Activation of mesolimbic reward system via laterodorsal tegmental nucleus and hypothalamus in exercise-induced hypoalgesia.

Ventral tegmental area (VTA) dopamine (DA) neurons are the primary source of dopamine in target structures that constitute the mesolimbic reward system. Previous studies demonstrated that voluntary wheel running (VWR) by neuropathic pain (NPP) model mice produces exercise-induced hypoalgesia (EIH), and that activation of mesolimbic reward system may lead to EIH. However, the neuronal mechanism by which the mesolimbic reward system is activated by VWR is unknown. Here, we found that VWR produces EIH effects and reverses the marked reduction in activated lateral VTA (lVTA)-DA neurons induced by NPP. The proportions of activated laterodorsal tegmental nucleus (LDT)-cholinergic and lateral hypothalamus-orexin neurons were significantly enhanced by VWR. Retrograde tracing and dual immunostaining revealed that VWR activates lVTA-projecting LDT-cholinergic/non-cholinergic and lateral hypothalamic area (LHA)-orexin/non-orexin neurons. Therefore, EIH effects may be produced, at least in part, by activation of the mesolimbic reward system via activation of LDT and LHA neurons.

Regulation of AMP-activated protein kinase signaling by AFF4 protein, member of AF4 (ALL1-fused gene from chromosome 4) family of transcription factors, in hypothalamic neurons.

In the hypothalamus, fasting induces a member of the AF4 family of transcription factors, AFF4, which was originally identified as a fusion partner of the mixed-lineage leukemia gene in infant acute lymphoblastic leukemia. However, the roles of AFF4 in the hypothalamus remain unclear. We show herein that expression of AFF4 increased upon addition of ghrelin and fasting in the growth hormone secretagogue receptor-expressing neurons of the hypothalamus. In the growth hormone secretagogue receptor-expressing hypothalamic neuronal cell line GT1-7, ghrelin markedly induced expression of AFF4 in a time- and dose-dependent manner. Overexpression of AFF4 in GT1-7 cells specifically induced expression of the AMP-activated protein kinase (AMPK) α2 subunit but failed to induce other AMPK subunits and AMPK upstream kinases. The promoter activity of the AMPKα2 gene increased upon addition of AFF4, suggesting that AFF4 regulates transcription of the AMPKα2 gene. Additionally, AFF4 also increased the phosphorylation of acetyl-CoA carboxylase α (ACCα), a downstream target of AMPK. In GT1-7 cells, ghrelin phosphorylated ACCα through AMPKα phosphorylation in the early phase (15 min) of the activation. However, ghrelin-induced expression of AMPKα2 and phosphorylation of ACCα in the late phase (2 h) of the activation were independent of AMPKα phosphorylation. Attenuation of expression of AFF4 by its siRNA in GT1-7 cells decreased ghrelin-induced AMPKα2 expression and ACCα phosphorylation in the late phase of the activation. AFF4 may therefore help to maintain activation of AMPK downstream signaling under conditions of prolonged stimulation with ghrelin, such as during fasting.

Regulation of ghrelin signaling by a leptin-induced gene, negative regulatory element-binding protein, in the hypothalamic neurons.

Leptin, the product of the ob gene, plays important roles in the regulation of food intake and body weight through its receptor in the hypothalamus. To identify novel transcripts induced by leptin, we performed cDNA subtraction based on selective suppression of the polymerase chain reaction by using mRNA prepared from the forebrain of leptin-injected ob/ob mice. One of the genes isolated was a mouse homolog of human negative regulatory element-binding protein (NREBP). Its expression was markedly increased by leptin in the growth hormone secretagogue-receptor (GHS-R)-positive neurons of the arcuate nucleus and ventromedial hypothalamic nucleus. The promoter region of GHS-R contains one NREBP binding sequence, suggesting that NREBP regulates GHS-R transcription. Luciferase reporter assays showed that NREBP repressed GHS-R promoter activity in a hypothalamic neuronal cell line, GT1-7, and its repressive activity was abolished by the replacement of negative regulatory element in GHS-R promoter. Overexpression of NREBP reduced the protein expression of endogenous GHS-R without affecting the expression of ob-Rb in GT1-7 cells. To determine the functional importance of NREBP in the hypothalamus, we assessed the effects of NREBP on ghrelin action. Although phosphorylation of AMP-activated protein kinase α (AMPKα) was induced by ghrelin in GT1-7 cells, NREBP repressed ghrelin-induced AMPKα phosphorylation. These results suggest that leptin-induced NREBP is an important regulator of GHS-R expression in the hypothalamus and provides a novel molecular link between leptin and ghrelin signaling.

[Limbic system and autonomic nervous system].

The viral transneuronal labeling method using pseudorabies virus (PRV) is an ideal technique for identifying the central sites that regulate the sympathetic nervous system. Regions were identified in limbic system such as extended amygdaloid complex, lateral septum, infralimbic, insular, ventromedial temporal cortical regions, as well as in several hypothalamic and brain stem nuclei. Emotional stress causes rapid and transient expression of immediate early genes (IEGs) such as c-Fos in the brain, and the monitoring of IEGs has enabled the visualization of the neurocircuitry of stress. By a comparison of the data from the separate PRV and c-Fos neuroanatomical labeling techniques, the central sites which regulate emotional stress-induced sympathoadrenal activation can be deduced. Estrogen receptors are expressed in the brain, where estrogen modulates central nervous function and autonomic nervous function. Estrogen attenuated the stress-induced c-Fos expression in medial amygdaloid nucleus, paraventricular hypothalamic nucleus; these same regions contain central sympathetic neurons and neurons with immunoreactive estrogen receptors.

