Enhanced morphine analgesia in mice lacking beta-arrestin 2

The ability of morphine to alleviate pain is mediated through a heterotrimeric guanine nucleotide binding protein (G protein)-coupled heptahelical receptor (GPCR), the mu opioid receptor (muOR). The efficiency of GPCR signaling is tightly regulated and ultimately limited by the coordinated phosphorylation of the receptors by specific GPCR kinases and the subsequent interaction of the phosphorylated receptors with beta-arrestin 1 and beta-arrestin 2. Functional deletion of the beta-arrestin 2 gene in mice resulted in remarkable potentiation and prolongation of the analgesic effect of morphine, suggesting that muOR desensitization was impaired. These results provide evidence in vivo for the physiological importance of beta-arrestin 2 in regulating the function of a specific GPCR, the muOR. Moreover, they suggest that inhibition of beta-arrestin 2 function might lead to enhanced analgesic effectiveness of morphine and provide potential new avenues for the study and treatment of pain, narcotic tolerance, and dependence.     

Rad6-dependent ubiquitination of histone H2B in yeast

Although ubiquitinated histones are present in substantial levels in vertebrate cells, the roles they play in specific biological processes and the cellular factors that regulate this modification are not well characterized. Ubiquitinated H2B (uH2B) has been identified in the yeast Saccharomyces cerevisiae, and mutation of the conserved ubiquitination site is shown to confer defects in mitotic cell growth and meiosis. uH2B was not detected in rad6 mutants, which are defective for the ubiquitin-conjugating enzyme Ubc2, thus identifying Rad6 as the major cellular activity that ubiquitinates H2B in yeast.     

Limb and skin abnormalities in mice lacking IKKalpha

The gene encoding inhibitor of kappa B (IkappaB) kinase alpha (IKKalpha; also called IKK1) was disrupted by gene targeting. IKKalpha-deficient mice died perinatally. In IKKalpha-deficient fetuses, limb outgrowth was severely impaired despite unaffected skeletal development. The epidermal cells in IKKalpha-deficient fetuses were highly proliferative with dysregulated epidermal differentiation. In the basal layer, degradation of IkappaB and nuclear localization of nuclear factor kappa B (NF-kappaB) were not observed. Thus, IKKalpha is essential for NF-kappaB activation in the limb and skin during embryogenesis. In contrast, there was no impairment of NF-kappaB activation induced by either interleukin-1 or tumor necrosis factor-alpha in IKKalpha-deficient embryonic fibroblasts and thymocytes, indicating that IKKalpha is not essential for cytokine-induced activation of NF-kappaB.     

Continuous fatty acid oxidation and reduced fat storage in mice lacking acetyl-CoA carboxylase 2

Malonyl-coenzyme A (malonyl-CoA), generated by acetyl-CoA carboxylases ACC1 and ACC2, is a key metabolite in the regulation of energy homeostasis. Here, we show that Acc2-/- mutant mice have a normal life span, a higher fatty acid oxidation rate, and lower amounts of fat. In comparison to the wild type, Acc2-deficient mice had 10- and 30-fold lower levels of malonyl-CoA in heart and muscle, respectively. The fatty acid oxidation rate in the soleus muscle of the Acc2-/- mice was 30% higher than that of wild-type mice and was not affected by addition of insulin; however, addition of insulin to the wild-type muscle reduced fatty acid oxidation by 45%. The mutant mice accumulated 50% less fat in their adipose tissue than did wild-type mice. These results raise the possibility that pharmacological manipulation of ACC2 may lead to loss of body fat in the context of normal caloric intake.     

Photic induction of mPer1 and mPer2 in cry-deficient mice lacking a biological clock

Mice lacking mCry1 and mCry2 are behaviorally arrhythmic. As shown here, cyclic expression of the clock genes mPer1 and mPer2 (mammalian Period genes 1 and 2) in the suprachiasmatic nucleus and peripheral tissues is abolished and mPer1 and mPer2 mRNA levels are constitutively high. These findings indicate that the biological clock is eliminated in the absence of both mCRY1 and mCRY2 (mammalian cryptochromes 1 and 2) and support the idea that mammalian CRY proteins act in the negative limb of the circadian feedback loop. The mCry double-mutant mice retain the ability to have mPer1 and mPer2 expression induced by a brief light stimulus known to phase-shift the biological clock in wild-type animals. Thus, mCRY1 and mCRY2 are dispensable for light-induced phase shifting of the biological clock.     

