Central activation of catecholamine-independent lipolysis drives the end-stage catabolism of all adipose tissues.

Several adipose depots, including constitutive bone marrow adipose tissue (cBMAT), resist conventional lipolytic cues, making them metabolically non-responsive. However, under starvation, wasting, or cachexia, the body can eventually catabolize these stable adipocytes through unknown mechanisms. To study this, we developed a mouse model of brain-evoked depletion of all fat, including cBMAT, independent of food intake. Genetic, surgical, and chemical approaches demonstrated that depletion of stable fat required adipose triglyceride lipase-dependent lipolysis but was independent of local nerves, the sympathetic nervous system, and catecholamines. Instead, concurrent hypoglycemia and hypoinsulinemia activated a potent catabolic state by suppressing lipid storage and increasing catecholamine-independent lipolysis via downregulation of cell-autonomous lipolytic inhibitors Acvr1c, G0s2, and Npr3. This was also sufficient to delipidate classical adipose depots. Overall, this work defines unique adaptations of stable adipocytes to resist lipolysis in healthy states while isolating a potent in vivo neurosystemic pathway by which the body can rapidly catabolize all adipose tissues.

Norepinephrine Neurons in the Nucleus of the Solitary Tract Suppress Luteinizing Hormone Secretion in Female Mice.

Stress impairs fertility, at least in part, via inhibition of gonadotropin secretion. Luteinizing hormone (LH) is an important gonadotropin that is released in a pulsatile pattern in males and in females throughout the majority of the ovarian cycle. Several models of stress, including acute metabolic stress, suppress LH pulses via inhibition of neurons in the arcuate nucleus of the hypothalamus that coexpress kisspeptin, neurokinin B, and dynorphin (termed KNDy cells) which form the pulse generator. The mechanism for inhibition of KNDy neurons during stress, however, remains a significant outstanding question. Here, we investigated a population of catecholamine neurons in the nucleus of the solitary tract (NTS), marked by expression of the enzyme dopamine beta-hydroxylase (DBH), in female mice. First, we found that a subpopulation of DBH neurons in the NTS is activated (express c-Fos) during metabolic stress. Then, using chemogenetics, we determined that activation of these cells is sufficient to suppress LH pulses, augment corticosterone secretion, and induce sickness-like behavior. In subsequent studies, we identified evidence for suppression of KNDy cells (rather than downstream signaling pathways) and determined that the suppression of LH pulses was not dependent on the acute rise in glucocorticoids. Together these data support the hypothesis that DBH cells in the NTS are important for regulation of neuroendocrine and behavioral responses to stress.

Norepinephrine Drives Sleep Fragmentation Activation of Asparagine Endopeptidase, Locus Ceruleus Degeneration, and Hippocampal Amyloid-ß42 Accumulation.

Chronic sleep disruption (CSD), from insufficient or fragmented sleep and is an important risk factor for Alzheimer's disease (AD). Underlying mechanisms are not understood. CSD in mice results in degeneration of locus ceruleus neurons (LCn) and CA1 hippocampal neurons and increases hippocampal amyloid-ß42 (Aß42), entorhinal cortex (EC) tau phosphorylation (p-tau), and glial reactivity. LCn injury is increasingly implicated in AD pathogenesis. CSD increases NE turnover in LCn, and LCn norepinephrine (NE) metabolism activates asparagine endopeptidase (AEP), an enzyme known to cleave amyloid precursor protein (APP) and tau into neurotoxic fragments. We hypothesized that CSD would activate LCn AEP in an NE-dependent manner to induce LCn and hippocampal injury. Here, we studied LCn, hippocampal, and EC responses to CSD in mice deficient in NE [dopamine ß-hydroxylase (Dbh)-/-] and control male and female mice, using a model of chronic fragmentation of sleep (CFS). Sleep was equally fragmented in Dbh -/- and control male and female mice, yet only Dbh -/- mice conferred resistance to CFS loss of LCn, LCn p-tau, and LCn AEP upregulation and activation as evidenced by an increase in AEP-cleaved APP and tau fragments. Absence of NE also prevented a CFS increase in hippocampal AEP-APP and Aß42 but did not prevent CFS-increased AEP-tau and p-tau in the EC. Collectively, this work demonstrates AEP activation by CFS, establishes key roles for NE in both CFS degeneration of LCn neurons and CFS promotion of forebrain Aß accumulation, and, thereby, identifies a key molecular link between CSD and specific AD neural injuries.

