Lauren P. Shearman

mCRY1 and mCRY2 Are Essential Components of the Negative Limb of the Circadian Clock Feedback Loop

We determined that two mouse cryptochrome genes, mCry1 and mCry2, act in the negative limb of the clock feedback loop. In cell lines, mPER proteins (alone or in combination) have modest effects on their cellular location and ability to inhibit CLOCK:BMAL1 -mediated transcription. This suggested cryptochrome involvement in the negative limb of the feedback loop. Indeed, mCry1 and mCry2 RNA levels are reduced in the central and peripheral clocks of Clock/Clock mutant mice. mCRY1 and mCRY2 are nuclear proteins that interact with each of the mPER proteins, translocate each mPER protein from cytoplasm to nucleus, and are rhythmically expressed in the suprachiasmatic circadian clock. Luciferase reporter gene assays show that mCRY1 or mCRY2 alone abrogates CLOCK:BMAL1-E box-mediated transcription. The mPER and mCRY proteins appear to inhibit the transcriptional complex differentially.

Three period Homologs in Mammals: Differential Light Responses in the Suprachiasmatic Circadian Clock and Oscillating Transcripts Outside of Brain

We have cloned and characterized the mouse cDNA of a third mammalian homolog of the Drosophila period gene and designated it mPer3. The mPER3 protein shows approximately 37% amino acid identity with mPER1 and mPER2 proteins. The three mammalian PER proteins share several regions of sequence homology, and each contains a protein dimerization PAS domain. mPer3 RNA levels oscillate in the suprachiasmatic nuclei (SCN) and eyes. In the SCN, mPer3 RNA levels are not acutely altered by light exposure at different times during subjective night. This contrasts with the acute induction by light of mPer1 and mPer2 RNA levels during early and late subjective night. mPer3 is widely expressed in tissues outside of brain. In liver, skeletal muscle, and testis, mPer RNAs exhibit prominent, synchronous circadian oscillations. The results highlight the differential light responses among the three mammalian Per genes in the SCN and raise the possibility of circadian oscillators in mammals outside of brain and retina.

A Molecular Mechanism Regulating Rhythmic Output from the Suprachiasmatic Circadian Clock

We examined the transcriptional regulation of the clock-controlled arginine vasopressin gene in the suprachiasmatic nuclei (SCN). A core clock mechanism in mouse SCN appears to involve a transcriptional feedback loop in which CLOCK and BMAL1 are positive regulators and three mPeriod (mPer) genes are involved in negative feedback. We show that the RNA rhythm of each mPer gene is severely blunted in Clock/Clock mice. The vasopressin RNA rhythm is abolished in the SCN of Clock/Clock animals, leading to markedly decreased peptide levels. Luciferase reporter gene assays show that CLOCK-BMAL1 heterodimers act through an E box enhancer in the vasopressin gene to activate transcription; this activation can be inhibited by the mPER and mTIM proteins. These data indicate that the transcriptional machinery of the core clockwork directly regulates a clock-controlled output rhythm.

Two period Homologs: Circadian Expression and Photic Regulation in the Suprachiasmatic Nuclei

We have characterized a mammalian homolog of the Drosophila period gene and designated it Per2. The PER2 protein shows >40% amino acid identity to the protein of another mammalian per homolog (designated Per1) that was recently cloned and characterized. Both PER1 and PER2 proteins share several regions of homology with the Drosophila PER protein, including the protein dimerization PAS domain. Phylogenetic analysis supports the existence of a family of mammalian per genes. In the mouse, Per1 and Per2 RNA levels exhibit circadian rhythms in the SCN and eyes, sites of circadian clocks. Both Per1 and Per2 RNAs in the SCN are increased by light exposure during subjective night but not during subjective day. The results advance our knowledge of candidate clock elements in mammals.

Molecular Dissection of Two Distinct Actions of Melatonin on the Suprachiasmatic Circadian Clock

The pineal hormone melatonin elicits two effects on the suprachiasmatic nuclei (SCN): acute neuronal inhibition and phase-shifting. Melatonin evokes its biological effects through G protein-coupled receptors. Since the Mel1a melatonin receptor may transduce the major neurobiological actions of melatonin in mammals, we examined whether it mediates both melatonin effects on SCN function by using mice with targeted disruption of the Mel1a receptor. The Mel1a receptor accounts for all detectable, high affinity melatonin binding in mouse brain. Functionally, this receptor is necessary for the acute inhibitory action of melatonin on the SCN. Melatonin-induced phase shifts, however, are only modestly altered in the receptor-deficient mice; pertussis toxin still blocks melatonin-induced phase shifts in Mel1a receptor-deficient mice. The other melatonin receptor subtype, the Mel1b receptor, is expressed in mouse SCN, implicating it in the phase-shifting response. The results provide a molecular basis for two distinct, mechanistically separable effects of melatonin on SCN physiology.

