Growth hormone-releasing peptide-6 increases insulin-like growth factor-I mRNA levels and activates Akt in RCA-6 cells as a model of neuropeptide Y neurones.

Chronic systemic administration of growth hormone (GH)-releasing peptide-6 (GHRP-6), an agonist for the ghrelin receptor, to normal adult rats increases insulin-like growth factor (IGF)-I mRNA and phosphorylated Akt (pAkt) levels in various brain regions, including the hypothalamus. Because neuropeptide Y (NPY) neurones of the arcuate nucleus express receptors for ghrelin, we investigated whether these neurones increase their IGF-I and p-Akt levels in response to this agonist. In control rats, immunoreactive pAkt was practically undetectable; however, GHRP-6 increased p-Akt immunoreactivity in the arcuate nucleus, with a subset of neurones also being immunoreactive for NPY. Immunoreactivity for IGF-I was detected in NPY neurones in both experimental groups. To determine if activation of this intracellular pathway is involved in modulation of NPY synthesis RCA-6 cells, an embryonic rat hypothalamic neuronal cell line that expresses NPY was used. We found that GHRP-6 stimulates NPY and IGF-I mRNA synthesis and activates Akt in this cell line. Furthermore, inhibition of Akt activation by LY294002 treatment did not inhibit GHRP-6 induction of NPY or IGF-I synthesis. These results suggest that some of the effects of GHRP-6 may involve stimulation of local IGF-I production and Akt activation in NPY neurones in the arcuate nucleus. However, GHRP-6 stimulation of NPY production does not involve this second messenger pathway.

The corticotropin-releasing factor-hypocretin connection: implications in stress response and addiction.

The hypothalamic neuropeptides hypocretins (orexins) play a crucial role in the stability of arousal and alertness. Recent data have raised the hypothesis that hypocretin neurons are also part of the circuitries that mediate the hypothalamic stress response. In particular, we have recently demonstrated that corticotrophin-releasing factor (CRF)-immunoreactive terminals make direct synaptic contacts with hypocretin-expressing neurons and that numerous hypocretinergic neurons express the CRF-R1/2 receptors. Furthermore, CRF excites hypocretinergic cells ex vivo through CRF-R1 receptors. Activation of hypocretinergic neurons in response to acute stress is severely impaired in CRF-R1 knockout mice. Moreover, the stress response is impaired in hypocretin-deficient mice. We propose that upon stressor stimuli, CRF stimulates the release of hypocretins, and this circuit contributes to activation and maintenance of arousal associated with the stress response and addiction.

The number of lactotrophs is reduced in the anterior pituitary of streptozotocin-induced diabetic rats.

AIMS/HYPOTHESIS: Prolactin secretion is often reduced in insulin dependent diabetes mellitus, but little is known about the mechanism involved. Since changes in the hormonal environment modulate cell proliferation, death and cellular makeup of the anterior pituitary, we have analysed whether the number of lactotrophs is reduced in diabetic rats. METHODS: Streptozotocin induced diabetic rats were maintained hyperglycaemic for 2 months. Pituitary prolactin, growth hormone, Bcl-2, Bax and PCNA concentrations were analysed by western blot analysis. In situ hybridisation was used for quantification of prolactin and growth hormone mRNA containing cells. Cell death was detected by TUNEL labelling, alone and in combination with immunocytochemistry for prolactin or growth hormone. RESULTS: Diabetic rats had fewer lactotrophs ( p<0.01). This was coincident with a decrease in overall protein and prolactin content. An increase in pituitary cell death was found and some of the TUNEL labelling co-localised with prolactin immunostaining. No change in the concentration of Bcl-2 or Bax, proteins implicated in apoptosis, was detected. PCNA content was higher in the pituitaries of diabetic rats, suggesting increased proliferation. CONCLUSION/INTERPRETATION: Anterior pituitary cell turnover is affected in poorly controlled diabetes mellitus. A decrease in the number of lactotrophs, as a result of increased cell death, could underlie, at least in part, the reduction in prolactin secretion observed in diabetic animals.

Purification and characterization of insulin-mimetic inositol phosphoglycan-like molecules from grass pea (Lathyrus sativus) seeds.