Endothelial MEKK3-KLF2/4 signaling integrates inflammatory and hemodynamic signals during definitive hematopoiesis.

The hematopoietic stem cells (HSCs) that produce blood for the lifetime of an animal arise from RUNX1+ hemogenic endothelial cells (HECs) in the embryonic vasculature through a process of endothelial-to-hematopoietic transition (EHT). Studies have identified inflammatory mediators and fluid shear forces as critical environmental stimuli for EHT, raising the question of how such diverse inputs are integrated to drive HEC specification. Endothelial cell MEKK3-KLF2/4 signaling can be activated by both fluid shear forces and inflammatory mediators, and it plays roles in cardiovascular development and disease that have been linked to both stimuli. Here we demonstrate that MEKK3 and KLF2/4 are required in endothelial cells for the specification of RUNX1+ HECs in both the yolk sac and dorsal aorta of the mouse embryo and for their transition to intraaortic hematopoietic cluster (IAHC) cells. The inflammatory mediators lipopolysaccharide and interferon-γ increase RUNX1+ HECs in an MEKK3-dependent manner. Maternal administration of catecholamines that stimulate embryo cardiac function and accelerate yolk sac vascular remodeling increases EHT by wild-type but not MEKK3-deficient endothelium. These findings identify MEKK-KLF2/4 signaling as an essential pathway for EHT and provide a molecular basis for the integration of diverse environmental inputs, such as inflammatory mediators and hemodynamic forces, during definitive hematopoiesis.

M1-muscarinic receptors promote fear memory consolidation via phospholipase C and the M-current.

Neuromodulators released during and after a fearful experience promote the consolidation of long-term memory for that experience. Because overconsolidation may contribute to the recurrent and intrusive memories of post-traumatic stress disorder, neuromodulatory receptors provide a potential pharmacological target for prevention. Stimulation of muscarinic receptors promotes memory consolidation in several conditioning paradigms, an effect primarily associated with the M1 receptor (M1R). However, neither inhibiting nor genetically disrupting M1R impairs the consolidation of cued fear memory. Using the M1R agonist cevimeline and antagonist telenzepine, as well as M1R knock-out mice, we show here that M1R, along with ß2-adrenergic (ß2AR) and D5-dopaminergic (D5R) receptors, regulates the consolidation of cued fear memory by redundantly activating phospholipase C (PLC) in the basolateral amygdala (BLA). We also demonstrate that fear memory consolidation in the BLA is mediated in part by neuromodulatory inhibition of the M-current, which is conducted by KCNQ channels and is known to be inhibited by muscarinic receptors. Manipulating the M-current by administering the KCNQ channel blocker XE991 or the KCNQ channel opener retigabine reverses the effects on consolidation caused by manipulating ß2AR, D5R, M1R, and PLC. Finally, we show that cAMP and protein kinase A (cAMP/PKA) signaling relevant to this stage of consolidation is upstream of these neuromodulators and PLC, suggesting an important presynaptic role for cAMP/PKA in consolidation. These results support the idea that neuromodulatory regulation of ion channel activity and neuronal excitability is a critical mechanism for promoting consolidation well after acquisition has occurred.

Stress and glucocorticoids impair memory retrieval via ß2-adrenergic, Gi/o-coupled suppression of cAMP signaling.

