Metabolic States and Circadian Oscillators
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Metabolic States and Circadian Oscillators
In organisms as diverse as fruit flies and mammals, circadian rhythms are controlled by a transcriptional feedback system whose activity fluctuates as a function of the light-dark cycle. In mammals, the master clock (circadian pacemaker) resides in the SCN (Suprachiasmatic Nucleus) of the brains hypothalamus and this endogenous clock drives physiology and behavior. In the absence of external time cues, the SCN master clock generates cycles of approximately but not exactly 24 hours, and its phase must therefore be readjusted every day. This task depends on the retina, which detects changes in light intensity during the days light-dark cycle (the photoperiod) and transmits this information to the SCN neurons (Ref.1).

The mammalian circadian feedback loop in gene expression is established by the BMAL1 (Brain and Muscle ARNT-Like-1) and Clk (Clock) transcriptional activator proteins and by the Cry (Cryptochromes) transcriptional repressor proteins. Clk is a basic helix-loop-helix transcription factor that binds to E-box (CACGTG) DNA motifs when partnered with BMAL1 (Ref.2). The Clk: BMAL1 heterodimer stimulates the expression of other essential pacemaker components, such as the period proteins Per1 and Per2 and the Cry1 and Cry2. The Crys repress the transcription of target genes switched on by Clk: BMAL1 , and thereby establish a negative feedback loop in which Per and Cry gene expression switches off Clk: BMAL1 transcriptional activity (Ref.3). In some brain regions, NPAS2 (Neuronal PAS domain protein-2, also called MOP4) substitutes for Clk. BMAL1 can bind to Clk or NPAS2, and these heterodimers activate transcription. Alternatively, BMAL1 can form homodimers with itself that do not activate transcription. The formation of the Clk: BMAL1 and NPAS2: BMAL1 heterodimers and their binding to DNA is stimulated by reduced NADH (Nicotinamide Adenine Dinucleotide) and inhibited by oxidized NAD. These heterodimers enhance the expression of the Clk genes Cry and Per and the Clk output gene Ldh (Lactate Dehydrogenase)(Ref.4). Cry proteins repress Clk: NPAS2-mediated gene activation, possibly by oxidizing the NAD+ cofactors associated with these proteins. Conceivably, the negative action of Cry proteins on Clk-NPAS2 is reinforced by Ldh, which may increase the cellular concentration of NAD+. Ldh has the potential to generate either NAD or NADH in the reversible conversion between pyruvate and lactate. The DNA binding activity of both NPAS2: BMAL1 and Clk: BMAL1 , the components utilized by the cellular clock in the SCN, are exquisitely sensitive to the cells redox state. When bound to BMAL1 , both of the NADH and NAD cofactors recognize E-box motifs in DNA recognition sequences. The reduced and oxidized NAD (P) electron carriers have inverse effects on the proportion of NPAS2: BMAL1 (or Clk: BMAL1 ) heterodimers to BMAL1 : BMAL1 homodimers that bind to DNA. Although the heterodimeric interaction is greatly stimulated by NAD(P)H, it is strongly inhibited by NAD(P)+. As a consequence, only BMAL1 homodimers (whose affinity for DNA is not affected by NAD cofactors) occupy E-box motifs at a low NAD (P) H/NAD (P)+ ratio. Because BMAL1 homodimers are incapable of activating transcription, the susceptibility of the heterodimer to this redox potential establishes a molecular switch for activating the Clk: BMAL1 transcription complex. Redox electron transfer through NADH and NAD may also provide an elegant way for Crys to inhibit the activity of Clk and NPAS2. The interaction of Crys with NPAS2 provokes electron transfer from NPAS2-associated NAD (P) H to Cry-associated FAD (Flavin Adenine Dinucleotide), and finally to the heme cofactor bound by NPAS2 (Ref.5).

The unexpected relationship between circadian clock proteins and redox potential adds a new wrinkle to chronobiology research. The principal sites of NPAS2 expression include the somatosensory cortex, visual cortex, auditory cortex, olfactory tubercles, striatum, and accumbens nucleus. These regions of the forebrain process sensory information, including touch, pain, temperature, vision, hearing, and smell, as well as emotive behaviors such as fear and anxiety. During wakefulness, these NPAS2-expressing regions of the brain receive and process stimulatory neuronal activity that entrains the molecular clock in the manner similar to the retino-SCN pathway (Ref.6).