Linear motif specificity in signaling through p38α and ERK2 mitogen-activated protein kinases.

Mitogen-activated protein kinase (MAPK) cascades are essential for eukaryotic cells to integrate and respond to diverse stimuli. Maintaining specificity in signaling through MAPK networks is key to coupling distinct inputs to appropriate cellular responses. Docking sites-short linear motifs found in MAPK substrates, regulators, and scaffolds-can promote signaling specificity through selective interactions, but how they do so remains unresolved. Here, we screened a proteomic library for sequences interacting with the MAPKs extracellular signal-regulated kinase 2 (ERK2) and p38α, identifying selective and promiscuous docking motifs. Sequences specific for p38α had high net charge and lysine content, and selective binding depended on a pair of acidic residues unique to the p38α docking interface. Finally, we validated a set of full-length proteins harboring docking sites selected in our screens to be authentic MAPK interactors and substrates. This study identifies features that help define MAPK signaling networks and explains how specific docking motifs promote signaling integrity.

Sulforaphane reduces obesity by reversing leptin resistance.

The ascending prevalence of obesity in recent decades is commonly associated with soaring morbidity and mortality rates, resulting in increased health-care costs and decreased quality of life. A systemic state of stress characterized by low-grade inflammation and pathological formation of reactive oxygen species (ROS) usually manifests in obesity. The transcription factor nuclear factor erythroid-derived 2-like 2 (NRF2) is the master regulator of the redox homeostasis and plays a critical role in the resolution of inflammation. Here, we show that the natural isothiocyanate and potent NRF2 activator sulforaphane reverses diet-induced obesity through a predominantly, but not exclusively, NRF2-dependent mechanism that requires a functional leptin receptor signaling and hyperleptinemia. Sulforaphane does not reduce the body weight or food intake of lean mice but induces an anorectic response when coadministered with exogenous leptin. Leptin-deficient Lepob/ob mice and leptin receptor mutant Leprdb/db mice display resistance to the weight-reducing effect of sulforaphane, supporting the conclusion that the antiobesity effect of sulforaphane requires functional leptin receptor signaling. Furthermore, our results suggest the skeletal muscle as the most notable site of action of sulforaphane whose peripheral NRF2 action signals to alleviate leptin resistance. Transcriptional profiling of six major metabolically relevant tissues highlights that sulforaphane suppresses fatty acid synthesis while promoting ribosome biogenesis, reducing ROS accumulation, and resolving inflammation, therefore representing a unique transcriptional program that leads to protection from obesity. Our findings argue for clinical evaluation of sulforaphane for weight loss and obesity-associated metabolic disorders.

Histone deacetylase 6 inhibition restores leptin sensitivity and reduces obesity.

The adipose tissue-derived hormone leptin can drive decreases in food intake while increasing energy expenditure. In diet-induced obesity, circulating leptin levels rise proportionally to adiposity. Despite this hyperleptinemia, rodents and humans with obesity maintain increased adiposity and are resistant to leptin's actions. Here we show that inhibitors of the cytosolic enzyme histone deacetylase 6 (HDAC6) act as potent leptin sensitizers and anti-obesity agents in diet-induced obese mice. Specifically, HDAC6 inhibitors, such as tubastatin A, reduce food intake, fat mass, hepatic steatosis and improve systemic glucose homeostasis in an HDAC6-dependent manner. Mechanistically, peripheral, but not central, inhibition of HDAC6 confers central leptin sensitivity. Additionally, the anti-obesity effect of tubastatin A is attenuated in animals with a defective central leptin-melanocortin circuitry, including db/db and MC4R knockout mice. Our results suggest the existence of an HDAC6-regulated adipokine that serves as a leptin-sensitizing agent and reveals HDAC6 as a potential target for the treatment of obesity.

Endogenous cannabinoids are required for MC4R-mediated control of energy homeostasis.