Acute stress impairs the retrieval of hippocampus-dependent memory, and this effect is mimicked by exogenous administration of stress-responsive glucocorticoid hormones. It has been proposed that glucocorticoids affect memory by promoting the release and/or blocking the reuptake of norepinephrine (NE), a stress-responsive neurotransmitter. It has also been proposed that this enhanced NE signaling impairs memory retrieval by stimulating ß(1)-adrenergic receptors and elevating levels of cAMP. In contrast, other evidence indicates that NE, ß(1), and cAMP signaling is transiently required for the retrieval of hippocampus-dependent memory. To resolve this discrepancy, wild-type rats and mice with and without gene-targeted mutations were stressed or treated with glucocorticoids and/or adrenergic receptor drugs before testing memory for inhibitory avoidance or fear conditioning. Here we report that glucocorticoids do not require NE to impair retrieval. However, stress- and glucocorticoid-induced impairments of retrieval depend on the activation of ß(2) (but not ß(1))-adrenergic receptors. Offering an explanation for the opposing functions of these two receptors, the impairing effects of stress, glucocorticoids and ß(2) agonists on retrieval are blocked by pertussis toxin, which inactivates signaling by G(i/o)-coupled receptors. In hippocampal slices, ß(2) signaling decreases cAMP levels and greatly reduces the increase in cAMP mediated by ß(1) signaling. Finally, augmenting cAMP signaling in the hippocampus prevents the impairment of retrieval by systemic ß(2) agonists or glucocorticoids. These results demonstrate that the ß(2) receptor can be a critical effector of acute stress, and that ß(1) and ß(2) receptors can have quite distinct roles in CNS signaling and cognition.

Increased tumor necrosis factor-α, cleaved caspase 3 levels and insulin receptor substrate-1 phosphorylation in the ß1-adrenergic receptor knockout mouse.

PURPOSE: To investigate the role of ß1-adrenergic receptors on insulin like growth factor (IGF)-1 receptor signaling and apoptosis in the retina using ß1-adrenergic receptor knockout (KO) mice. METHODS: Western blotting and enzyme-linked immunosorbent assay analyses were done on whole retinal lysates from ß1-adrenergic receptor KO mice and wild-type littermates. In addition, vascular analyses of degenerate capillaries and pericyte ghosts were done on the retina of the ß1-adrenergic receptor KO mice versus littermates. RESULTS: Lack of ß1-adrenergic receptors produced a significant increase in both degenerate capillaries and pericyte ghosts. This was accompanied by an increase in cleaved caspase 3 and tumor necrosis factor α levels. IGF-1 receptor phosphorylation was not changed; however, protein kinase B (Akt) phosphorylation was significantly decreased. The decrease in Akt phosphorylation is likely caused by increased insulin receptor substrate-1 serine 307 (IRS-1(Ser307)) phosphorylation, which is inhibitory to IGF-1 receptor signaling. CONCLUSIONS: These studies further support the idea that maintenance of ß-adrenergic receptor signaling is beneficial for retinal homeostasis. Loss of ß1-adrenergic receptor signaling alters tumor necrosis factor α and apoptosis levels in the retina, as well as Akt and IGF-1 receptor phosphorylation. Since many of these same changes are observed in the diabetic retina, these data support that novel ß-adrenergic receptor agents may provide additional avenues for therapeutics.

Effects of insulin-like growth factor-1 (IGF-1) receptor signaling on rates of apoptosis in retina of dopamine beta hydroxylase (Dbh-/-) knockout mice.

We have investigated whether insulin-like growth factor-1 (IGF-1) receptor signaling alters rates of apoptosis in dopamine beta-hydroxylase (Dbh(-/-)) knockout mice. Retinal lysates from Dbh(-/-) and their heterozygote littermates (Dbh(+/-)) were used to examine the role of norepinephrine in the regulation of IGF-1 receptor signaling and apoptosis in the retina. Western blot analysis was done for protein levels of total and phosphorylated IGF-1 receptor, insulin receptor substrate-1 (IRS-1), insulin receptor substrate-2 (IRS-2), and Akt. A caspase 3 ELISA and dopamine ELISA were done on retinal lysates. To verify which regions of the retina were undergoing apoptosis, TUNEL labeling was performed. No changes in dopamine were noted between the KO and heterozygote mice. IGF-1 receptor phosphorylation was significantly decreased in Dbh(-/-) mice as compared to their heterozygote littermates (P<0.05 vs. heterozygous mice). IRS-1 protein phosphorylation was significantly decreased in KO mice (P<0.05 vs. heterozygous mice), while no significant changes were noted in IRS-2 protein phosphorylation. Akt protein phosphorylation was also reduced in the KO mice, likely leading to increased cleaved caspase 3 levels. The increase in apoptosis in the Dbh(-/-) mice occurred predominantly in the inner retina. Our results suggest that IGF-1 receptor signaling is reduced in the retina of mice with dysfunctional adrenergic receptor signaling. The data also indicate that IGF-1 receptor signaling occurs primarily through IRS-1, rather than IRS-2. The reduction in Akt phosphorylation, likely through reduced IGF-1 receptor signaling, could explain the increase in cleaved caspase 3, leading to apoptosis. These results suggest that alterations in adrenergic receptor signaling modulate IGF-1 receptor signaling, which can regulate apoptosis in the retina.

