Michio Niimi

Neuropeptide Y in central control of feeding and interactions with orexin and leptin

Neuropeptide (NPY) increases feeding when injected into the brain. In this study, we tested the hypothesis that its action might be related to feeding regulation of the orexin and leptin systems in rats. Intracerebroventricular administration of NPY (1 nmol/5 µL) stimulated feeding in rats. Injection of an antibody to orexin-A inhibited feeding, suggesting that endogenous orexin exerts a stimulatory tone on feeding. Intracerebroventricular injection of orexin antiserum before injection of NPY significantly attenuated the feeding response to NPY. On the other hand, ip pretreatment with leptin (2 mg/kg) significantly decreased food intake and inhibited NPY-induced feeding. We then examined whether orexin-containing neurons are activated under the stimulation of feeding in response to intracerebroventricular NPY or suppression of feeding in response to ip leptin, using Fos-like immunoreactivity (FLI) as a marker of neural activation. We observed that FLI was induced in the paraventricular, supraoptic, and dorsomedial nuclei as well as the lateral hypothalamic area (LHA) following administration of NPY. Double staining with anti-Fos and antiorexin antibodies revealed that 23.4% of the orexin-containing neurons in the LHA expressed FLI after NPY injection. Approximately 7.8% of the orexin-positive neurons in the LHA coexpressed Fos after leptin plus NPY. Our data indicate that a functional interaction among NPY, orexin, and leptin exists that may contribute to the central regulation of appetite.

A Mutant High-Density Lipoprotein Receptor Inhibits Proliferation of Human Breast Cancer Cells

High-density lipoprotein (HDL) stimulates the growth of many types of cells, including those of breast cancer. High levels of HDL are associated with an increased risk of breast cancer development. A scavenger receptor of the B class (SR-BI)/human homolog of SR-BI, CD36, and LIMPII analogous-1 (CLA-1) facilitates the cellular uptake of cholesterol from HDL and thus augments cell growth. Furthermore, HDL is also believed to have antiapoptotic effects on various cell types, and this feature adds to its ability to promote cell growth. These collaborative roles of HDL and CLA-1 prompted us to assess the function of these components on human breast cancer cells. In this study, we created a mutant CLA-1 (mCLA) that lacked the COOH-terminal tail to determine its potential role in breast cancer cell growth. Expression of mCLA inhibited the proliferation of breast cancer cell line MCF-7. This inhibitory action of mCLA required the transcriptional factor activator protein-1 (AP-1), and the mutant receptor also affected the antiapoptotic features of HDL. The effect of HDL on AP-1 activation and [3H]thymidine incorporation was abrogated by wortmannin, a specific inhibitor of phosphoinositide 3-kinase. Furthermore, the dominant negative mutant of Akt abolished the ability of HDL to activate AP-1. These findings raise the possibility that the inhibitors of the effects of HDL may be of therapeutic value for breast cancer.

Molecular cloning and gene expression of growth hormone-releasing peptide receptor in rat tissues.

We cloned a fragment of the rat GH-releasing peptide (GHRP) receptor homologue and examined the tissue distribution of GHRP receptor mRNA in rats. Sequence analysis showed that the open reading frame is well conserved between rat and human with 96% identity in a 364-amino acid overlap. By reverse transcription-polymerase chain reaction we detected GHRP receptor mRNAs in the rat brain including the hypothalamus, anterior pituitary, and renal pelvis in twenty-eight tissues tested. Microdissection revealed that GHRP receptor mRNAs were localized predominantly in the arcuate nucleus and ventromedial hypothalamus.

Immunohistochemical Identification of Galanin and Growth Hormone-Releasing Factor-Containing Neurons Projecting to the Median Eminence of the Rat

Through the combined demonstration of the retrograde transport of True blue and the immunohistochemical staining of galanin (GAL), the GAL neurons that project to the median eminence were identified. Moreover, the distribution of GAL and growth hormone-releasing factor (GRF) was analyzed in the arcuate nucleus (ARC) with an elution-restaining procedure. Following the injection of True blue into the median eminence, GAL-positive cells labeled with True blue were found mainly in the ARC. In addition, a few GAL neurons in the periventricular, the paraventricular and the supraoptic nuclei were labeled with True blue. The elution-restaining results revealed that many retrogradely labeled GAL neurons in the ARC contained GRF. These findings suggest that a subpopulation of GAL neurons in the ARC sending axons to the median eminence produces, stores and releases a GRF-like peptide.

Induction of Fos protein in the rat hypothalamus elicited by insulin-induced hypoglycemia

To evaluate the responses to insulin-induced hypoglycemia of neurons in vivo, we studied Fos protein induction in the brain by means of immunohistochemistry. The induction of Fos protein was maximum after the first injection of insulin for 3 h. This induction was found in the parvocellular division of paraventricular nucleus (PVN), the periventricular, dorsomedial and arcuate nuclei and the lateral hypothalamic area of the hypothalamus. These findings show the activation of specific subsets of neurons in areas of the hypothalamus following hypoglycemic stimulation.

Central administration of neuromedin U activates neurons in ventrobasal hypothalamus and brainstem.

Neuromedin U (NMU) is a peptide isolated from the porcine spinal cord. Recently, two receptors for NMU have been identified and characterized. A recent study indicated that NMU is an anorectic chemical in the brain. The present study shows that NMU has an action in the brain to inhibit food intake in rats. Intracerebroventricular injection of NMU inhibited dark-phase feeding. Animals injected with NMU showed a strong increase in Fos-immunoreactive nuclei in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus, and in the parabrachial nucleus of the brain stem. Double immunohistochemistry revealed that a high number of oxytocin-immunoreactive neurons in the PVN and SON contained Fos after intracerebroventricular injection of NMU. In addition, a small proportion of vasopressinergic cells within the PVN and SON were found to contain Fos. The effect of NMU on the hypothalamus and brain stem contributes to the inhibitory effects of NMU on feeding behavior.