Hypothalamic regulation of feeding and energy expenditure is a fundamental and evolutionarily conserved neurophysiological process critical for survival. Dysregulation of these processes, due to environmental or genetic causes, can lead to a variety of pathological conditions ranging from obesity to anorexia. Melanocortins and endogenous cannabinoids (eCBs) have been implicated in the regulation of feeding and energy homeostasis; however, the interaction between these signaling systems is poorly understood. Here, we show that the eCB 2-arachidonoylglycerol (2-AG) regulates the activity of melanocortin 4 receptor (MC4R) cells in the paraventricular nucleus of the hypothalamus (PVNMC4R) via inhibition of afferent GABAergic drive. Furthermore, the tonicity of eCBs signaling is inversely proportional to energy state, and mice with impaired 2-AG synthesis within MC4R neurons weigh less, are hypophagic, exhibit increased energy expenditure, and are resistant to diet-induced obesity. These mice also exhibit MC4R agonist insensitivity, suggesting that the energy state-dependent, 2-AG-mediated suppression of GABA input modulates PVNMC4R neuron activity to effectively respond to the MC4R natural ligands to regulate energy homeostasis. Furthermore, post-developmental disruption of PVN 2-AG synthesis results in hypophagia and death. These findings illustrate a functional interaction at the cellular level between two fundamental regulators of energy homeostasis, the melanocortin and eCB signaling pathways in the hypothalamic feeding circuitry.

The melanocortin-3 receptor is a pharmacological target for the regulation of anorexia.

Ablation of hypothalamic AgRP (Agouti-related protein) neurons is known to lead to fatal anorexia, whereas their activation stimulates voracious feeding and suppresses other motivational states including fear and anxiety. Despite the critical role of AgRP neurons in bidirectionally controlling feeding, there are currently no therapeutics available specifically targeting this circuitry. The melanocortin-3 receptor (MC3R) is expressed in multiple brain regions and exhibits sexual dimorphism of expression in some of those regions in both mice and humans. MC3R deletion produced multiple forms of sexually dimorphic anorexia that resembled aspects of human anorexia nervosa. However, there was no sexual dimorphism in the expression of MC3R in AgRP neurons, 97% of which expressed MC3R. Chemogenetic manipulation of arcuate MC3R neurons and pharmacologic manipulation of MC3R each exerted potent bidirectional regulation over feeding behavior in male and female mice, whereas global ablation of MC3R-expressing cells produced fatal anorexia. Pharmacological effects of MC3R compounds on feeding were dependent on intact AgRP circuitry in the mice. Thus, the dominant effect of MC3R appears to be the regulation of the AgRP circuitry in both male and female mice, with sexually dimorphic sites playing specialized and subordinate roles in feeding behavior. Therefore, MC3R is a potential therapeutic target for disorders characterized by anorexia, as well as a potential target for weight loss therapeutics.

The role of interindividual licking received and dopamine genotype on later-life licking provisioning in female rat offspring.

INTRODUCTION: Rat mothers exhibit natural variations in care that propagate between generations of female offspring. However, there is limited information on genetic variation that could influence this propagation. METHODS: We assessed early-life maternal care received by individual female rat offspring, later-life maternal care provisioning, and dopaminergic activity in the maternal brain in relation to naturally occurring genetic polymorphisms linked to the dopaminergic system. We also conducted a systematic analysis of other genetic variants potentially related to maternal behavior in our Long-Evans rat population. RESULTS: While we did not find a direct relationship between early-life licking received and later-life licking provisioning, this relationship was indirectly affected by dopamine levels in the nucleus accumbens and dependent on variation in the dopamine receptor 2 gene (rs107017253). More specifically, female rat offspring with the A/G genotype showed a positive relationship between average licking received and dopamine levels in the nucleus accumbens of the maternal brain; there was no relationship with female rat offspring with the A/A genotype. The higher dopamine levels in the nucleus accumbens corresponded with higher maternal licking provisioning from postnatal days 2-9. We also discovered and validated several new variants that were predicted by our systematic analysis. CONCLUSION: Our findings suggest that genetic variation influences the relationship between early-life maternal care received and the dopaminergic system of the maternal brain, which can indirectly influence later-life maternal care provisioning.

