Lipogenesis is exquisitely regulated by nutritional/hormonal states. Transcription of fatty acid synthase (FAS), a central enzyme in lipogenesis, is low in fasting but increases drastically with feeding. In transcriptional activation of FAS by feeding/insulin, USF constitutively bound to the −65 E-box is required. Here, we show that USF functions as a molecular switch by recruiting various interacting proteins during the fasting/feeding transition. During feeding/insulin, USF-1 recruits and is phosphorylated by DNA-PK, which is dephosphorylated/activated by PP1. Phosphorylation of USF-1 allows recruitment of and acetylation by P/CAF, resulting in the FAS promoter activation. In fasting, USF-1 is deacetylated by HDAC9 causing the promoter inactivation. DNA break/repair components associated with USF also bring about transient DNA breaks during feeding-induced FAS activation. In DNA-PK deficient SCID mice, feeding induced USF-1 phosphorylation/acetylation, DNA-breaks, and FAS activation leading to lipogenesis are impaired, resulting in decreased liver and circulating triglyceride levels. Our study demonstrates that DNA-PK mediates the feeding/insulin-dependent lipogenic gene activation.

Summary Uncoupling protein 1 (UCP1) mediates non-shivering thermogenesis and, upon cold exposure, is induced in BAT and subcutaneous white adipose tissue (iWAT). Here, by high-throughput screening using the UCP1 promoter, we identify Zfp516 as a novel transcriptional activator of UCP1 as well as PGC1α thereby promoting a BAT program. Zfp516 itself is induced by cold and sympathetic stimulation through the cAMP-CREB/ATF2 pathway. Zfp516 directly binds to the proximal region of the UCP1 promoter, not to the enhancer region where other transcription factors bind, and interacts with PRDM16 to activate the UCP1 promoter. Although ablation of Zfp516 causes embryonic lethality, knockout embryos still show drastically reduced BAT mass. Overexpression of Zfp516 in adipose tissue promotes browning of iWAT even at room temperature, increasing body temperature, energy expenditure, and preventing diet-induced obesity. Zfp516 may represent a future target for obesity therapeutics.

Canonical transient receptor potential (TRPC) channels are Ca 2؉ -permeable nonselective cation channels that are widely expressed in numerous cell types. Seven different members of TRPC channels have been isolated. The activity of these channels is regulated by the filling state of intracellular Ca 2؉ stores and͞or diacylglycerol and͞or Ca 2؉ ͞ calmodulin. However, no evidence is available as to whether TRPC channels are regulated by direct phosphorylation on the channels. In the present study, TRPC isoform 3 (TRPC3) gene was overexpressed in HEK293 cells that were stably transfected with protein kinase G (PKG). We found that the overexpressed TRPC3 mediated store-operated Ca 2؉ influx and that this type of Ca 2؉ influx was inhibited by cGMP. The inhibitory effect of cGMP was abolished by KT5823 or H8. Point mutations at two consensus PKG phosphorylation sites (T11A and S263Q) of TRPC3 channel markedly reduced the inhibitory effect of cGMP. In addition, TRPC3 proteins were purified from HEK293 cells that were transfected with either wild-type or mutant TRPC3 constructs, and in vitro PKG phosphorylation assay was carried out. It was found that wild-type TRPC3 could be directly phosphorylated by PKG in vitro and that the phosphorylation was abolished in the presence of KT5823. The phosphorylation signal was greatly reduced in mutant protein T11A or S263Q. Taken together, TRPC3 channels could be directly phosphorylated by PKG at position T11 and S263, and this phosphorylation abolished the store-operated Ca 2؉ influx mediated by TRPC3 channels in HEK293 cells.

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Abstract-TRPC1 (transient receptor potential canonical 1) is a Ca 2ϩ -permeable cation channel involved in diverse physiological function. TRPC1 may associate with other proteins to form a signaling complex, which is crucial for channel function. In the present study, we investigated the interaction between TRPC1 and large conductance Ca 2ϩ -sensitive K ϩ channel (BK Ca ). With the use of potentiometric fluorescence dye DiBAC 4 (3), we found that store-operated Ca 2ϩ influx resulted in membrane hyperpolarization of vascular smooth muscle cells (VSMCs). The hyperpolarization was inhibited by an anti-TRPC1 blocking antibody T1E3 and 2 BK Ca channel blockers, charybdotoxin and iberiotoxin. These data were confirmed by sharp microelectrode measurement of membrane potential in VSMCs of intact arteries. Furthermore, T1E3 treatment markedly enhanced the membrane depolarization and contraction of VSMCs in response to several contractile agonists including phenylephrine, endothelin-1, and U-46619. In coimmunoprecipitation experiments, an antibody against BK Ca ␣-subunit [BK Ca (␣)] could pull down TRPC1, and moreover an anti-TRPC1 antibody could reciprocally pull down BK Ca (␣). Double-labeling immunocytochemistry showed that TRPC1 and BK Ca were colocalized in the same subcellular regions, mainly on the plasma membrane, in VSMCs. These data suggest that, TRPC1 physically associates with BK Ca in VSMCs and that Ca 2ϩ influx through TRPC1 activates BK Ca to induce membrane hyperpolarization. The hyperpolarizing effect of TRPC1-BK Ca coupling could serve to reduce agonist-induced membrane depolarization, thereby preventing excessive contraction of VSMCs to contractile agonists. (Circ Res. 2009;104:670-678.)