Expression patterns of clock genes in the pancreas of the rat

Introduction: All higher vertebrates have developed mechanisms to measure time and regulate daytime-dependent body functions during evolution. Thus, „biological clocks“ have evolved which function as rhythm generators. Within mammals, the central circadian oscillator is situated in a paired neuronal structure of the hypothalamus, the suprachiasmatic nucleus (SCN). Since the biological clock generates periods of circa, but not quite exactly 24 hr, the phase needs to be synchronized. The most important synchronizer for SCN-generated rhythms is the daylight. Besides the central circadian oscillator, „second-order clocks“ in organs like the liver or pancreas have been described. Synchronization of clocks of the periphery proceeds by largely unknown zeitgeber factors other than light. The circadian rhythm itself is generated by the action of so-called clock genes with either transcriptionally positive (e.g. Bmal1/Clock) or negative (e.g. Per1, Cry1) function. The genes influence each others expression, forming a circadian loop and they also regulate the expression of clock-controlled output genes.

Materials and Methods: After six week old Wistar rats were anaesthetised, the pancreas was removed immediately and stored in a conservation liquid RNAlater (Ambion Inc.). RNA extraction of the tissue was performed with Trizol™ (Invitrogen).

For quantitative evaluation of the mRNA, the cDNA was amplified after reverse transcription, through 40 cycles under standard conditions in a real-time PCR device (Corbett Res. Inc.) using specific primers. As a standard reference for expression levels, the mRNA of the house-keeping gene β-Actin was measured.

Results: Quantitative evaluation of the clock gene transcripts of Per1 and Bmal1 in a circadian series displayed a characteristic profile with an expression maximum for Per1- and an expression minimum for Bmal1 transcripts at T11. This anti-phasic profile of the two clock genes is characteristic for a functional circadian oscillator. In addition, the expression of the clock genes Per2, Cry1, Clock and Tim was monitored. In contrast to Per2 and Cry1, Clock and Tim transcripts did not display a circadian rhythm. The analysis of clock-gene-controlled output genes Dbp and RevErba revealed a high amplitude for both transcripts in-phase with Per1.

Discussion: The results of antagonistic circadian expression patterns of the crucial clock genes Per1 and Bmal1 and of the circadian expression of Per2 and Cry1 speak in favour of a functional peripheral oscillator in the rat pancreas. Since the clock-controlled output genes Dbp and RevErba also display a circadian expression, this fact indicates that the heterodimeric transcription factor BMAL1/CLOCK regulates the transcription of Dbp and RevErba through E-box-elements of their promoters. Taken together, the results indicate, that the circadian pattern of insulin secretion, as observed with isolated rat pancreatic islets in perifusion, can be explained by the action of a peripheral circadian oscillator. The synchronising factors of this oscillator are as yet unknown The indolamine melatonin might, however, be a zeitgeber of importance. Whether the modern lifestyle of men leads to disruption of circadian processes and desynchronization of the pancreatic oscillator is an open question and needs to be discussed with respect to the development of type 2 diabetes.