Akt-mediated phosphorylation of EZH2 suppresses methylation of lysine 27 in histone H3

Enhancer of Zeste homolog 2 (EZH2) is a methyltransferase that plays an important role in many biological processes through its ability to trimethylate lysine 27 in histone H3. Here, we show that Akt phosphorylates EZH2 at serine 21 and suppresses its methyltransferase activity by impeding EZH2 binding to histone H3, which results in a decrease of lysine 27 trimethylation and derepression of silenced genes. Our results imply that Akt regulates the methylation activity, through phosphorylation of EZH2, which may contribute to oncogenesis.     

H2S induces a suspended animation-like state in mice

Mammals normally maintain their core body temperature (CBT) despite changes in environmental temperature. Exceptions to this norm include suspended animation-like states such as hibernation, torpor, and estivation. These states are all characterized by marked decreases in metabolic rate, followed by a loss of homeothermic control in which the animal's CBT approaches that of the environment. We report that hydrogen sulfide can induce a suspended animation-like state in a nonhibernating species, the house mouse (Mus musculus). This state is readily reversible and does not appear to harm the animal. This suggests the possibility of inducing suspended animation-like states for medical applications.     

Deficit in attachment behavior in mice lacking the mu-opioid receptor gene

Endogenous opioid binding to micro receptors is hypothesized to mediate natural rewards and has been proposed to be the basis of infant attachment behavior. Here, we report that micro-opioid receptor knockout mouse pups emit fewer ultrasonic vocalizations when removed from their mothers but not when exposed to cold or male mice odors. Moreover these knockout pups do not show a preference toward their mothers' cues and do not show ultrasonic calls potentiation after brief maternal exposure. Results from this study may indicate a molecular mechanism for diseases characterized by deficits in attachment behavior, such as autism or reactive attachment disorder.     

Extended longevity in mice lacking the insulin receptor in adipose tissue

Caloric restriction has been shown to increase longevity in organisms ranging from yeast to mammals. In some organisms, this has been associated with a decreased fat mass and alterations in insulin/insulin-like growth factor 1 (IGF-1) pathways. To further explore these associations with enhanced longevity, we studied mice with a fat-specific insulin receptor knockout (FIRKO). These animals have reduced fat mass and are protected against age-related obesity and its subsequent metabolic abnormalities, although their food intake is normal. Both male and female FIRKO mice were found to have an increase in mean life-span of approximately 134 days (18%), with parallel increases in median and maximum life-spans. Thus, a reduction of fat mass without caloric restriction can be associated with increased longevity in mice, possibly through effects on insulin signaling.     

Impaired nociception and pain sensation in mice lacking the capsaicin receptor

The capsaicin (vanilloid) receptor VR1 is a cation channel expressed by primary sensory neurons of the "pain" pathway. Heterologously expressed VR1 can be activated by vanilloid compounds, protons, or heat (>43 degrees C), but whether this channel contributes to chemical or thermal sensitivity in vivo is not known. Here, we demonstrate that sensory neurons from mice lacking VR1 are severely deficient in their responses to each of these noxious stimuli. VR1-/- mice showed normal responses to noxious mechanical stimuli but exhibited no vanilloid-evoked pain behavior, were impaired in the detection of painful heat, and showed little thermal hypersensitivity in the setting of inflammation. Thus, VR1 is essential for selective modalities of pain sensation and for tissue injury-induced thermal hyperalgesia.     

ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex

The conserved histone variant H2AZ has an important role in the regulation of gene expression and the establishment of a buffer to the spread of silent heterochromatin. How histone variants such as H2AZ are incorporated into nucleosomes has been obscure. We have found that Swr1, a Swi2/Snf2-related adenosine triphosphatase, is the catalytic core of a multisubunit, histone-variant exchanger that efficiently replaces conventional histone H2A with histone H2AZ in nucleosome arrays. Swr1 is required for the deposition of histone H2AZ at specific chromosome locations in vivo, and Swr1 and H2AZ commonly regulate a subset of yeast genes. These findings define a previously unknown role for the adenosine triphosphate-dependent chromatin remodeling machinery.     

Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase

Fat tissue is the most important energy depot in vertebrates. The release of free fatty acids (FFAs) from stored fat requires the enzymatic activity of lipases. We showed that genetic inactivation of adipose triglyceride lipase (ATGL) in mice increases adipose mass and leads to triacylglycerol deposition in multiple tissues. ATGL-deficient mice accumulated large amounts of lipid in the heart, causing cardiac dysfunction and premature death. Defective cold adaptation indicated that the enzyme provides FFAs to fuel thermogenesis. The reduced availability of ATGL-derived FFAs leads to increased glucose use, increased glucose tolerance, and increased insulin sensitivity. These results indicate that ATGL is rate limiting in the catabolism of cellular fat depots and plays an important role in energy homeostasis.     

Impaired prion replication in spleens of mice lacking functional follicular dendritic cells

In scrapie-infected mice, prions are found associated with splenic but not circulating B and T lymphocytes and in the stroma, which contains follicular dendritic cells (FDCs). Formation and maintenance of mature FDCs require the presence of B cells expressing membrane-bound lymphotoxin-alpha/beta. Treatment of mice with soluble lymphotoxin-beta receptor results in the disappearance of mature FDCs from the spleen. We show that this treatment abolishes splenic prion accumulation and retards neuroinvasion after intraperitoneal scrapie inoculation. These data provide evidence that FDCs are the principal sites for prion replication in the spleen.     

Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta)

Glucose homeostasis depends on insulin responsiveness in target tissues, most importantly, muscle and liver. The critical initial steps in insulin action include phosphorylation of scaffolding proteins and activation of phosphatidylinositol 3-kinase. These early events lead to activation of the serine-threonine protein kinase Akt, also known as protein kinase B. We show that mice deficient in Akt2 are impaired in the ability of insulin to lower blood glucose because of defects in the action of the hormone on liver and skeletal muscle. These data establish Akt2 as an essential gene in the maintenance of normal glucose homeostasis.     

Role of histone H3 lysine 27 methylation in Polycomb-group silencing

Polycomb group (PcG) proteins play important roles in maintaining the silent state of HOX genes. Recent studies have implicated histone methylation in long-term gene silencing. However, a connection between PcG-mediated gene silencing and histone methylation has not been established. Here we report the purification and characterization of an EED-EZH2 complex, the human counterpart of the Drosophila ESC-E(Z) complex. We demonstrate that the complex specifically methylates nucleosomal histone H3 at lysine 27 (H3-K27). Using chromatin immunoprecipitation assays, we show that H3-K27 methylation colocalizes with, and is dependent on, E(Z) binding at an Ultrabithorax (Ubx) Polycomb response element (PRE), and that this methylation correlates with Ubx repression. Methylation on H3-K27 facilitates binding of Polycomb (PC), a component of the PRC1 complex, to histone H3 amino-terminal tail. Thus, these studies establish a link between histone methylation and PcG-mediated gene silencing.     

Role of histone H3 lysine 27 methylation in X inactivation

The Polycomb group (PcG) protein Eed is implicated in regulation of imprinted X-chromosome inactivation in extraembryonic cells but not of random X inactivation in embryonic cells. The Drosophila homolog of the Eed-Ezh2 PcG protein complex achieves gene silencing through methylation of histone H3 on lysine 27 (H3-K27), which suggests a role for H3-K27 methylation in imprinted X inactivation. Here we demonstrate that transient recruitment of the Eed-Ezh2 complex to the inactive X chromosome (Xi) occurs during initiation of X inactivation in both extraembryonic and embryonic cells and is accompanied by H3-K27 methylation. Recruitment of the complex and methylation on the Xi depend on Xist RNA but are independent of its silencing function. Together, our results suggest a role for Eed-Ezh2-mediated H3-K27 methylation during initiation of both imprinted and random X inactivation and demonstrate that H3-K27 methylation is not sufficient for silencing of the Xi.