The sympathetic tone mediates leptin's inhibition of insulin secretion by modulating osteocalcin bioactivity

The osteoblast-secreted molecule osteocalcin favors insulin secretion, but how this function is regulated in vivo by extracellular signals is for now unknown. In this study, we show that leptin, which instead inhibits insulin secretion, partly uses the sympathetic nervous system to fulfill this function. Remarkably, for our purpose, an osteoblast-specific ablation of sympathetic signaling results in a leptin-dependent hyperinsulinemia. In osteoblasts, sympathetic tone stimulates expression of Esp, a gene inhibiting the activity of osteocalcin, which is an insulin secretagogue. Accordingly, Esp inactivation doubles hyperinsulinemia and delays glucose intolerance in ob/ob mice, whereas Osteocalcin inactivation halves their hyperinsulinemia. By showing that leptin inhibits insulin secretion by decreasing osteocalcin bioactivity, this study illustrates the importance of the relationship existing between fat and skeleton for the regulation of glucose homeostasis.     

TIM-4 expressed on APCs induces T cell expansion and survival

TIM (T cell, Ig, mucin) proteins can regulate T cell immune responses. Tim-4 mRNA is not expressed in T cells, but exclusively in APCs. Tim-4 is a ligand for Tim-1 and Tim-4.Ig fusion protein was shown to either inhibit or expand T cells. However, the molecular basis for such opposite effects was not defined. By generating mAbs, we show that expression of Tim-4 protein is restricted to CD11c(+) and CD11b(+) cells and is up-regulated upon activation. We show that Tim-4 specifically phosphorylates Tim-1 and induces T cell expansion by enhancing cell division and reducing apoptosis. Tim-4 also induces the phosphorylation of signaling molecules LAT, Akt, and ERK1/2 in T cells. Tim-4, expressed on APCs, is a costimulatory molecule that promotes T cell expansion and survival by cross-linking Tim-1 on T cells.     

Retinoic acid increases Foxp3+ regulatory T cells and inhibits development of Th17 cells by enhancing TGF-beta-driven Smad3 signaling and inhibiting IL-6 and IL-23 receptor expression

The de novo generation of Foxp3+ regulatory T (Treg) cells in the peripheral immune compartment and the differentiation of Th17 cells both require TGF-beta, and IL-6 and IL-21 are switch factors that drive the development of Th17 cells at the expense of Treg cell generation. The major vitamin A metabolite all-trans retinoic acid (RA) not only enforces the generation of Treg cells but also inhibits the differentiation of Th17 cells. Herein we show that RA enhances TGF-beta signaling by increasing the expression and phosphorylation of Smad3, and this results in increased Foxp3 expression even in the presence of IL-6 or IL-21. RA also inhibits the expression of IL-6Ralpha, IRF-4, and IL-23R and thus inhibits Th17 development. In vitro, RA significantly promotes Treg cell conversion, but in vivo during the development of experimental autoimmune encephalomyelitis it does not increase the frequency of Treg cells in the face of an ongoing inflammation. However, RA suppresses the disease very efficiently by inhibiting proinflammatory T cell responses, especially pathogenic Th17 responses. These data not only identify the signaling mechanisms by which RA can affect both Treg cell and Th17 differentiation, but they also highlight that in vivo during an autoimmune reaction, RA suppresses autoimmunity mainly by inhibiting the generation of effector Th17 cells.     

Latency-associated peptide identifies a novel CD4+CD25+ regulatory T cell subset with TGFbeta-mediated function and enhanced suppression of experimental autoimmune encephalomyelitis

