Effect of a high-fat diet and leptin on STAT3 phosphorylation in hippocampal astrocytes.

OBJECTIVE: The aim of the current study was to evaluate the influence of HFD on the functionality of LepR by quantifying phosphorylated levels of 705Tyr-STAT3 in hippocampus astrocytes from mice that consumed an HFD either during the juvenile or the adult period. METHODS: Five- and eight-week-old male mice, fed during 8 weeks with either control chow or HFD, received a single dose of leptin and their brains were prepared for immunofluorescence to identify double-positive GFAP/p705Tyr-STAT3 cells. RESULTS: HFD intake led to increased pSTAT3 immunoreactivity in GFAP+ cells in the CA1/CA3 hippocampus areas. The effect was observed both in adolescent and adult mice. Leptin increased pSTAT3 immunoreactivity in control animals but was devoid of effect in HFD mice. HFD itself has no effect on the number of GFAP+ cells. CONCLUSIONS: Our data show that regular intake of HFD enhances STAT3 signaling in CA1/CA3 astrocytes, an effect that could be linked to the increase of leptin triggered by HFD. The increase of pSTAT3 might be integral to homeostatic mechanisms aimed at maintaining hippocampus function.

Acute up-regulation of the rat brain somatostatin receptor-effector system by leptin is related to activation of insulin signaling and may counteract central leptin actions.

Leptin and somatostatin (SRIF) have opposite effects on food seeking and ingestive behaviors, functions partially regulated by the frontoparietal cortex and hippocampus. Although it is known that the acute suppression of food intake mediated by leptin decreases with time, the counter-regulatory mechanisms remain unclear. Our aims were to analyze the effect of acute central leptin infusion on the SRIF receptor-effector system in these areas and the implication of related intracellular signaling mechanisms in this response. We studied 20 adult male Wister rats including controls and those treated intracerebroventricularly with a single dose of 5 µg of leptin and sacrificed 1 or 6h later. Density of SRIF receptors was unchanged at 1h, whereas leptin increased the density of SRIF receptors at 6h, which was correlated with an elevated capacity of SRIF to inhibit forskolin-stimulated adenylyl cyclase activity in both areas. The functional capacity of SRIF receptors was unaltered as cell membrane levels of αi1 and αi2 subunits of G inhibitory proteins were unaffected in both brain areas. The increased density of SRIF receptors was due to enhanced SRIF receptor subtype 2 (sst2) protein levels that correlated with higher mRNA levels for this receptor. These changes in sst2 mRNA levels were concomitant with increased activation of the insulin signaling, c-Jun and cyclic AMP response element-binding protein (CREB); however, activation of signal transducer and activator of transcription 3 was reduced in the cortex and unchanged in the hippocampus and suppressor of cytokine signaling 3 remained unchanged in these areas. In addition, the leptin antagonist L39A/D40A/F41A blocked the leptin-induced changes in SRIF receptors, leptin signaling and CREB activation. In conclusion, increased activation of insulin signaling after leptin infusion is related to acute up-regulation of the SRIF receptor-effector system that may antagonize short-term leptin actions in the rat brain.

Activation of microglia in specific hypothalamic nuclei and the cerebellum of adult rats exposed to neonatal overnutrition.

Much attention has been drawn to the possible involvement of hypothalamic inflammation in the pathogenesis of metabolic disorders, especially in response to a high-fat diet. Microglia, the macrophages of the central nervous system, can be activated by proinflammatory signals resulting in the local production of specific interleukins and cytokines, which in turn could exacerbate the pathogenic process. Because obesity itself is considered to be a state of chronic inflammation, we evaluated whether being overweight results in microglial activation in the hypothalamus of rats on a normal diet. Accordingly, we used a model of neonatal overnutrition that entailed adjustment of litter size at birth (small litters: four pups/dam versus normal litters: 12 pups/dam) and resulted in a 15% increase in bodyweight and increased circulating leptin levels at postnatal day 60. Rats that were overnourished during neonatal life had an increased number of activated microglia in specific hypothalamic areas such as the ventromedial hypothalamus, which is an important site for metabolic control. However, this effect was not confined to the hypothalamus because significant microglial activation was also observed in the cerebellar white matter. There was no change in circulating tumour necrosis factor (TNF) α levels or TNFα mRNA levels in either the hypothalamus or cerebellum. Interleukin (IL)6 protein levels were higher in both the hypothalamus and cerebellum, with no change in IL6 mRNA levels. Because circulating IL6 levels were elevated, this rise in central IL6 could be a result of increased uptake. Thus, activation of microglia occurs in adult rats exposed to neonatal overnutrition and a moderate increase in weight gain on a normal diet, possibly representing a secondary response to systemic inflammation. Moreover, this activation could result in local changes in specific hypothalamic nuclei that in turn further deregulate metabolic homeostasis.