Epac signaling is required for hippocampus-dependent memory retrieval.

Previously we uncovered a critical role for norepinephrine and beta(1)-adrenergic signaling in hippocampus-dependent memory retrieval. Because the beta(1) receptor couples to G(s), we examine here whether cAMP is also required for contextual memory retrieval. Using pharmacologic and genetic approaches to manipulate cAMP and downstream signaling, we demonstrate that cAMP and two of its targets, protein kinase A (PKA) and exchange protein activated by cAMP (Epac), are both required for retrieval. These findings demonstrate that cAMP signaling through Epac (as well as PKA) plays an essential role in cognition.

Cytosolic catechols inhibit alpha-synuclein aggregation and facilitate the formation of intracellular soluble oligomeric intermediates.

Aberrant aggregation of alpha-synuclein (alpha-syn) to form fibrils and insoluble aggregates has been implicated in the pathogenic processes of many neurodegenerative diseases. Despite the dramatic effects of dopamine in inhibiting the formation of alpha-syn fibrils by stabilization of oligomeric intermediates in cell-free systems, no studies have examined the effects of intracellular dopamine on alpha-syn aggregation. To study this process and its association with neurodegeneration, intracellular catechol levels were increased to various levels by expressing different forms of tyrosine hydroxylase, in cells induced to form alpha-syn aggregates. The increase in the steady-state dopamine levels inhibited the formation of alpha-syn aggregates and induced the formation of innocuous oligomeric intermediates. Analysis of transgenic mice expressing the disease-associated A53T mutant alpha-syn revealed the presence of oligomeric alpha-syn in nondegenerating dopaminergic neurons that do contain insoluble alpha-syn. These data indicate that intraneuronal dopamine levels can be a major modulator of alpha-syn aggregation and inclusion formation, with important implications on the selective degeneration of these neurons in Parkinson's disease.

Signals from the sympathetic nervous system regulate hematopoietic stem cell egress from bone marrow.

Hematopoietic stem and progenitor cells (HSPC), attracted by the chemokine CXCL12, reside in specific niches in the bone marrow (BM). HSPC migration out of the BM is a critical process that underlies modern clinical stem cell transplantation. Here we demonstrate that enforced HSPC egress from BM niches depends critically on the nervous system. UDP-galactose ceramide galactosyltransferase-deficient (Cgt(-/-)) mice exhibit aberrant nerve conduction and display virtually no HSPC egress from BM following granulocyte colony-stimulating factor (G-CSF) or fucoidan administration. Adrenergic tone, osteoblast function, and bone CXCL12 are dysregulated in Cgt(-/-) mice. Pharmacological or genetic ablation of adrenergic neurotransmission indicates that norepinephrine (NE) signaling controls G-CSF-induced osteoblast suppression, bone CXCL12 downregulation, and HSPC mobilization. Further, administration of a beta(2) adrenergic agonist enhances mobilization in both control and NE-deficient mice. Thus, these results indicate that the sympathetic nervous system regulates the attraction of stem cells to their niche.

Norepinephrine regulates hepatic innate immune system in leptin-deficient mice with nonalcoholic steatohepatitis.