Inter-individual maternal care received and genotype interactions affect dopaminergic phenotypes in female rat offspring.

Rat mothers exhibit natural variations in care and can shape offspring adult behaviour and their maternal care by affecting the dopaminergic system. We explored whether genotype and gene × environment interactions are involved in these processes in nulliparous female offspring. We assessed maternal licking/grooming toward individual female pups during the first week postpartum and dopamine-related behaviour of the offspring in adulthood. Behaviours explored included strategy shifting, impulsive action and sucrose preference. Single nucleotide polymorphisms in the dopamine receptor 2, dopamine transporter and catechol-O-methyltransferase genes were examined in relation to offspring behaviour and baseline dopamine turnover in select brain regions. Dopamine receptor 2 (RS107017253) variation moderated, or interacted with, the relationship between early-life licking received and behaviour. Specifically, offspring with the A/A genotype showed a significant correlation between early-life licking received and behaviour. Offspring with the A/G and G/G genotypes did not show this relationship. Dopamine transporter gene variation affected offspring behaviour regardless of early-life licking received. Our findings suggest that genotype can directly affect dopamine-related behaviours and alter the sensitivity of offspring to the maternal environment. This could be informative on how maternal care is transmitted between generations of female offspring.

Leptin Receptor Signaling in Sim1-Expressing Neurons Regulates Body Temperature and Adaptive Thermogenesis.

Leptin signals to regulate food intake and energy expenditure under conditions of normative energy homeostasis. The central expression and function of leptin receptor B (LepRb) have been extensively studied during the past two decades; however, the mechanisms by which LepRb signaling dysregulation contributes to the pathophysiology of obesity remains unclear. The paraventricular nucleus of the hypothalamus (PVN) plays a crucial role in regulating energy balance as well as the neuroendocrine axes. The role of LepRb expression in the PVN in regard to the regulation of physiological function of leptin has been controversial. The single-minded homolog 1 gene (Sim1) is densely expressed in the PVN and in parts of the amygdala, making Sim1-Cre mice a useful model for examining molecular mechanisms regulating PVN function. In this study, we characterized the physiological role of LepRb in Sim1-expressing neurons using LepRb-floxed × Sim1-Cre mice. Sim1-specific LepRb-deficient mice were surprisingly hypophagic on regular chow but gained more weight upon exposure to a high-fat diet than did their control littermates. We show that Sim1-specific deletion of a single LepRb gene copy caused decreased surface and core body temperatures as well as decreased energy expenditure in ambient room temperatures in both female and male mice. Furthermore, cold-induced adaptive (nonshivering) thermogenesis is disrupted in homozygous knockout mice. A defective thermoregulatory response was associated with defective cold-induced upregulation of uncoupling protein 1 in brown adipose tissue and reduced serum T4. Our study provides novel functional evidence supporting LepRb signaling in Sim1 neurons in the regulation of body weight, core body temperature, and cold-induced adaptive thermogenesis.

Both maternal care received and genotype influence stress-related phenotype in female rats.

Rat dams differ naturally in the level of maternal care they provide to their offspring within the same litter. We explored possible mechanisms of differential maternal care focused on genetic variation. We examined single nucleotide polymorphisms in the glucocorticoid receptor, FK506-binding protein, and serotonin transporter genes in two separate cohorts, and the relationship between differential maternal care received, genotype, and offspring phenotype. Allelic variation in all three genes was significantly associated with levels of maternal care received by offspring and behavioral and endocrine stress responses in adulthood. Differences in pup behavior were also associated with allelic variation in these genes. Together, these results indicate that the dam/pup interaction is dynamic and implicate the genotype of the offspring in influencing the level of maternal care received. They further suggest that some genotypes may have a dampening effect on the impact of maternal care on stress-related phenotypes in adulthood.

Within- and between-litter maternal care alter behavior and gene regulation in female offspring.

