SUMMARY Emerging evidence suggests that microbes resident in the human intestine represent a key environmental factor contributing to obesity-associated disorders. Here we demonstrate that the gut microbiota-initiated trimethylamine-N-oxide (TMAO)-generating pathway is linked to obesity and energy metabolism. In multiple clinical cohorts, systemic levels of TMAO were observed to strongly associate with type 2 diabetes. In addition, circulating TMAO levels were associated with obesity traits in the different inbred strains represented in the Hybrid Mouse Diversity Panel. Further, antisense oligonucleotide-mediated knockdown or genetic deletion of the TMAO-producing enzyme, flavin-containing monooxygenase 3 (FMO3), conferred protection against obesity in mice. Complimentary mouse and human studies indicate a negative regulatory role for FMO3 in the beiging of white adipose tissue. Collectively, our studies reveal a link between the TMAO-producing enzyme FMO3 and obesity and the beiging of white adipose tissue.

Heat shock transcription factor 1 (HSF-1) activates the transcription of heat shock genes in eukaryotes. Under normal physiological growth conditions, HSF-1 is a monomer. Its transcriptional activity is repressed by constitutive phosphorylation. Upon activation, HSF-1 forms trimers, acquires DNA binding activity, increases transcriptional activity, and appears as punctate granules in the nucleus. In this study, using bromouridine incorporation and confocal laser microscopy, we demonstrated that newly synthesized pre-mRNAs colocalize to the HSF-1 punctate granules after heat shock, suggesting that these granules are sites of transcription. We further present evidence that glycogen synthase kinase 3␤ (GSK-3␤) and extracellular signal-regulated kinase mitogen-activated protein kinase (ERK MAPK) participate in the down regulation of HSF-1 transcriptional activity. Transient increases in the expression of GSK-3␤ facilitate the disappearance of HSF-1 punctate granules and reduce hsp-70 transcription after heat shock. We have also shown that ERK is the priming kinase for GSK-3␤. Taken together, these results indicate that GSK-3␤ and ERK MAPK facilitate the inactivation of activated HSF-1 after heat shock by dispersing HSF-1 from the sites of transcription.The nuclear translocation, DNA binding, and transcriptional activities of most mammalian transcription factors are regulated by phosphorylation. In many cases, multiple protein kinases can act on a single transcription factor (reviewed in reference 27). Heat shock transcription factor 1 (HSF-1) is subject to complex regulation by phosphorylation. HSF-1 binds to conserved regulatory sequences known as heat shock elements (HSEs) and controls the expression of heat shock proteins in response to chemical, environmental, and physiological stresses (1,36,42,61,68,74).Under normal physiological growth conditions, mammalian HSF-1 exists in a latent, monomeric form (4,55,66,69); is constitutively phosphorylated (4, 10, 43, 55); and is distributed in both the cytoplasm and nucleus. The functional role of phosphorylation in HSF-1 regulation is unclear. Strong evidence suggests that constitutive phosphorylation of HSF-1 negatively regulates HSF-1 activity (9, 31, 32, 43). Upon heat shock, the latent form of HSF-1 is translocated into the nucleus, forms trimers, is hyperphosphorylated, and appears as punctate granules (4,44,51,55). The function of hyperphosphorylation (4,10,46,55) or the role of HSF-1 punctate granules is not known. Punctate granules have been suggested to be important for some activity of HSF-1, perhaps its DNA binding activity (58).Phosphorylated forms of HSF-1 have been extensively studied by phosphopeptide mapping as well as mutational analysis (30)(31)(32)71). The data suggest that HSF-1 is phosphorylated on multiple serine residues and, perhaps, a threonine residue. Constitutive phosphorylation of serine 307, which is located distal to the transcriptional activation domain, negatively regulates HSF-1 function, since mutation of serine 307 to alanine (31, 71)...

The activation of heat shock transcription factor-1 (HSF-1) after treatment of mammalian cells with stresses such as heat shock, heavy metals, or ethanol induces the synthesis of heat shock proteins. HSF-1 is phosphorylated at normal growth temperature and is hyperphosphorylated upon stress. We recently presented evidence that HSF-1 can be phosphorylated by the mitogen activated protein kinase, ERK1, and that such phosphorylation appears to negatively regulate the activity of HSF-1. In this report, we have tested the ability of ERK1 to phosphorylate various HSF-1 deletion mutants. Our results show that ERK1 phosphorylation is dependent on a region of HSF-1 extending from amino acids 280 to 308. This region contains three serine residues that are potential ERK1 phosphorylation sites. The region falls within a previously defined regulatory domain of HSF-1. The possibility of protein kinases other than ERK1 phosphorylating HSF-1 was also examined using in-gel kinase assays. The results show that HSF-1 can be phosphorylated in a ras-dependent manner by other members of the MAP kinase family such as JNK and p38 protein kinases and possibly others.

The aim of this study was to examine the role of the phosphatidylinositol 3-kinase (PI3K)/serine/threonine kinase Akt signaling pathway in mediating interactions between angiotensin II (Ang II) and insulin-like growth factor-1 (IGF-1) in regulation of inducible nitric oxide synthase (iNOS) in vascular smooth muscle cells (VSMCs). Exposure to 100 nM IGF-1 for 10 min resulted in increased insulin-receptor substrate-1 associated PI3K activity and Akt kinase activity, whereas 100 nM Ang II pretreatment for 5 min strikingly decreased these IGF-1 effects. NOS activity was also increased in VSMCs following exposure to IGF-1 (10 min up to 24 h). Pretreatment with Ang II for 5 min reduced IGF-1-induced NOS activity. IGF-1 treatment for 24 hr increased iNOS gene transcription, and Ang II pretreatment reduced this stimulation of iNOS gene expression by attenuating PI3K/Akt signaling. These results implicate PI3K/ Akt pathways in Ang II/IGF-1 regulation of iNOS in VSMCs.