physiological processes, such as cholesterol metabolism. We examined the impact of CSE on transcriptional regulation mediated peroxisome proliferator-activated receptors (PPARs) and its interaction with cofactors to elucidate differences in the molecular mechanism between CSE and other agonists of PPARs. We constructed several mutant PPARs (mPPARs) with amino acid substitution in the ligand-binding domain, which according to the molecular modeling, may affect the binding of agonists. In transient expression assays, each wild-type peroxisome proliferator-activated receptor (PPAR) mediated transcription stimulated by CSE was faintly yet significantly elevated compared to the control. The CSE-induced transcriptional activation was abolished in the H323A, H323Y, S342A, and H449A mPPARγs, although the activation elevated by pioglitazone was reserved. In the mPPARγ with Y473A and mPPARß/δs with H286Y and Y436A, the pioglitazone-induced or L165041-activated transcriptional elevations were decreased and were lower than that of CSE-induced stimulation. These results suggested that CSE activated both mutant PPARs to be selectively different from those ligands. Mammalian two-hybrid assay illustrated that CSE could mildly recruit SRC1 or GRIP1 to the wild-type PPARγ. Representative ingredients, such as acrolein and crotonaldehyde present in CSE, could stimulate PPAR isoforms even at the toxicological concentrations and might possibly contribute to stimulatory effects. CSE mildly regulates the cholesterol metabolism-related genes, such as low density lipoprotein (LDL) receptor and Liver X receptor (LXR)ß. In conclusion, these CSE effects the nuclear hormone receptors and their cofactors thereby disturbing metabolic phenomena. Therefore, CSE might be involved in cholesterol metabolism.

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.

Chondroitin 4-O-Sulfotransferase Is Indispensable for Sulfation of Chondroitin and Plays an Important Role in Maintaining Normal Life Span and Oxidative Stress Responses in Nematodes.

Chondroitin sulfate (CS)/chondroitin (Chn) chains are indispensable for embryonic cell division and cytokinesis in the early developmental stages in Caenorhabditis elegans and mice, whereas heparan sulfate (HS) is essential for axon guidance during nervous system development. These data indicate that the fundamental functions of CS and HS are conserved from worms to mammals and that the function of CS/Chn differs from that of HS. Although previous studies have shown that C. elegans produces HS and non-sulfated Chn, whether the organism produces CS remains unclear. Here, we demonstrate that C. elegans produces a small amount of 4-O-sulfated Chn and report the identification of C41C4.1, an orthologue of the human chondroitin 4-O-sulfotransferase gene. Loss of C41C4.1 in C. elegans resulted in a decline in 4-O-sulfation of CS and an increase in the number of sulfated units in HS. C41C4.1 deletion mutants exhibited reduced survival rates after synchronization with sodium hypochlorite. Collectively, these results show for the first time that CS glycans are present in C. elegans and that the Chn 4-O-sulfotransferase responsible for the sulfation plays an important role in protecting nematodes from oxidative stress.

Impairment of embryonic cell division and glycosaminoglycan biosynthesis in glucuronyltransferase-I-deficient mice.

We have revealed that in Caenorhabditis elegans, non-sulfated chondroitin is required for normal cell division and cytokinesis at an early developmental stage, whereas heparan sulfate is essential for embryonic morphogenesis in the later stages of development. To clarify the roles of chondroitin sulfate and heparan sulfate in early embryogenesis in mammals, we generated glucuronyltransferase-I (GlcAT-I) knock-out mice by gene targeting. GlcAT-I is an enzyme required for the synthesis of both chondroitin sulfate and heparan sulfate. Here we report that mice with a deletion of GlcAT-I showed remarkable reduction of the synthesis of chondroitin sulfate and heparan sulfate and embryonic lethality before the 8-cell stage because of failed cytokinesis. In addition, treatment of wild-type 2-cell embryos with chondroitinase ABC had marked effects on cell division, although many heparitinase-treated embryos normally developed to blastocysts. Taken together, these results suggest that chondroitin sulfate in mammals, as with non-sulfated chondroitin in C. elegans, is indispensable for embryonic cell division.