CD4(+)CD25(+) regulatory T cells (Tregs) are essential for maintaining self-tolerance and immune homeostasis. Here we characterize a novel subset of CD4(+)CD25(+) Tregs that express latency-associated peptide (LAP) on their cell surface (CD4(+)CD25(+)LAP(+) cells). CD4(+)CD25(+)LAP(+) cells express elevated levels of Foxp3 and Treg-associated molecules (CTLA4, glucocorticoid-induced TNFR-related gene), secrete TGFbeta, and express both cell surface TGFbeta and surface receptors for TGFbeta. In vitro, the suppressive function of CD4(+)CD25(+)LAP(+) cells is both cell contact and soluble factor dependent; this contrasts with CD4(+)CD25(+)LAP(-) cells, which are mainly cell contact dependent. In a model of experimental autoimmune encephalomyelitis, CD4(+)CD25(+)LAP(+) cells exhibit more potent suppressive activity than CD4(+)CD25(+)LAP(-) cells, and the suppression is TGFbeta dependent. We further show that CD4(+)CD25(+)LAP(+) cells suppress myelin oligodendrocyte glycoprotein-specific immune responses by inducing Foxp3 and by inhibiting IL-17 production. Our findings demonstrate that CD4(+)CD25(+) Tregs are a heterogeneous population and that the CD4(+)CD25(+) subset that expresses LAP functions in a TGFbeta-dependent manner and has greater in vivo suppressive properties. Our work helps elucidate the ambiguity concerning the role of TGFbeta in CD4(+)CD25(+) Treg-mediated suppression and indicates that LAP is an authentic marker able to identify a TGFbeta-expressing CD4(+)CD25(+) Treg subset.     

The effects of age on radiation resistance and oxidative stress in adult Drosophila melanogaster

Drosophila melanogaster (fruit fly) is a well-established model organism for genetic studies of development and aging. We examined the effects of lethal ionizing radiation on male and female adult Drosophila of different ages, using doses of radiation from 200 to 1500 Gy. Fifty percent lethality 2 days postirradiation (LD(50/2)) in wild-type 1-day-old adult fruit flies was approximately 1238 Gy for males and 1339 Gy for females. We observed a significant age-dependent decline in the radiation resistance of both males and females. Radiation damage is postulated to occur by the generation of oxygen radicals. An age-related decline in the ability of flies to resist an agent that induces oxygen radicals, paraquat, was observed when comparing 10- and 20-day adults. Female flies are more resistant to paraquat than male flies. Oxidative stress mediated by paraquat was additive with sublethal exposures to radiation in young adults. Therefore, the ability to repair the damage caused by oxygen radicals seems to decline with the age of the flies. Because Drosophila adults are largely post-mitotic, our data suggest that adult Drosophila melanogaster can serve as an excellent model to study the factors responsible for radiation resistance in post-mitotic tissue and age-dependent changes in this resistance.     

GSK-3beta-dependent destabilization of cyclin D1 mediates replicational stress-induced arrest of cell cycle

Chemotherapeutic agents are well known to induce growth arrest of cancerous cells by inducing DNA damage/replicational stress and engaging cellular apoptotic machinery. Our studies on hydroxyurea (HU) recognized cyclin D1 destabilization as the initiator of growth arrest at G(1)/S-phase independent of other cell cycle regulators. Cyclin D1 degradation was associated with its phosphorylation at Thr286 by glycogen synthase kinase-3beta and inactivation of Akt kinase. Overexpression of the cyclin D1(T286A) mutant, or constitutively active Akt, conferred stability to cyclin D1 and helped bypass cell cycle arrest. Thus, growth arrest by HU seems to involve destabilization of cyclin D1 in addition to its well-established role as ribonucleotide reductase inhibitor.     

Courtship interactions stimulate rapid changes in GnRH synthesis in male ring doves

