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beta-Adducin is a cytoskeletal protein that interacts with the actin filaments to suppress actin polymerization and facilitate actin-spectrin binding. We have previously shown that beta-adducin is phosphorylated by Fyn at tyrosine489 in the rat brain and bound to its Src-homology 2 domain. In the present study, we examined the immunohistochemical localization of the tyrosine489-phosphorylated form of beta-adducin (pY489-beta-adducin) in the rat brain. Among brain regions, highest immunoreactivity was located in the hypothalamic tanycytes that are of glial origin lining around the third cerebral ventricle. Their immunoreactive processes extended into the arcuate nucleus, ventromedial hypothalamus and the median eminence. In addition, the pY489-beta-adducin immunoreactivity in the tanycytes was enhanced after fasting for 36-48 h, being associated with a morphological change of the DARPP-32-immunoreactivity. Intraperitoneal injection of 2-deoxy-d-glucose also enhances pY489-beta-adducin immunoreactivity in the tanycytes, along with increased food intake. These results suggest that tyrosine phosphorylation of beta-adducin in the tanycytes is involved in hypothalamic regulation of food intake and energy homeostasis.

Previously, we observed that olfactory stimulation with scent of grapefruit oil (SGFO) enhances sympathetic nerve activities and suppresses gastric vagal (parasympathetic) nerve activity (GVNA), increases plasma glycerol concentration and body temperature, and decreases appetite in rats. Here, we show that olfactory stimulation with SGFO for 10 min elevates renal sympathetic nerve activity (RSNA) and blood pressure (BP) and lowers GVNA in urethane-anesthetized rats. Olfactory stimulation with limonene, a major component of grapefruit oil, also elicited increases in RSNA and BP in urethane-anesthetized rats. Anosmic treatment with ZnSO(4) eliminated both the effects of SGFO and scent of limonene on RSNA and BP. Intracerebral administration of diphenhydramine, a histaminergic H1-antagonist, abolished SGFO- or scent of limonene-mediated increases in RSNA and BP as well as the decrease in GVNA. Moreover, bilateral lesions of the hypothalamic suprachiasmatic nucleus (SCN) eliminated the SGFO- and limonene-mediated increases in RSNA and BP and decrease in GVNA, but bilateral lesions of the cerebral cortex did not have any affect on these parameters. These findings suggest that scent of grapefruit oil and its active component, limonene, affect autonomic neurotransmission and blood pressure through central histaminergic nerves and the suprachiasmatic nucleus.

Circadian rhythms of mammals are generated by a circadian oscillation of master pacemaker genes in the suprachiasmatic nucleus of the hypothalamus (SCN), and entrained by environmental factors such as 24-h light-dark cycles. We have previously shown that light exposure during the dark period enhanced tyrosine phosphorylation of brain immunoglobulin-like molecule with tyrosine-based activation motifs (BIT) in the rat SCN. To elucidate the functional roles of BIT in the circadian clock, we stimulated BIT using an anti-BIT monoclonal antibody (mAb) 1D4, which reacts with its extracellular region and induces phosphorylation of its intracellular tyrosine residues. Administration of mAb 1D4 into the third cerebral ventricle induced tyrosine phosphorylation of BIT in the SCN. Behavioral analyses showed that the SCN-injection of the antibody at CT15 induced a phase delay of the circadian rhythm of locomotor activity, and that at CT20 induced a phase advance. Pretreatment with MK801, a non-competitive antagonist of NMDARs, diminished the 1D4-induced phase shift at CT20, but not at CT15. These results suggest that BIT is involved in the entrainment of circadian rhythms through the function of NMDARs and non-NMDARs.

