Circadian Rhythms, SCN, and Metabolism

Definition: Circadian rhythms are biochemical, cellular, or physiological patterns synchronized to a 24-hour clock (e.g., body temperature, blood pressure, melatonin, reaction times).
Master Clock: The Suprachiasmatic Nucleus (SCN), located in the hypothalamus, serves as the master clock coordinating all circadian rhythms.
Discovery: First measured in mammals (rats) in 1912 (locomotor activity). The SCN was identified as the origin in the 1970s through ablation and transplantation studies.

Synchronization: Circadian rhythms are synchronized to a 24-hour period by external cues called Zeitgebers (time givers).
Photic Cues: Light is the most powerful Zeitgeber. Light exposure during normal activity has little effect; light at night shifts rhythms.
Non-Photic Cues: Include exercise, social interaction, stress, and regulated feeding time. These can also adjust the clock.
Photic Signaling: Driven by glutamate, binding to SCN cell receptors, altering phosphorylation and period length.
Non-Photic Signaling: Involves other brain regions and neurotransmitters like serotonin and neuropeptide Y.
Phase Response Curve: Describes how light pulses at different times can cause phase delays (waking later) or phase advances (waking earlier) in an animal's activity.

Genetic Basis: Circadian rhythms are genetically driven patterns of transcription and translation.
Clock Genes: First cell-autonomous rhythms described in 1995. Key protein products are bMAL and Clock.
Feedback Loop: bMAL and Clock bind to E-box promoter regions, increasing transcription/translation of Period (Per) and Cryptochrome (Cry) genes.
Negative Feedback: High levels of Period and Cryptochrome are phosphorylated by casein kinase 1 epsilon, leading to their degradation. When Per/Cry levels are high, they negatively inhibit bMAL/Clock transcription/translation, creating a 24-hour oscillatory cycle.

Novel Discovery (O'Neil & Reddy, 2011): Demonstrated circadian rhythms of the metabolic protein peroxiredoxin in enucleated red blood cells, suggesting a metabolic clock independent of transcription/translation.
Metabolic Links:
- Restricted feeding can prevent weight gain in animals on a high-fat diet, altering metabolism.
- Degradation of Cryptochrome 1 (Cry1) in liver cells increases metabolism (e.g., gluconeogenesis).
- Disruption of circadian rhythms (e.g., shift work, misaligned feeding) is linked to metabolic dysfunction (e.g., type 2 diabetes), cardiovascular events, and cancer.
- High-fat diets can disrupt circadian rhythms themselves.

Goal: Investigate if red blood cells can communicate metabolic parameters to SCN cells.
Methodology:
- Measuring peroxiredoxin activity in red blood cells (found dampening in high glucose medium).
- Utilizing SCN 2.2 cells (modified fibroblast line mimicking SCN function) in culture.
- Using Transwell inserts to physically separate red blood cells and SCN cells while allowing communication.
- Transfection: SCN 2.2 cells are transfected with a plasmid (containing luciferase linked to a circadian gene like period) and selected using blasticidin resistance to measure metabolism.
- Future Steps: Measure SCN 2.2 cell metabolism using an ATP kit, then assess if red blood cells alter SCN cell metabolism. This would show the SCN responding to metabolism, rather than solely dictating it.