Rat dams show natural variations in maternal care, licking and grooming (LG), that are associated with distinct behavioral and neural phenotypes in offspring. However, there has been limited research on the effects of differences in LG received by female pups and of variations in maternal care within the litter. Here, we investigated LG received by measuring active maternal care after pup retrieval of female offspring. We then examined locomotor activity, open field exploration, and restraint stress reactivity in adult female offspring. We also investigated the expression of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) and DNA methylation of the GR17 promoter in the hippocampus. High compared with low LG siblings and female offspring from high compared with low LG dams showed increased locomotor activity. High compared with low LG siblings also showed reduced anxiety behavior regardless of the overall level of LG received in the litter. Unexpectedly, both the lowest licked offspring from low LG litters and the highest licked offspring from high LG litters showed suppressed corticosterone (CORT) responses to stress. However, high LG offspring within litters also showed increased expression of the GR gene, which was negatively correlated with the CORT response to restraint. DNA methylation at 2 CpG sites within GR17 promoter was significantly higher in high LG offspring. These differences in the response to maternal care both within- and between-litters were distinct in part from previous reports of between litter effects, potentially a result of the sex studied or the methods used to observe maternal care.

Cocaine exposure prior to pregnancy alters the psychomotor response to cocaine and transcriptional regulation of the dopamine D1 receptor in adult male offspring.

There is evidence that maternal experience prior to pregnancy can play an important role in behavioral, physiological, and genetic programming of offspring. Likewise, exposure to cocaine in utero can result in marked changes in central nervous system function of offspring. In this study, we examined whether exposure of rat dams to cocaine prior to pregnancy subsequently alters indices of behavior, physiology, and gene expression in offspring. Multiple outcome measures were examined in adult male offspring: (1) behavioral expression of cocaine-induced psychomotor activation; (2) levels of corticosterone in response to immobilization stress; and (3) expression of multiple genes, including dopamine receptor D1 (DRD1) and D2 (DRD2), glucocorticoid receptor (GR), and corticotropin-releasing factor (CRF), in functionally relevant brain regions. Adult Sprague-Dawley females were exposed to cocaine (15-30 mg/kg, i.p.) or saline for 10 days, and were then mated to drug naïve males of the same strain. Separate groups of adult male offspring were tested for their acute psychomotor response to cocaine (0, 15, 30 mg/kg, i.p.), corticosterone responsivity to 20 min of immobilization stress, and expression of multiple genes using quantitative PCR. Offspring of dams exposed to cocaine prior to conception exhibited increased psychomotor sensitivity to cocaine, and upregulated gene expression of DRD1 in the medial prefrontal cortex (mPFC). Neither stress-induced corticosterone levels nor gene expression of GR or CRF genes were altered. These data suggest that cocaine exposure before pregnancy can serve to enhance psychomotor sensitivity to cocaine in offspring, possibly via alterations in dopamine function that include upregulation of the DRD1.

The effects of vitamin D3 during pregnancy and lactation on offspring physiology and behavior in sprague-dawley rats.

Recent findings show that developmental vitamin D deficiency leads to altered brain morphology and behavioral development in the rat offspring. We examined the effects of different dietary vitamin D levels in rat dams on behavior and biochemistry of the offspring. Females were divided into five conditions and received diets containing 0, 1,5, 3.3, 6.0, or 10.0 IU/g of vitamin D3 from mating to weaning. Offspring were tested as juveniles and as adults for anxiety, social learning and behavior, and locomotion. Results show that both deficient and excessive levels of vitamin D3 in juveniles lead to altered physiology and behavior. In juveniles but not adults, variations in vitamin D were related to variations in measures of anxiety and marginally, activity levels. For social behaviors, both juveniles and adults were affected by mothers' diets. In general, offspring of animals receiving abnormal concentrations of vitamin D showed the most deficits.

In-vitro evidence for efficacy of antimicrobial mouthrinses.

