The mammalian transcription factors CLOCK and BMAL1 are essential components of the molecular clock that coordinate behavior and metabolism with the solar cycle. Genetic or environmental perturbation of circadian cycles contributes to metabolic disorders including type 2 diabetes. To study the impact of the cell-autonomous clock on pancreatic β-cell function, we examined islets from mice with either intact or disrupted BMAL1 expression both throughout life and limited to adulthood. We found pronounced oscillation of insulin secretion that was synchronized with the expression of genes encoding secretory machinery and signaling factors that regulate insulin release. CLOCK/BMAL1 co-localized with the pancreatic transcription factor PDX1 within active enhancers distinct from those controlling rhythmic metabolic gene networks in liver. β-cell clock ablation in adult mice caused severe glucose intolerance. Thus cell-type specific enhancers underlie the circadian control of peripheral metabolism throughout life and may help explain its deregulation in diabetes.

Highlights d NAD + induces transcription of stress and metabolic genes through the circadian clock d BMAL1 is required for chromatin remodeling and HSF1 recruitment in response to NAD + d NAD + regulates PER2 acetylation and localization to control feedback repression d NAD + counters age-related decline in circadian function

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In mammals, circadian rhythms are entrained to the light cycle and drive daily oscillations in levels of NAD+, a cosubstrate of the class III histone deacetylase sirtuin 1 (SIRT1) that associates with clock transcription factors. Although NAD+ also participates in redox reactions, the extent to which NAD(H) couples nutrient state with circadian transcriptional cycles remains unknown. Here we show that nocturnal animals subjected to time-restricted feeding of a calorie-restricted diet (TRF-CR) only during night-time display reduced body temperature and elevated hepatic NADH during daytime. Genetic uncoupling of nutrient state from NADH redox state through transduction of the water-forming NADH oxidase from Lactobacillus brevis (LbNOX) increases daytime body temperature and blood and liver acyl-carnitines. LbNOX expression in TRF-CR mice induces oxidative gene networks controlled by brain and muscle Arnt-like protein 1 (BMAL1) and peroxisome proliferator-activated receptor alpha (PPARα) and suppresses amino acid catabolic pathways. Enzymatic analyses reveal that NADH inhibits SIRT1 in vitro, corresponding with reduced deacetylation of SIRT1 substrates during TRF-CR in vivo. Remarkably, Sirt1 liver nullizygous animals subjected to TRF-CR display persistent hypothermia even when NADH is oxidized by LbNOX. Our findings reveal that the hepatic NADH cycle links nutrient state to whole-body energetics through the rhythmic regulation of SIRT1.

Forward genetic screens have been highly successful in revealing roles of genes and pathways in complex biological events. Traditionally these screens have focused on isolating mutants with the greatest phenotypic deviance, with the hopes of discovering genes that are central to the biological event being investigated. Behavioral screens in mice typically use simple activity-based assays as endophenotypes for more complex emotional states of the animal. They generally set the selection threshold for a putative mutant at 3 SDs (z score of 3) from the average behavior of normal animals to minimize false-positive results. Behavioral screens using a high threshold for detection have generally had limited success, with high false-positive rates and subtle phenotypic differences that have made mapping and cloning difficult. In addition, targeted reverse genetic approaches have shown that when genes central to behaviors such as open field behavior, psychostimulant response, and learning and memory tasks are mutated, they produce subtle phenotypes that differ from wild-type animals by 1 to 2 SDs (z scores of 1 to 2). We have conducted a second-generation (G2) dominant Nethyl-N-nitrosourea (ENU) screen especially designed to detect subtle behavioral mutants for open field activity and psychostimulant response behaviors. We successfully detect mutant lines with only 1 to 2 SD shifts in mean response compared with wild-type control animals and present a robust statistical and methodological framework for conducting such forward genetic screens. Using this methodology we have screened 229 ENU mutant lines and have identified 15 heritable mutant lines. We conclude that for screens in mice that use activity-based endophenotypic measurements for complex behavioral states, this G2 screening approach yields better results.

338178.120. Freely available online through the Genes & Development Open Access option.

The mechanism of circadian oscillations in mammals is cell autonomous and is generated by a set of genes that form a transcriptional autoregulatory feedback loop. While these ''clock genes'' are well conserved among animals, their specific functions remain to be fully understood and their roles in central versus peripheral circadian oscillators remain to be defined. We utilized the in vivo inducible tetracycline-controlled transactivator (tTA) system to regulate Clock gene expression conditionally in a tissue-specific and temporally controlled manner. Through the use of Secretogranin II to drive tTA expression, suprachiasmatic nucleus-and brain-directed expression of a tetO::Clock D19 dominantnegative transgene lengthened the period of circadian locomotor rhythms in mice, whereas overexpression of a tetO::Clock wt wild-type transgene shortened the period. Low doses (10 lg/ml) of doxycycline (Dox) in the drinking water efficiently inactivated the tTA protein to silence the tetO transgenes and caused the circadian periodicity to return to a wild-type state. Importantly, low, but not high, doses of Dox were completely reversible and led to a rapid reactivation of the tetO transgenes. The rapid time course of tTA-regulated transgene expression demonstrates that the CLOCK protein is an excellent indicator for the kinetics of Dox-dependent induction/repression in the brain. Interestingly, the daily readout of circadian period in this system provides a real-time readout of the tTA transactivation state in vivo. In summary, the tTA system can manipulate circadian clock gene expression in a tissuespecific, conditional, and reversible manner in the central nervous system. The specific methods developed here should have general applicability for the study of brain and behavior in the mouse.