DNA-PK: Relaying the insulin signal to USF in lipogenesis

Abstract

To meet the constant energy requirement in the face of highly variable food supply, mammals employ intricate and precise mechanisms for energy storage. When total energy intake is in excess of energy expenditure such as after a meal, excess carbohydrates are converted to fatty acids (de novo lipogenesis). Excess fatty acids are then converted to triacylglycerol to be stored in adipose tissue and released as oxidative fuels for other tissues during times of energy need such as fasting and exercise. In sustaining the balance between energy excess and energy deficiency, the process of lipogenesis is tightly controlled by nutritional and hormonal conditions1. Thus, enzymes involved in fatty acid and fat synthesis are tightly and coordinately regulated during fasting/feeding: Activities of these enzymes are very low in fasting due to the increase in glucagon/cAMP levels. Conversely, in the fed condition, especially after a high carbohydrate meal, activities of these enzymes drastically increase as blood glucose and insulin levels rise1. Fatty acid synthase (FAS), a central lipogenic enzyme, plays a crucial role in de novo lipogenesis by catalyzing all of the seven reactions involved in fatty acid synthesis. While FAS is not known to be regulated by allosteric effectors or by covalent modification, its transcription is exquisitely regulated by fasting/feeding and by insulin1. The FAS promoter thus provides an excellent model system to dissect the transcriptional activation of lipogenesis by feeding/insulin. In early studies of insulin regulation of the FAS promoter, we found that Upstream Stimulatory Factor (USF) binding to the -65 E-box is required for transcriptional activation by insulin2,3,4. The critical role of USF in the activation of the FAS promoter by insulin was further verified by overexpressing dominant negative or wild type forms of USF2,3. The induction of FAS by fasting/feeding was significantly impaired in USF knockout mice5. Functional analysis and chromatin immunoprecipitation (ChIP) in mice transgenic for various 5′ deletions and mutations of the FAS promoter-CAT reporter gene showed that USF binding to the -65 E-box is required for feeding/insulin-mediated FAS promoter activation in vivo6. Notably, USF binding was detected in both fasted and fed states. On the FAS promoter, USF recruits another transcription factor SREBP-1, whose level increases upon insulin treatment via the PI3K pathway7, to bind the -150 SRE and mediate insulin/feeding responsiveness8. Although early studies of ectopically expressed SREBP-1 in cultured cells has been shown to bind the -65 E-box9, the functional analysis and chromatin immunoprecipitation (ChIP) in mice transgenic for various 5′ deletions and mutations of the FAS promoter-CAT reporter gene clearly showed SREBP-1 binds the -150 SRE, but not -65 E-box to activate the FAS promoter during feeding/insulin treatment in vivo6. Although SRBEP-1c binding to the -150 SRE is critical for the feeding/insulin response, SREBP-1c itself cannot bind the SRE without being recruited by USF, which is constitutively bound to the -65 E-box4, 6,10. Many lipogenic promoters contain a closely spaced E-box and SRE in the proximal promoter region, and a similar mechanism for activation of several lipogenic genes has been documented previosuly4. Possibly, the SREBP-1c promoter is also regulated by USF and SREBP-1c in response to feeding/insulin. Thus, USF, along with SREBP-1c, plays a critical role in mediating the transcriptional activation of lipogenesis in response to feeding/insulin. Studies have shown that LXR may play a role in the transcriptional regulation of lipogenesis by activating SREBP-1c transcription11. LXR has also been reported to directly regulate the FAS promoter in cultured cells12. A carbohydrate response element (ChoRE) where ChREBP can bind has also been reported to be present far upstream of the FAS promoter region13. Nevertheless, FAS promoter-reporter transgenic mice studies showed that the FAS promoter that contains both an E-box and SRE, but lacks a LXRE or ChoRE, is sufficient for high level activation of the FAS promoter during fasting/feeding suggesting that binding of LXR or ChREBP may not be critical in vivo6. Regardless, questions remain in understanding the FAS promoter activation involving USF and SREBP. Apart from HAT/HDAC, are other coactivators required for activation? What chromatin remodeling machinery and mediators are recruited to the FAS promoter? Are there common mechanisms to explain the transcriptional regulation of other coordinately regulated lipogenic genes? Is chromatin folding involved in sharing transcription machineries among lipogenic gene promoters? Further studies are necessary to understand the details of the transcriptional activation of lipogenic genes. While many metabolic effects of insulin are mediated through protein phosphorylation via the well characterized PI3K cascade which activates PKB/Akt, insulin can also