Nitric oxide involvement in lipid emulsion-induced vascular pain in anesthetized rats.

We examined the vascular pain induced by arterial infusion of 20% lipid emulsion by using a flexor reflex model in anesthetized rats. Arterial infusion of 20% lipid emulsion at doses of 0.6 to 2 ml/2 min induced flexor reflexes that were late in onset, persistent, and intense compared with those induced by 2.7% amino acid and 7.5% glucose solution, 5% sodium chloride solution, 1% propofol, and capsaicin. The flexor reflex induced by 20% lipid emulsion was significantly inhibited by preinjected procaine hydrochloride (4 mg/rat, i.a.) but not by the critical dose of indomethacin (10 mg/kg, i.p.). These results suggest that the flexor reflex might reflect a 20% lipid emulsion-induced vascular pain response and that the site of action of noxious agents involved in this event might be a vascular bed, but the production of prostanoids through cyclooxygenase might not be involved in the action mechanisms. The 20% lipid emulsion-induced vascular pain was significantly inhibited by preinjection of 10 mg/kg N(G)-nitro-l-arginine methyl ester hydrochloride (l-NAME), a nitric oxide (NO) synthase inhibitor, and the inhibition by l-NAME was recovered by the addition of sodium nitroprusside (30 microg/kg/min), which is an endothelium-independent NO donor to 20% lipid emulsion. These results indicate that increased NO production is responsible for 20% lipid emulsion-induced vascular pain. In summary, the arterial infusion of 20% lipid emulsion induced a delayed, persistent and intense flexor reflex, presumably indicating vascular pain in rats that might be induced by NO production through the activation of NO synthase.

Attenuation of fasting-induced phosphorylation of mitogen-activated protein kinases (ERK/p38) in the mouse hypothalamus in response to refeeding.

Nutritional status modify the expression of hypothalamic neuropeptides through various signal molecules, including mitogen-activated protein kinases (MAPKs) and cAMP/calcium-responsive element-binding protein (CREB), for the regulation of energy balance. Previously, we demonstrated fasting-induced activation of extracellular signal-regulated kinase 1/2 (ERK) and p38 mitogen-activated protein kinase (p38) in the murine hypothalamus. To study how caloric intake after food deprivation influences intracellular signal transduction, we investigated the phosphorylation of ERK and p38 in the murine hypothalamus of refed mice. In the arcuate nucleus, refeeding significantly attenuated fasting-induced phosphorylation of ERK and CREB. In the paraventricular nucleus, fasting-induced phosphorylation of ERK and p38 was also significantly decreased by refeeding. Thus, refeeding rapidly reduced the fasting-induced phosphorylation of ERK, p38, and CREB, suggesting that the activation of these signal molecules in the hypothalamus is precisely regulated with feeding status.

Increase of galanin-like immunoreactivity in rat hypothalamic arcuate neurons after peripheral nerve injury.

Galanin and galanin receptors are widely distributed within the central nervous system, and may play important roles in pain signaling and modulation. In the present study, we examined the galanin immunoreactivity (IR) in the hypothalamus and the amygdala following peripheral nerve injury. Four weeks after the operation, the ipsilateral mechanical threshold in the spared nerve injury (SNI) group (0.87 +/- 0.33 g) was significantly lower than that in the sham group (12.53 +/- 3.41 g; P < 0.05). In the SNI group, the number of galanin-IR neurons per section in the arcuate nucleus (Arc) of the hypothalamus was 10.2 +/- 1.7, significantly higher than that in the sham group (5.6 +/- 1.0; P < 0.05). These data suggest that the galanin-ergic neurons in the Arc may be involved in the functional modulation of descending pain modulation system following peripheral nerve injury.

Stimulation of 3T3-L1 adipogenesis by signal transducer and activator of transcription 5.

Signal transducer and activator of transcription 5 (Stat5) mediates signaling of many cytokines and growth factors. Here we show that Stat5 functions as an initial mediator of adipogenesis. The preadipocyte cell line 3T3-L1 undergoes adipocyte differentiation upon appropriate hormonal induction. We found that Stat5A and Stat5B were strongly activated at an early stage of 3T3-L1 differentiation. To investigate physiological roles of Stat5 in adipogenesis, we have constructed 3T3-L1 cell lines in which either an exogenous wild type (wt) or dominant negative (dn) form of Stat5A expression was controlled under the doxycycline-regulatable promoter. Precocious induction of wt-Stat5A in adipocyte differentiation promoted accumulation of triglycerides within the cells. In contrast, induction of dn-Stat5A attenuated lipid accumulation. Northern blot analyses revealed that the expression of proadipogenic transcription factors was influenced in a complementary fashion by ectopic expression of either wt- or dn-Stat5A. Notably, Stat5 regulated expression of peroxisome proliferator-activated receptor-gamma, which plays crucial roles in adipogenesis. We have also generated transgenic mice in which dn-Stat5A is expressed in an adipose tissue-specific fashion and found attenuation of peroxisome proliferator-activated receptor-gamma and of many adipocyte-related genes. These results highlight a novel role of Stat5 in adipocyte differentiation.