Melanin-concentrating hormone 1 receptor-deficient mice are lean, hyperactive, and hyperphagic and have altered metabolism

Melanin-concentrating hormone (MCH) is a cyclic 19-aa hypothalamic neuropeptide derived from a larger prohormone precursor of MCH (Pmch), which also encodes neuropeptide EI (NEI) and neuropeptide GE (NGE). Pmch-deficient (Pmch−/−) mice are lean, hypophagic, and have an increased metabolic rate. Transgenic mice overexpressing Pmch are hyperphagic and develop mild obesity. Consequently, MCH has been implicated in the regulation of energy homeostasis. The MCH 1 receptor (MCH1R) is one of two recently identified G protein-coupled receptors believed to be responsible for the actions of MCH. We evaluated the physiological role of MCH1R by generating MCH1R-deficient (Mch1r−/−) mice. Mch1r−/− mice have normal body weights, yet are lean and have reduced fat mass. Surprisingly, Mch1r−/− mice are hyperphagic when maintained on regular chow, and their leanness is a consequence of hyperactivity and altered metabolism. Consistent with the hyperactivity, Mch1r−/− mice are less susceptible to diet-induced obesity. Importantly, chronic central infusions of MCH induce hyperphagia and mild obesity in wild-type mice, but not in Mch1r−/− mice. We conclude that MCH1R is a physiologically relevant MCH receptor in mice that plays a role in energy homeostasis through multiple actions on locomotor activity, metabolism, appetite, and neuroendocrine function.

Neither Agouti-Related Protein nor Neuropeptide Y Is Critically Required for the Regulation of Energy Homeostasis in Mice

ABSTRACT Agouti-related protein (AgRP), a neuropeptide abundantly expressed in the arcuate nucleus of the hypothalamus, potently stimulates feeding and body weight gain in rodents. AgRP is believed to exert its effects through the blockade of signaling by α-melanocyte-stimulating hormone at central nervous system (CNS) melanocortin-3 receptor (Mc3r) and Mc4r. We generated AgRP-deficient (Agrp−/−) mice to examine the physiological role of AgRP. Agrp−/− mice are viable and exhibit normal locomotor activity, growth rates, body composition, and food intake. Additionally, Agrp−/− mice display normal responses to starvation, diet-induced obesity, and the administration of exogenous leptin or neuropeptide Y (NPY). In situ hybridization failed to detect altered CNS expression levels for proopiomelanocortin, Mc3r, Mc4r, or NPY mRNAs in Agrp−/− mice. As AgRP and the orexigenic peptide NPY are coexpressed in neurons of the arcuate nucleus, we generated AgRP and NPY double-knockout (Agrp−/−;Npy−/−) mice to determine whether NPY or AgRP plays a compensatory role in Agrp−/− or NPY-deficient (Npy−/−) mice, respectively. Similarly to mice deficient in either AgRP or NPY, Agrp−/−;Npy−/− mice suffer no obvious feeding or body weight deficits and maintain a normal response to starvation. Our results demonstrate that neither AgRP nor NPY is a critically required orexigenic factor, suggesting that other pathways capable of regulating energy homeostasis can compensate for the loss of both AgRP and NPY.

Targeted Disruption of the mPer3 Gene: Subtle Effects on Circadian Clock Function

ABSTRACT Neurons in the mammalian suprachiasmatic nucleus (SCN) contain a cell-autonomous circadian clock that is based on a transcriptional-translational feedback loop. The basic helix-loop-helix–PAS proteins CLOCK and BMAL1 are positive regulators and drive the expression of the negative regulators CRY1 and CRY2, as well as PER1, PER2, and PER3. To assess the role of mouse PER3 (mPER3) in the circadian timing system, we generated mice with a targeted disruption of the mPer3 gene. Western blot analysis confirmed the absence of mPER3-immunoreactive proteins in mice homozygous for the targeted allele. mPer1,mPer2, mCry1, and Bmal1 RNA rhythms in the SCN did not differ between mPER3-deficient and wild-type mice. Rhythmic expression of mPer1 and mPer2 RNAs in skeletal muscle also did not differ between mPER3-deficient and wild-type mice. mPer3 transcripts were rhythmically expressed in the SCN and skeletal muscle of mice homozygous for the targeted allele, but the level of expression of the mutant transcript was lower than that in wild-type controls. Locomotor activity rhythms in mPER3-deficient mice were grossly normal, but the circadian cycle length was significantly (0.5 h) shorter than that in controls. The results demonstrate that mPer3 is not necessary for circadian rhythms in mice.

Molecular Analysis of Mammalian Timeless

We cloned the mouse cDNA of a mammalian homolog of the Drosophila timeless (tim) gene and designated it mTim. The mTim protein shows five homologous regions with Drosophila TIM. mTim is weakly expressed in the suprachiasmatic nuclei (SCN) but exhibits robust expression in the hypophyseal pars tuberalis (PT). mTim RNA levels do not oscillate in the SCN nor are they acutely altered by light exposure during subjective night. mTim RNA is expressed at low levels in several peripheral tissues, including eyes, and is heavily expressed in spleen and testis. Yeast two-hybrid assays revealed an array of interactions between the various mPER proteins but no mPER-mTIM interactions. The data suggest that PER-PER interactions have replaced the function of PER-TIM dimers in the molecular workings of the mammalian circadian clock.

Discovery of N-[(1S,2S)-3-(4-Chlorophenyl)-2- (3-cyanophenyl)-1-methylpropyl]-2-methyl-2- {[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide (MK-0364), a novel, acyclic cannabinoid-1 receptor inverse agonist for the treatment of obesity.

The discovery of novel acyclic amide cannabinoid-1 receptor inverse agonists is described. They are potent, selective, orally bioavailable, and active in rodent models of food intake and body weight reduction. A major focus of the optimization process was to increase in vivo efficacy and to reduce the potential for formation of reactive metabolites. These efforts led to the identification of compound 48 for development as a clinical candidate for the treatment of obesity.