Regulation of circadian gene expression in liver by systemic signals and hepatocyte oscillators.

The mammalian circadian timing system has a hierarchical structure, in that a master pacemaker located in the suprachiasmatic nuclei (SCN) coordinates slave oscillators present in virtually all body cells. In both the SCN and peripheral organs, the rhythm-generating oscillators are self-sustained and cell-autonomous, and it is likely that the molecular makeup of master and slave oscillators is nearly identical. However, due to variations in period length, the phase coherence between peripheral oscillators in intact animals must be established by daily signals emanating directly or indirectly from the SCN master clock. The synchronization of individual cellular clocks in peripheral organs is probably accomplished by immediate-early genes that interpret the cyclic systemic signals and convey this phase information to core clock components. This model predicts that circadian gene expression in peripheral organs can be influenced either by systemic signals emanating from the SCN master clock, local oscillators, or both. We developed a transgenic mouse strain in which hepatocyte clocks are only operative when the tetracycline analog doxycycline is added to the food or drinking water. The genome-wide mapping of genes whose cyclic expression in liver does not depend on functional hepatocyte oscillators unveiled putative signaling pathways that may participate in the phase entrainment of peripheral clocks.

The CAG repeat polymorphism in the AR gene affects high density lipoprotein cholesterol and arterial vasoreactivity.

Genomic effects of T are exerted via the AR. The length of the polymorphic CAG repeat sequence in the AR gene is inversely correlated with the transcriptional regulation of target genes by T. In 110 healthy men (20-50 yr), we investigated the interactions among this polymorphism, serum levels of sex hormones, cardiovascular risk factors, and flow-mediated and nitrate-induced vasodilatation of the brachial artery. The number of CAG repeat had no significant correlations with serum concentrations of total or free T. Stepwise multiple regression analysis revealed positive correlations of the number of CAG repeat with serum levels of high density lipoprotein cholesterol (partial r = 0.44; P < 0.001) and flow-mediated vasodilatation (partial r = 0.37; P < 0.001). The association of CAG repeat with high density lipoprotein (HDL) cholesterol was independent of body fat content and serum levels of free T, which both had significant negative correlations with HDL cholesterol. The association of CAG repeat with flow-mediated vasodilatation was independent of cigarette smoking and serum levels of free T and low density lipoprotein cholesterol, which also were correlated with flow-mediated vasodilatation. We conclude that a low number of CAG repeat in the AR gene implies a greater chance for low levels of HDL cholesterol and reduced endothelial response to ischemia, which are both important risk factors for coronary heart disease.

Analysis of circadian liver gene expression by ADDER, a highly sensitive method for the display of differentially expressed mRNAs.

We describe a novel and highly sensitive method for the differential display of mRNAs, called ADDER (Amplification of Double-stranded cDNA End Restriction fragments). The technique involves the construction and PCR amplification of double-stranded cDNA restriction fragments complementary to 3'-terminal mRNA sequences. Aliquots of these cDNA fragments are then amplified by touchdown PCR with 192 pairs of display primers (16 upstream primers and 12 downstream primers) that differ in their ultimate and penultimate nucleotides and the PCR products are compared by size-fractionation on urea-polyacrylamide sequencing gels. By using the ADDER technology for the comparison of liver RNAs harvested at different times around the clock we detected nearly 300 cDNA fragments complementary to mRNAs with circadian accumulation profiles and sequenced 51 of them. The majority of these cDNAs correspond to genes which were not previously known to be rhythmically expressed. A large fraction of the identified genes encoded factors involved in the processing and detoxification of nutrients. This suggests that a primary purpose of circadian transcription in the liver is the anticipation of food processing and detoxification. Several genes involved in human disease were also identified, including the one encoding presenilin II, a protein implicated in the development of Alzheimer's DISEASE:

The CAG repeat polymorphism in the androgen receptor gene affects bone density and bone metabolism in healthy males.

OBJECTIVE: Bone metabolism and bone density (BD) are influenced by sex hormones. Testosterone (T) action is exerted through the androgen receptor (AR). We investigated the potential impact of the CAG repeat (CAGR) polymorphism within the AR gene on BD and bone metabolism in healthy younger males. PATIENTS AND MEASUREMENTS: The number of CAGRs in 110 healthy men aged 20-50 years was determined by sequence analysis. We assessed BD by the radiation-free method of quantitative ultrasound (QUS) of the phalanges. Serum levels of bone-specific alkaline phosphatase (BAP) and urine secretion of free deoxypyridinoline (DPD, corrected for creatinine), serum levels of sex hormones, body fat content and lifestyle factors were determined. RESULTS: In stepwise multiple regression models controlling for age, body fat content and lifestyle factors, the number of CAGRs was an independent negative predictor of BD (partial r = - 0.286, P = 0.001), whereas it was positively associated with markers of bone turnover (for BAP: partial r = 0.32, P= 0.001; for DPD: partial r = 0-241, P = 0.013). Levels of free T and oestradiol showed an independent and positive association with BD; age contributed significantly to lower BD. Age and free T were negatively associated with markers of bone turnover, whereas oestradiol showed a positive correlation with BAP and DPD. ANOVA in groups according to age and the CAGR length suggested an increased age-dependent bone loss in subjects with a CAGR length of 22-31 compared with 14-21 CAGRs (overall P < 0.01). CONCLUSIONS: A high number of CAG repeats within the androgen receptor gene attenuates testosterone effects on bone density and bone metabolism. This seems to be associated with accelerated age-dependent bone loss.

Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus.

In mammals, circadian oscillators exist not only in the suprachiasmatic nucleus, which harbors the central pacemaker, but also in most peripheral tissues. It is believed that the SCN clock entrains the phase of peripheral clocks via chemical cues, such as rhythmically secreted hormones. Here we show that temporal feeding restriction under light-dark or dark-dark conditions can change the phase of circadian gene expression in peripheral cell types by up to 12 h while leaving the phase of cyclic gene expression in the SCN unaffected. Hence, changes in metabolism can lead to an uncoupling of peripheral oscillators from the central pacemaker. Sudden large changes in feeding time, similar to abrupt changes in the photoperiod, reset the phase of rhythmic gene expression gradually and are thus likely to act through a clock-dependent mechanism. Food-induced phase resetting proceeds faster in liver than in kidney, heart, or pancreas, but after 1 wk of daytime feeding, the phases of circadian gene expression are similar in all examined peripheral tissues.