ARTICLE REVIEW PSYC525

and lifelong absence of FXR as occurs in the FXR-null model, can result in abnormal metabolic effects that are quite different from those caused by acute, transient antagonism of this receptor. Because the FXR-null mouse was produced using Cre–loxP technology, conditional disruption of this allele after normal development has occurred can now be used to help resolve this issue. An alternative explanation is that the site(s) of pharmacological action of guggulsterone do not include all of the tissues in which FXR is functional, such as the liver and gut (i.e. although FXR synthesis is uniformly absent from all tissues of the FXR-null mouse model, guggulsterone might antagonize FXR only within a subset of these sites). In the absence of in vivo data regarding the modulation of FXR target gene expression by guggulsterone, this is difficult to judge. Thus, it remains a possibility that the effects of orally-administered guggulsterone occur primarily at the level of the gut (i.e. versus gut and liver), for instance, by affecting cholesterol absorption and bile-acid reuptake processes regulated by FXR, rather than the hepatic biosynthesis and transport of bile acids. Again, the conditional nature of

the strategy used to create the FXR-null mouse model allows for tissue-specific deletion of the FXR gene and might help resolve this issue.

As reinforced by the recent work of Urizar et al. [3], as well as by the present therapeutic use of bile-acid binding resins for hypercholesterolemia, there exists an intimate linkage between bile acid and cholesterol metabolism. Recent demonstrations that FXR is also involved in the regulation of genes (e.g. encoding apolipoprotein A-I, apolipoprotein C-II and phospholipids transfer protein) [4–6] more closely linked with lipid rather than bile-acid homeostasis, presents additional avenues by which FXR ligands could be beneficial for the treatment of disorders of lipid metabolism. As suggested by the work of Urizar et al. [3] and others (e.g. [7]), careful and comprehensive study of the effects of natural products, such as guggulsterone, on the function of nuclear hormone receptors, is likely to yield additional agents with desirable therapeutic effects.

References

1 Sinal, C.J. et al. (2000) Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis. Cell 102, 731–744

2 Singh, R.B. et al. (1994) Hypolipidemic and antioxidant effects of Commiphora mukul as an adjunct to dietary therapy in patients with hypercholesterolemia. Cardiovasc. Drugs Ther. 8, 659–664

3 Urizar, N.L. et al. (2002) A natural product that lowers cholesterol as an antagonist ligand for FXR. Science 296, 1703–1706

4 Claudel, T. et al. (2002) Bile acid-activated nuclear receptor FXR suppresses apolipoprotein A-I transcription via a negative FXR response element. J. Clin. Invest. 109, 961–971

5 Kast, H.R. et al. (2001) Farnesoid X-activated receptor induces apolipoprotein C-II transcription: a molecular mechanism linking plasma triglyceride levels to bile acids. Mol. Endocrinol. 15, 1720–1728

6 Urizar, N.L. et al. (2000) The farnesoid X-activated receptor mediates bile acid activation of phospholipid transfer protein gene expression. J. Biol. Chem. 275, 39313–39317

7 Wu, J. et al. (2002) The hypolipidemic natural product guggulsterone acts as an antagonist of the bile acid receptor. Mol. Endocrinol. 16, 1590–1597

Christopher J. Sinal

Dept of Pharmacology, Dalhousie University, Halifax, Nova Scotia Canada B3H 4H7.

Frank J. Gonzalez

Laboratory of Metabolism, National Cancer Institute, Bethesda, MD 20892, USA. e-mail: [email protected]

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276 Research Update

Time to consider new brain clock signals

Alastair V. Ferguson and G. Trevor Cottrell

The suprachiasmatic nucleus is well

recognized as a central nervous system

controller of circadian rhythms. The

mechanisms underlying the ability of

neurons in this region to generate such

rhythms are, at best, poorly understood.

Two recently published reports suggest

that circadian change in the expression

of L-type Ca2++ channels, and a novel

protein prokineticin, might play essential

roles in molding crucial circadian output

from this nucleus.

Circadian rhythms provide mechanisms that regulate crucial physiological functions, presumably to meet varying functional needs throughout the day. This preset programming can influence all aspects of human physiology, including corticosteroid release, cardiovascular function, feeding behavior, locomotor activity and

wakefulness. Disruption of the circadian cycle by lack of sleep, abnormal meal schedules, sudden changes in the light–dark cycle, or continuous exposure to light or dark can result in significant disruption of many physiological systems, which can detrimentally affect activities of daily living.

For many years, our understanding of the central control of circadian rhythms has focused on the suprachiasmatic nucleus (SCN) – a unique area of the brain in which neurons display rhythmic and synchronous firing patterns attuned to a 24-h diurnal cycle, with activity highest during relative daytime. The SCN is known to receive visual inputs that allow intrinsic resetting of the circadian cycle in response to changes in the external light–dark cycle, and sends efferent connections to numerous other

central nervous system (CNS) regions, regulating arousal, cardiovascular function, feeding behavior and hormone secretion. Current dogma accepts the unique intrinsic ability of the SCN to function as a master circadian clock in addition to its ability to synchronize activity both of the neurons contained within this region and in other crucial CNS regulatory centers. Although the molecular mechanisms that regulate the intrinsic master circadian clock have been fairly well characterized, the mechanisms underlying rhythmicity and synchronization of cell excitability in the SCN are less clear. Two recent reports have provided intriguing new evidence regarding both potential cellular correlates of intrinsic rhythmicity, and the chemical messengers that communicate such

circadian signals within SCN and at distal targets of SCN neurons.