Ghrelin treatment protects lactotrophs from apoptosis in the pituitary of diabetic rats.

Poorly controlled diabetes is associated with hormonal imbalances, including decreased prolactin production partially due to increased lactotroph apoptosis. In addition to its metabolic actions, ghrelin inhibits apoptosis in several cell types. Thus, we analyzed ghrelin's effects on diabetes-induced pituitary cell death and hormonal changes. Six weeks after onset of diabetes in male Wistar rats (streptozotocin 70 mg/kg), minipumps infusing saline or 24 nmol ghrelin/day were implanted (jugular). Rats were killed two weeks later. Ghrelin did not modify body weight or serum glucose, leptin or adiponectin, but increased total ghrelin (P<0.05), IGF-I (P<0.01) and prolactin (P<0.01) levels. Ghrelin decreased cell death, iNOS and active caspase-8 (P<0.05) and increased prolactin (P<0.05), Bcl-2 (P<0.01) and Hsp70 (P<0.05) content in the pituitary. In conclusion, ghrelin prevents diabetes-induced death of lactotrophs, decreasing caspase-8 activation and iNOS content and increasing anti-apoptotic pathways such as pituitary Bcl-2 and Hsp70 and serum IGF-I concentrations.

Death of hypothalamic astrocytes in poorly controlled diabetic rats is associated with nuclear translocation of apoptosis inducing factor.

Astrocytes in the hypothalamus of poorly controlled diabetic rats are reduced in number, due to increased apoptosis and decreased proliferation, and undergo morphological changes, including a decrease in projections. These changes are associated with modifications in synaptic proteins and most likely affect neuroendocrine signalling and function. The present study aimed to determine the intracellular mechanisms underlying this increase in hypothalamic cell death. Adult male Wistar rats were injected with streptozotocin (70 mg/kg, i.p) and controls received vehicle. Rats were killed at 1, 4, 6 and 8 weeks after diabetes onset (glycaemia > 300 mg/dl). Cell death, as detected by enzyme-linked immunosorbent assay, increased at 4 weeks of diabetes. Immunohistochemistry and terminal dUTP nick-end labelling (TUNEL) assays indicated that these cells corresponded to glial fibrillary acidic protein (GFAP) positive cells. No significant change in fragmentation of caspases 2, 3, 6, 7, 8, 9, or 12 was observed with western blot analysis. However, enzymatic assays indicated that caspase 3 activity increased significantly after 1 week of diabetes and decreased below control levels thereafter. In the hypothalamus, cell bodies lining the third ventricle, fibres radiating from the third ventricle and GFAP positive cells expressed fragmented caspase 3, with this labelling increasing at 1 week of diabetes. However, because no nuclear labelling was observed and this increase in activity did not correlate temporally with the increased cell death, this caspase may not be involved in astrocyte death. By contrast, nuclear translocation of apoptosis inducing factor (AIF) increased significantly in astrocytes in parallel with the increase in death and AIF was found in TUNEL positive cells. Thus, nuclear translocation of AIF could underlie the increased death, whereas fragmentation of caspase 3 could be associated with the morphological changes found in hypothalamic astrocytes of diabetic rats.

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.

Oestrogen requires the insulin-like growth factor-I receptor for stimulation of prolactin synthesis via mitogen-activated protein kinase.

Sex steroids and growth factors interact at the intracellular level in a variety of tissues to control numerous physiological functions. Oestrogen is known to stimulate prolactin synthesis and secretion, but the effect of insulin-like growth factor (IGF)-I is less clear. We used GH3 cells, a somatolactotroph cell line, to study the interaction of 17beta-oestradiol (E(2)) and IGF-I on prolactin protein levels and the intracellular mechanisms involved. Cell cultures were treated with E(2) (10 nM) and/or IGF-I (10 ng/ml) for 8 h. The real-time reverse transcriptase-polymerase chain reaction, Western blot and enzyme-immunoassay were used to determine changes in prolactin mRNA and protein levels. At this time-point, there were no significant changes in cell number, prolactin mRNA expression, or the amount of secreted prolactin. However, E(2) increased intracellular prolactin concentrations. IGF-I alone had no effect, but blocked the stimulatory effect of E(2). MAPK (ERK1/2) activation, as determined by Western blot analysis, increased with both E(2) and IGF-I, but not with the combination of these factors. The MAPK inhibitor PD98059 blocked the ability of E(2) to increase intracellular prolactin concentrations. Similarly, the IGF-I receptor antagonist, JB1, blocked the effect of E(2) on prolactin synthesis and MAPK activation, as did the oestrogen receptor antagonist ICI182 780. These results suggest that, to stimulate prolactin synthesis, E(2) activates the MAPK cascade and that this requires the presence of both oestrogen and IGF-I receptors.