In mammals, the brain usually uses glucose as a sole energy source. Thus, under a central glucopenic condition after intracranial injection of 2-deoxy- d -glucose (2DG), an inhibitor of glucose utilization, it has been shown that rats elevate their blood glucose level through excitation of the sympathetic nerves. Experiments were conducted with rats to examine the role of the hypothalamic suprachiasmatic nucleus (SCN) in the hyperglycemic response to intracerebroventricular injection of either 2DG or vasoactive intestinal peptide (VIP). It was observed that, (1) intracerebroventricular injection of a VIP-antagonist inhibited the hyperglycemic and hyperglucagonemic responses to the intracranial injection of 2DG; (2) bilateral electrolytic lesioning of the SCN suppressed the hyperglycemic and hyperglucagonemic responses to intracranial injection of 2DG, and intracerebroventricular injection of VIP restored these responses to 2DG; and (3) bilateral electrolytic lesioning of the SCN also suppressed the hyperglycemic and hyperglucagonemic responses to the VIP injection, and additional intracerebroventricular injection of 2DG caused hyperglycemia. These findings indicate that in rats with bilateral lesions of the SCN intracranial injection of 2DG is able to elicit hyperglycemia when VIP was administered intracranially, and suggest that neurons containing VIP-like immunoreactive substance (VIP-neurons) in the SCN have an important role in the mechanism of hyperglycemia elicitation following intracranial injection of 2DG. Moreover, these findings show that 2DG and VIP are able to realize their functions through acting on the brain sites outside the SCN.

Mammalian Per proteins are thought to be important in the mechanism of circadian rhythm. We identified a novel protein PIPS (Per1 interacting protein of the suprachiasmatic nucleus) with the yeast two-hybrid system using PAS domain of rat Per1 (rPer1) as a bait. PIPS is about a 180-kDa protein and expressed mainly in the brain, especially in the hypothalamus including the suprachiasmatic nuclei (SCN). PIPS interacts with mouse Per1 (mPer1) in vitro and in cultured cells transfected with both molecules. Furthermore, it was found that mPer1 translocated PIPS into the nuclei in the cultured cells. Thus, these findings suggest a possibility that PIPS is involved in the feedback loop or output mechanism of circadian rhythm through interacting with Per1 in the SCN.

In rats there is a direct neural connection called the retinohypothalamic tract (RHT) from retinal ganglion cells to the ventrolateral part of the suprachiasmatic nucleus (SCN), which has neurons containing vasoactive intestinal polypeptide (VIP)-like substance. Previously, we observed that bilateral orbital enucleation (blinding) caused temporary supression of the hyperglycemic response to intracranial injection of 2-deoxy- d -glucose (2DG) from week 4 to 6 after blinding. Moreover, bilateral lesions of the SCN had a similar effect. From these findings, we supposed that the neurons responsible for the hyperglycemic response to 2DG were present in the SCN, that after blinding these neurons temporarily lost their activity, and that this functional change was reflected in the morphology of the SCN. To investigate this possibility, we examined the morphological changes of the SCN by Nissl staining and immunohistochemical studies with anti-VIP and anti-peptide histidine isoleucine (PHI) antibodies in blinded rats, and the relationship between these morphological changes and the hyperglycemic response to 2DG. After surgical blinding, we observed following changes. (1) The optic chiasm became thinner. (2) The SCN became displaced rostrally. (3) The density of neurons in the middle to caudal part of the SCN, where the retinal ganglion cells projected, decreased markedly without change in cell number during the period when the hyperglycemic response to intracranial injection of 2DG was temporarily suppressed after blinding. The first and second changes seemed to reflect reduction of fibers and axon terminals of retinal ganglion cells and their innervation, respectively. As the third change was parallel with suppression of the hyperglycemic response to 2DG injection, it may reflect functional change of the neurons in the SCN that are responsible for the hyperglycemia due to 2DG.

The role of the suprachiasmatic nucleus (SCN) in photoperiod-dependent hyperglycemia due to d -mannitol (MA) and d -glucose (GL) was investigated using SCN-lesioned rats. In control rats, intracranial injection of MA and GL caused hyperglycemia only in the light period and dark period, respectively. In SCN-lesioned rats, administration of MA and GL caused no change in plasma glucose level, irrespective of the lighting condition. SCN-lesioned rats have higher plasma insulin levels than sham-operated rats through this experiment. From these findings, we propose that the SCN is involved in a glucose homeostasis and regulation of insulin secretion.