OBJECTIVE: The objective of this study was to compare the antimicrobial activity of commercially available antiseptic mouthrinses against saliva-derived plaque biofilms in static and flow-through biofilm systems in vitro. METHODS: Nine mouthrinses were tested in a recirculating flow-through biofilm model (RFTB) with viability assessment by ATP bioluminescence. In addition, five mouthrinses were evaluated in a batch chamber slide biofilm (BCSB) model, using live/dead staining and confocal laser scanning microscopy. RESULTS: In the RFTB model, essential oil (EO) and chlorhexidine (CHX)-containing rinses showed equivalent antimicrobial activity and were more effective than a range of cetyl pyridinium chloride (CPC1) formulations. In the BCSB model, twice-daily mouthrinse exposure demonstrated that the EO rinse was significantly more effective than rinses containing amine and stannous fluorides (AFSF), a combination of CPC/CHX, and another CPC formulation (CPC2). EO showed biofilm kill comparable to the CHX rinse. CONCLUSIONS: The present studies have shown that mouthrinses vary significantly in their capability to kill plaque biofilm bacteria in BCSB and RFTB models. The EO mouthrinse demonstrated superior antiplaque biofilm activity to AFSF, CPC/CHX, and CPC rinses and comparable activity to CHX. The methods tested may be of value for the in-vitro screening of antiseptic rinses with different modes of antimicrobial action.

Lipid raft compartmentalization of urokinase receptor signaling in human neutrophils.

Urokinase plasminogen activator (uPA) receptors (uPAR) can be engaged for activation signaling either by aggregation or by binding exogenous uPA. These signaling mechanisms require uPAR to associate with two distinct adhesion proteins, L-selectin and complement receptor 3 (CR3), respectively. uPAR contains a glycosylphosphatidylinositol anchor, suggesting that it is concentrated within glycosphingolipid-enriched microdomains, or "lipid rafts". This study was undertaken to determine the extent to which uPAR-mediated signaling is compartmentalized to lipid rafts. Human neutrophil uPAR was cross-linked or stimulated with uPA after pretreatment with the lipid raft-disrupting agents, methyl-beta-cyclodextrin or filipin III. Both agents suppressed increases in intracellular Ca(2+) concentrations ([Ca(2+)](i)) triggered by cross-linking, but did not affect [Ca(2+) ](i) in response to uPA. Neutrophil membranes were separated into lipid raft and non-raft fractions, revealing the presence of uPAR and L-selectin, but the virtual absence of CR3 alpha chain in lipid rafts, either constitutively or in response to uPAR aggregation. Fluorescence resonance energy transfer experiments confirmed close proximity of a lipid raft marker to both uPAR and L-selectin, but not CR3. We conclude that uPAR can engage distinct signaling pathways involving different partner proteins that are functionally and physically segregated from one another in both lipid raft and non-raft domains of the plasma membrane.

Dental calculus composition following use of essential-oil/ZnCl2 mouthrinse.

PURPOSE: To test the hypothesis that anticalculus agents cannot completely inhibit calculus formation but can influence the types of calcium phosphate which form, i.e., they can influence the composition of the inorganic component of human dental calculus (HDC). MATERIALS AND METHODS: The composition of HDC specimens obtained from a 16-week multi-center clinical study using three regimens were analyzed, investigators blinded. The treatment regimens were: (a) standard dentifrice (SD), (b) pyrophosphate antitartar dentifrice, and (c) SD with Tartar Control Listerine Antiseptic mouthrinse (containing essential oils and 0.09% zinc chloride). 25 individual samples and eight pooled samples from each group were analyzed using X-ray diffraction, infrared spectroscopy, and scanning electron microscopy. RESULTS: (1) relative frequency of occurrence for: (a) bacteria: Group A = 100%, Group B = 60%, and Group C = 25%; (b) Carbonate hydroxyapatite (CHA): Groups A, B, and C = 100%; (c) dicalcium phosphate dihydrate (DCPD): Group A = 55%; Group B = 45%; Group C = 80%; (2) The relative amount of DCPD is inversely proportional to that of CHA in HDC: the higher the amount of DCPD, the lower the amount of CHA. Group C regimen with essential oil/ZnCl2 mouthrinse and standard dentifrice showed a significant anti-microbial effect and favored the formation of DCPD, the most soluble Ca-P.