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.

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.

Involvement of insulin-like growth factor-I in inner ear organogenesis and regeneration.

The verterbrate inner ear is an excellent model system to study signalling mechanisms in embryonic development. During the last years, insulin-like growth factor-I (IGF-I) has attracted attention in relation to the regulation of inner ear ontogenesis. IGF-I and its high-affinity tyrosine-kinase receptor are expressed during early stages of inner ear development. IGF-I is a powerful mitogen for the otic vesicle, where it stimulates cell-division and mitogenic signalling cascades. Later in development, IGF-I also promotes survival and neurogenesis of the otic neurones in the cochleovestibular ganglion (CVG). The actions of IGF-I are associated with the generation of lipidic messengers and the activation of Raf kinase, which results in the rapid induction of the expression of the proliferative cell nuclear antigen (PCNA) and the nuclear proto-oncogenes c-fos and c-jun. Regulation of organogenesis involves a dynamic balance of the mechanisms regulating cell division, differentiation and death. A model is proposed where this balance is the consequence of the action of IGF-I and NGF, which converge in Raf activation or suppression. The combinatorial expression of jun and Fos family members in particular domains of the otic vesicle would be the final result of such cascade. Some of these mechanisms may be also implicated in otic regeneration.

Nerve growth factor and ceramides modulate cell death in the early developing inner ear.

Regulation of normal development involves a dynamic balance of the mechanisms regulating cell division, differentiation and death. We have investigated the signalling mechanisms involved in regulation of the balance between cell proliferation and apoptotic cell death in the otic vesicle. The sphingomyelin pathway signals apoptosis for nerve growth factor upon binding to p75 receptors. It is initiated by sphingomyelin hydrolysis to generate the second messenger ceramide. In the present study, we show that nerve growth factor stimulates sphingomyelin hydrolysis and the concomitant ceramide release in organotypic cultures of otic vesicles. Both nerve growth factor and ceramide induce apoptotic responses to a different extent. Ceramide-induced apoptosis was suppressed by insulin-like growth factor-I which is a strong promoter of cell growth and morphogenesis for the developing inner ear. In contrast, ceramide-1-phosphate protected the explants from apoptosis induced by serum withdrawal but did not antagonise ceramide-induced cell death. This study suggests that sphingomyelin-derived second messengers might be key modulators of programmed cell death during development.

Pattern of methionine adenosyltransferase isoenzyme expression during rat liver regeneration after partial hepatectomy.

The present report investigates the pattern of expression of liver-specific and extrahepatic methionine adenosyltransferase (MAT) isoenzymes in regenerating rat liver after partial hepatectomy. The results show that there is a switch in the expression of these isoenzymes that is coincident with maximal cell proliferation in the remaining liver lobes. Extrahepatic MAT levels increase about three times 4 h after hepatectomy, reaching a maximum 36 h later. This is accompanied by a rapid and transient increase in total MAT activity and levels of related metabolites S-adenosyl-L-methionine and S-adenosyl-L-homocysteine. Liver-specific MAT levels are reversibly down-regulated within 24-48 h post surgery. Our results indicate that MAT isoenzyme expression is tightly regulated during liver regeneration after two-third hepatectomy. The implications of these observations for evaluation of the degree of liver regeneration are briefly discussed.

Stimulation of DNA synthesis by natural ceramide 1-phosphate.

We found that natural (long-chain) ceramide 1-phosphate can be dispersed into aqueous solution when dissolved in an appropriate mixture of methanol/dodecane (49:1, v/v). This solvent mixture facilitates the interaction of this phosphosphingolipid with cells. Under these conditions, incubation of EGFR T17 fibroblasts with natural ceramide 1-phosphate caused a potent stimulation of DNA synthesis. This effect was accompanied by an increase in the levels of proliferating-cell nuclear antigen. Concentrations of natural ceramide 1-phosphate that stimulated the synthesis of DNA did not inhibit adenylate cyclase activity, nor did they stimulate phospholipase D. Natural ceramide 1-phosphate did not alter the cellular phosphorylation state of tyrosine residues or of mitogen-activated protein kinase. Furthermore, natural ceramide 1-phosphate failed to induce the expression of the proto-oncogenes c-myc and c-fos. Both the stimulation of DNA synthesis and the induction of proliferating-cell nuclear antigen by natural ceramide 1-phosphate were inhibited by natural ceramides. This work suggests that the use of methanol and dodecane to deliver natural ceramide 1-phosphate to cells may be useful for elucidation of the biological function(s) and mechanism(s) of action of ceramide 1-phosphate.