The first of these [1] provides insights into the ionic mechanisms linking the circadian clock to bioelectric outputs. In their rat SCN slice preparation, Pennartz et al. were able to record daytime oscillations in the membrane potential of SCN neurons that resulted from Ca2+ currents carried by L-type Ca2+ channels. Blockade of this Ca2+ current resulted in a decrease in action potential frequency and amplitude during the daytime cycle. Voltage oscillations and increased Ca2+ current were not observed in slice preparations isolated during relative nighttime periods. These experiments clearly demonstrate circadian changes in Ca2+-channel expression or regulation in rat SCN, and suggest potential involvement of other ion channels in the circadian regulation of neuronal excitability. Further study of relationships between circadian rhythms and bioelectric properties in SCN cells will probably be necessary to unravel the complex interactions underlying the rhythmic properties of this nucleus.

This publication opens the door to future experiments aimed at identifying the specific mechanisms of increased Ca2+ current in daytime neurons. Where Ca2+ current is the product of channel number, probability of open time and unitary current, it is likely that the twofold Ca2+-current increase observed in this study is a function of channel number, which would, in turn, imply transcriptional or translational control of channel expression. Such regulation might best be studied in one of the numerous mouse clock gene-knockout animals, which would then present the common 21st-century challenge of adapting these experimental models to the mouse. Additionally, it would be intriguing to know if SCN neurons of rats habituated to constant day or night cycles, or in which the retinohypothalamic tract has been ablated, show similar day or night patterns of Ca2+-channel rhythmicity, thus establishing whether such patterns are intrinsic features of this nucleus. It would also be worth determining if blockade or upregulation of L-type Ca2+

currents in SCN influences circadian rhythms. Such observations would

further solidify the potential causality of the relationship between the circadian clock and Ca2+-channel expression. It is important to reiterate, however, that the L-type Ca2+ channel alone is not responsible for the increased neuronal firing frequency observed in daytime cells. Therefore, there are probably other essential factors that establish the circadian changes in excitability in SCN neurons.

This brings us to a second recent report by Cheng et al. [2], who used molecular approaches to identify a novel secretory peptide produced by the SCN. This molecule, prokineticin (PK2), is rhythmically expressed in the SCN, with daytime levels ~50-fold greater than those at nighttime. This study demonstrates that PK2 expression is directly upregulated by clock genes (Clock–Bmal heterodimers), inhibited by period genes (Per and Cry), and inhibited in clock gene-knockout animals. The mice in this study were raised in constant darkness, and although they did not require light input for rhythmic PK2-expression regulation, light exposure did induce rapid PK2 expression during late-phase night cycles. Also of importance is the observation that PK2 receptors (PKR2) are localized in SCN target areas throughout the brain, in addition to in the SCN itself.

These results are exciting because they demonstrate a clear mechanism of PK2-expression regulation by the clock genes, as well as a mechanism of positive- feedback amplification of PK2 by its own receptor to induce large fluctuations in expression levels that are coincident with the circadian cycle. Thus, PK2 could be a crucial messenger that synchronizes neural activity within the SCN by its local modulatory actions, perhaps on ion channels (e.g. L-type Ca2+ channels); however, potential involvement of gap junctions, adhesion molecules and other neurohormones cannot be ruled out. The additional localization of PKR2 in SCN- target regions also suggests the possibility that PK2 might be a neurotransmitter and/or modulator that controls the excitability of crucial output neurons in these regions.

These two recent studies are provocative because they provide two different mechanisms for output regulation from the SCN to the same

target cells. It is interesting to speculate what effect this combined cellular stimulation would have on both acute excitability of the target neuron and the long-term responsiveness of the neuron to other inputs. Within the SCN, similar questions arise. A crucial element of SCN function is its ability to rhythmically synchronize the circadian cycles. These cycles are controlled by intracellular signaling molecules such as Ca2+ and cAMP. The external cues for triggering an increase in such signaling molecules have not been clearly defined; however, the work by Pennartz et al. shows a mechanism for the increased intracellular Ca2+ observed during the daytime phase, and further studies of the PKR2 could show a similar effect, with PK2 increasing cellular Ca2+ and/or cAMP. Furthermore, PK2 and L-type Ca2+ channels could act synergistically to enhance their own expression during daytime cycles.

In summary, these papers provide evidence for two separate proteins, the expression of which is altered in the SCN in response to intrinsic molecular signaling of the circadian clock. Such observations, although clearly not identifying the ‘holy grail’ of a single mechanism generating circadian rhythmicity in the SCN, clearly do emphasize the multiplicity of contributors to the development of the rhythm in this essential central controller of circadian regulation. Future studies need to be directed not only towards clarification of the precise roles of both L-type Ca2+ channels and PK2 in circadian function, but also towards identification of additional contributors to the precise and essential control of circadian changes in neuronal excitability within this CNS nucleus.

References

1 Pennartz, C.M.A. et al. (2002) Diurnal modulation of pacemaker potentials and calcium current in the mammalian circadian clock. Nature 416, 286–290

2 Cheng, M.Y. et al. (2002) Prokineticin 2 transmits the behavioural circadian rhythm of the suprachiasmatic nucleus. Nature 417, 405–410

Alastair V. Ferguson*

G. Trevor Cottrell

Dept of Physiology, Queen’s University, Kingston, Ontario, Canada K7L 3N6. *e-mail: [email protected]

TRENDS in Endocrinology & Metabolism Vol.13 No.7 September 2002

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277Research Update