EFFECT OF CONTINUOUS INTRAVENOUS INJECTION OF INTERLEUKIN-6 AND PRETREATMENT WITH CYCLOOXYGENASE INHIBITOR ON BRAIN C-FOS EXPRESSION IN THE RAT

The aim of the present study was to assess potential brain sites of stimulation by peripheral interleukin (IL)-6 of the hypothalamo-pituitary-adrenal (HPA) axis in the rat, using c-fos protein as a marker of cellular activation. Involvement of prostaglandins in IL-6-induced ACTH secretion and c-fos expression was also investigated. IL-6 was infused continuously (40 ng/min) for 90 min to conscious male rats. Blood samples were taken before the infusion and at 30 and 90 min for measurement of plasma ACTH. Expression of c-fos in the brain was examined by immunohistochemistry. Administration of IL-6 significantly elevated plasma ACTH levels at 30 min (495 +/- 105 vs. 117 +/- 17 pg/ml in controls, p < 0.05). Elevated levels were still present at 90 min (596 +/- 139 vs. 113 +/- 20 pg/ml in controls, p < 0.05). Infusion of IL-6 (3.6 micrograms/rat) markedly triggered c-fos expression in hypothalamic paraventricular (PVN) and supraoptic nuclei (SON), as well as in the central amygdaloid nucleus (CeA), the nucleus tractus solitarius and the locus coeruleus. Pretreatment with the cyclooxygenase inhibitor indomethacin (10 mg/kg, i.v.) suppressed the ACTH response induced by IL-6. The number of IL-6-induced immunoreactive cells in the PVN was significantly reduced by indomethacin pretreatment (p < 0.01), but the number of IL-6-induced c-fos-positive cells in the SON and CeA remained unchanged. These findings suggest that circulating IL-6 may exert central actions by acting directly or indirectly on brain neurons. In addition, the ability of IL-6 to activate the HPA axis may depend upon the release of prostaglandins, probably in the brain.

Effect of Excitatory Amino Acid Receptor Agonists on Secretion of Growth Hormone as Assessed by the Reverse Hemolytic Plaque Assay

Recent findings indicate that excitatory amino acids (EAAs) can modulate growth hormone (GH) secretion in several mammalian species in vivo and in vitro. In this study, we examined the effects of EAA receptor antagonists [N-methyl-D,L-aspartate (NMDA), kainic acid, L-glutamate] on GH secretion by the reverse hemolytic plaque assay (RHPA). Anterior pituitary cells of adult male Sprague-Dawley rats were enzymatically dispersed and subjected to RHPA. EAA receptor agonists increased the mean plaque area in a dose-dependent manner: the maximal increase was observed at 10 microM and increased the fraction of somatotrophs forming large plaques. NMDA (10 microM) did not increase the mean plaque area in the presence of the NMDA receptor antagonists 10 microM AP-7 and 10 microM MK-801. Coincubation of kainic acid with the non-NMDA receptor antagonist CNQX blocked the kainic-acid-stimulated increase in GH secretion. The addition of MK-801, AP-7 or CNQX to glutamate caused a partial reduction of the mean plaque area. Ten micromoles per liter glutamate with 10 nM GH-releasing hormone (GHRH) produced an additive effect on GHRH-induced GH release. Somatostatin suppressed the stimulatory action of glutamate. We speculate that glutamate plays a role in the regulation of GH secretion.

Effect of Central and Continuous Intravenous Injection of lnterleukin-1βon Brain c-fosExpression in the Rat: Involvement of Prostaglandins

Utilizing immunohistochemistry for the c-fos protein to detect neuronal activity, we examined the effects of continuous intravenous and intracerebroventricular infusion of interleukin (IL)-1 beta in the rat brain, and the involvement of prostaglandins (PGs) in IL-1 beta-induced c-fos expression. Continuous intravenous infusion of IL-1 beta (10 ng/min) markedly augmented c-fos expression in the paraventricular (PVN) and the supraoptic (SON) nuclei of the hypothalamus as well as in the central amygdaloid nucleus (CeA). The number of IL-1 beta-induced c-fos-positive cells in the PVN and SON was significantly lower in rats pretreated with indomethacin than in vehicle-treated rats. However, the number of IL-1 beta-induced c-fos-positive cells in the CeA remained unchanged. c-fos protein was induced after intracerebroventricular infusion of IL-1 beta (200 ng) in the PVN, SON, and arcuate nuclei of the hypothalamus, and in the CeA. The induction of c-fos immunoreactivity by central administration of IL-1 beta was blocked by indomethacin (500 micrograms/rat), except in the CeA. These findings suggest that PGs are involved in the complex transmission of signals from circulating or central IL-1 beta to hypothalamic neurons.

Identification of Dopamine and Growth Hormone-Releasing Factor-Containing Neurons Projecting to the Median Eminence of the Rat by Combined Retrograde Tracing and Immunohistochemistry

The topographical location of tyrosine hydroxylase (TH) neurons, a marker for dopamine neurons in the hypothalamus, that project to the median eminence was identified with immunofluorescence staining in combination with retrograde transblue, indicating that they projected to the median eminence. Only a few retrogradely labeled TH cells were observed in the periventricular nucleus and the lateral basal hypothalamus. The elution restaining procedure revealed that an average of 32% of the labeled TH cells in the ventrolateral portion of the ARC contained GRF. These findings support the hypothesis of co-expression of dopamine with GRF from the ARC.