It is not known why natural killer T (NKT) cells, which modulate liver injury by regulating local cytokine production, are reduced in leptin-deficient ob/ob mice. NKT cells express adrenoceptors. Thus, we hypothesize that the low norepinephrine (NE) activity of ob/ob mice promotes depletion of liver NKT cells, thereby sensitizing ob/ob livers to lipopolysaccharide (LPS) toxicity. To evaluate this hypothesis, hepatic NKT cells were quantified in wild-type mice before and after treatment with NE inhibitors, and in dopamine beta-hydroxylase knockout mice (which cannot synthesize NE) and ob/ob mice before and after 4 weeks of NE supplementation. Decreasing NE activity consistently reduces liver NKT cells, while increasing NE has the opposite effect. Analysis of hepatic and thymic NKT cells in mice of different ages demonstrate an age-related accumulation of hepatic NKT cells in normal mice, while liver NKT cells become depleted after birth in ob/ob mice, which have increased apoptosis of hepatic NKT cells. NE treatment inhibits apoptosis and restores hepatic NKT cells. In ob/ob mice with reduced hepatic NKT cells, hepatic T and NKT cells produce excessive T helper (Th)-1 proinflammatory cytokines and the liver is sensitized to LPS toxicity. NE treatment decreases Th-1 cytokines, increases production of Th-2 cytokines, and reduces hepatotoxicity. Studies of CD1d-deficient mice, which lack the receptor required for NKT cell development, demonstrate that they are also unusually sensitive to LPS hepatotoxicity. In conclusion, low NE activity increases hepatic NKT cell apoptosis and depletes liver NKT cells, promoting proinflammatory polarization of hepatic cytokine production that sensitizes the liver to LPS toxicity.

Potency of catecholamines and other L-tyrosine derivatives at the cloned mouse adrenergic receptors.

The adrenergic system is a neuromodulatory system whose endogenous ligands are considered to be the catecholamines norepinephrine (NE) and epinephrine (E). Evidence suggests that the catecholamine dopamine (DA) may also activate adrenergic signaling. Further, tyramine (TA) and octopamine (OA) are other monoamines that can be produced in catecholaminergic cells when tyrosine hydroxylase activity is low or absent, as in some genetic mouse models of adrenergic function. Here, we systematically examine the ability of these L-tyrosine-derived monoamines to activate all 10 known isoforms of the cloned mouse adrenergic receptors expressed in Chinese hamster ovary cells. In comparison to NE or E, DA is nearly as efficacious in this system but is from 1 to 4 orders of magnitude less potent. In comparison to DA, OA has roughly equivalent potency but is usually only a partial agonist. TA is either very weak or lacks agonism. Of note, all three mouse alpha(1) receptors increase cAMP, in contrast to results reported for human alpha(1d) receptors. In addition, a 12-amino acid hemagglutinin epitope tag added to the N-terminus of alpha(2) receptors selectively enhances the potency of NE approximately 10- to 100-fold, indicating that caution should be applied when interpreting physiological results from experiments using modified receptors.

Genetic alterations that inhibit in vivo pressure-overload hypertrophy prevent cardiac dysfunction despite increased wall stress.

BACKGROUND: A long-standing hypothesis has been that hypertrophy is compensatory and by normalizing wall stress acts to maintain normal cardiac function. Epidemiological data, however, have shown that cardiac hypertrophy is associated with increased mortality, thus casting doubt on the validity of this hypothesis. METHODS AND RESULTS: To determine whether cardiac hypertrophy is necessary to preserve cardiac function, we used 2 genetically altered mouse models that have an attenuated hypertrophic response to 8 weeks of pressure overload. End-systolic wall stress (sigma(es)) obtained by sonomicrometry after 1 week of pressure overload showed complete normalization of sigma(es) in pressure-overloaded wild-type mice (287+/-39 versus sham, 254+/-34 g/cm2), whereas the blunted hypertrophic response in the transgenic mice was inadequate to normalize sigma(es) (415+/-81 g/cm2, P<0.05). Remarkably, despite inadequate normalization of sigma(es), cardiac function as measured by serial echocardiography showed little deterioration in either of the pressure-overloaded genetic models with blunted hypertrophy. In contrast, wild-type mice with similar pressure overload showed a significant increase in chamber dimensions and progressive deterioration in cardiac function. Analysis of downstream signaling pathways in the late stages of pressure overload suggests that phosphoinositide 3-kinase may play a pivotal role in the transition from hypertrophy to heart failure. CONCLUSIONS: These data suggest that under conditions of pressure overload, the development of cardiac hypertrophy and normalization of wall stress may not be necessary to preserve cardiac function, as previously hypothesized.