BACKGROUND: Signal transduction through the hydrolysis of glycosyl-phosphatidylinositol (GPI) leading to the release of the water-soluble inositol phosphoglycan (IPG) molecules has been demonstrated to be important for mediating some of the actions of insulin and insulin-like growth factor-I (IGF-I). MATERIALS AND METHODS: In the present study, GPI from grass pea (Lathyrus sativus) seeds has been purified and partially characterized on the basis of its chromatographic properties and its compositional analysis. RESULTS: The results indicate that it shows similarities to GPI previously isolated from other sources such as rat liver. IPG was generated from L. sativus seed GPI by hydrolysis with a GPI-specific phospholipase D (GPI-PLD). This IPG inhibited protein kinase A (PKA) in an in vitro assay, caused cell proliferation in explanted cochleovestibular ganglia (CVG), and decreased 8-Br-cAMP-induced phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression in cultured hepatoma cells. CONCLUSIONS: Our data indicate that L. sativus seed IPG possess insulin-mimetic activities. This may explain why L. sativus seeds have been used in some traditional medicines to ameliorate diabetic symptoms.

Short-chain ceramide regulates hepatic methionine adenosyltransferase expression.

BACKGROUND: The metabolism of methionine plays an important role in regulating hepatic cellular function. Methionine adenosyltransferase (MAT) is the enzyme that catalyses the biosynthesis of S-adenosylmethionine (AdoMet) from ATP and methionine. Liver-specific MAT I/III levels are down-regulated in the regenerating rat liver after partial hepatectomy. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) are two cytokines fundamental for liver regeneration. TNF-alpha stimulates sphingomyelin metabolism and ceramide generation in a variety of cell systems. AIMS: The role of exogenous cell-permeable ceramide in modifying MAT I/III mRNA levels and its association with TNF-alpha and IL-6 actions were investigated in rat hepatocytes and H35 hepatoma cells. RESULTS: C2-ceramide (N-acetylsphingosine) at 1-10 microM decreased MAT I/III expression. The effect was maximum after 2 h of treatment and it was maintained up to 24 h. MAT I/III protein levels also decreased. IL-6 (1-10 ng/ml) potentiated C2-ceramide effects in cultured hepatocytes while decreasing by itself MAT I/III levels with a similar time-response curve in both cell types. C2-ceramide actions were not associated with an increase in cell death. TNF-alpha was also a potent antagonist for MAT I/III expression, at 1-20 ng/ml decreased MAT I/III levels and induced endogenous ceramide generation. The decrease of MAT I/III mRNA levels (in all the cases) was not due to a decrease in mRNA half-life which suggests a regulation at the transcriptional level. Finally, the decrease in MAT I/III mRNA levels correlated to a decrease in MAT activity. CONCLUSION: This work demonstrates that short-chain ceramide can be used as a novel exogenous agonist that can modulate hepatic methionine metabolism in association with cytokines.

Role of diffusible and transcription factors in inner ear development: implications in regeneration.

Organogenesis involves a dynamic balance of the mechanisms regulating cell division, differentiation and death. The development of the chicken embryo inner ear offers a well-characterised model at the morphological level to study which signals are implicated in the modulation of cellular activation and commitment. The early developmental decisions that control the origin of the inner ear elements are just beginning to be identified by complementary in vivo and in vitro studies. Insulin-like growth factor-I (IGF-I) and nerve growth factor (NGF) are among the best characterised diffusible factors acting during inner ear development. Although the cellular actions of these factors are beginning to be understood, the signalling pathways triggered by them still remain largely unknown. In this context, viral vehicles can be used to deliver genes and then analyse their functional roles during inner ear development. A model is proposed where the actions of IGF-I and NGF contribute to the combinatorial expression of Jun and Fos family members in particular domains of the otic vesicle. Some of these mechanisms may be also implicated in otic regeneration.

Towards the in vitro reconstitution of caveolae. Asymmetric incorporation of glycosylphosphatidylinositol (GPI) and gangliosides into liposomal membranes.

Large unilamellar vesicles consisting of phospholipids with or without cholesterol have been prepared containing GPI and/or gangliosides asymmetrically located in the outer leaflet of the bilayer. Such asymmetric distribution of GPI and gangliosides is found in 'rafts' and caveolae. Using these vesicles, GPI can be readily hydrolysed by phospholipases. Both cholesterol and ganglioside are seen to inhibit, in an additive way, the hydrolytic activity of GPI-specific phospholipase D.