Many birds and mammals show changes in the hypothalamo-pituitary-gonadal (HPG) axis in response to social or sexual interactions between breeding partners. While alterations in GnRH neuronal activity play an important role in stimulating these changes, it remains unclear if acute behaviorally-induced alterations in GnRH release are accompanied by parallel changes in GnRH synthesis. To investigate this relationship, we examined changes in the activity of GnRH neurons in the brains of male ring doves following brief periods of courtship interactions with females. Such interactions have been previously shown to increase plasma LH in courting male doves at 24 h, but not at 1 h, after pairing with females. In the first study, males allowed to court females for 2 h had 60% more cells that showed immunocytochemical labeling for GnRH-I in the preoptic area (POA) of the hypothalamus than did control males that remained isolated from females. To determine whether an increase in GnRH gene expression preceded this increase in GnRH immunoreactivity in the POA, changes in the number of cells with detectable GnRH-I mRNA in the POA were measured by in situ hybridization following a 1 h period of courtship interactions with females. In this second study, courting males exhibited 40% more cells with GnRH-I in this region than did isolated control males. GnRH-immunoreactive neurons in two other diencephalic regions failed to show these courtship-induced changes. Plasma LH was not elevated after 1 or 2 h of courtship. These results demonstrate that the release of GnRH-I in the POA that is presumably responsible for courtship-induced pituitary and gonadal activation is accompanied by a rapid increase in GnRH synthesis that occurs before plasma LH levels increase. We suggest that this increase in GnRH synthesis is necessary to support the extended period of HPG axis activation that is seen in this species during the 5–10 day period of courtship and nest building activity.     

Na-K-Cl Cotransporter-1 in the mechanism of ammonia-induced astrocyte swelling

Brain edema and the consequent increase in intracranial pressure and brain herniation are major complications of acute liver failure (fulminant hepatic failure) and a major cause of death in this condition. Ammonia has been strongly implicated as an important factor, and astrocyte swelling appears to be primarily responsible for the edema. Ammonia is known to cause cell swelling in cultured astrocytes, although the means by which this occurs has not been fully elucidated. A disturbance in one or more of these systems may result in loss of ion homeostasis and cell swelling. In particular, activation of the Na-K-Cl cotransporter (NKCC1) has been shown to be involved in cell swelling in several neurological disorders. We therefore examined the effect of ammonia on NKCC activity and its potential role in the swelling of astrocytes. Cultured astrocytes were exposed to ammonia (NH(4)Cl; 5 mm), and NKCC activity was measured. Ammonia increased NKCC activity at 24 h. Inhibition of this activity by bumetanide diminished ammonia-induced astrocyte swelling. Ammonia also increased total as well as phosphorylated NKCC1. Treatment with cyclohexamide, a potent inhibitor of protein synthesis, diminished NKCC1 protein expression and NKCC activity. Since ammonia is known to induce oxidative/nitrosative stress, and antioxidants and nitric-oxide synthase inhibition diminish astrocyte swelling, we also examined whether ammonia caused oxidation and/or nitration of NKCC1. Cultures exposed to ammonia increased the state of oxidation and nitration of NKCC1, whereas the antioxidants N-nitro-l-arginine methyl ester and uric acid all significantly diminished NKCC activity. These agents also reduced phosphorylated NKCC1 expression. These results suggest that activation of NKCC1 is an important factor in the mediation of astrocyte swelling by ammonia and that such activation appears to be mediated by NKCC1 abundance as well as by its oxidation/nitration and phosphorylation.     

Activation of endothelial nitric oxide synthase is critical for erythropoietin-induced mobilization of progenitor cells

The present study aimed to define the ability of erythropoietin (EPO) to mobilize hematopoietic stem cells (c-kit(+)/sca-1(+)/lin-1(-); KSL-cells) and hematopoietic progenitor cells (CD34(+) cells), including vascular endothelial growth factor receptor 2 expressing hematopoietic progenitor cells (CD34(+)/Flk-1(+) cells). We also sought to determine the role of endothelial nitric oxide synthase (eNOS) in EPO-induced mobilization. Wild type (WT) and eNOS(-/-) mice were injected bi-weekly with recombinant erythropoietin (EPO, 1000U/kg, s.c.) for 14 days. EPO increased the number of KSL, CD34(+), CD34(+)/Flk-1(+) cells in circulating blood of wild type mice. These effects of EPO were abolished in eNOS(-/-) mice. Our results demonstrate that, EPO stimulates mobilization of hematopoietic stem and progenitor cells. This effect of EPO is critically dependent on activation of eNOS.