Minireview: The Neuroendocrine Regulation of Puberty: Is the Time Ripe for a Systems Biology Approac

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第一篇 Minireview: The Neuroendocrine Regulation of Puberty: Is the Time Ripe for a Systems Biology Approac

【关键词】  puberty

division of neuroscience (s.r.o., a.l., c.m., c.r., a.-s.p., v.m., a.e.m.), oregon national primate research center/oregon health and science university, beaverton, oregon 97006

hospital for children and adolescents (s.h.), university of leipzig, 04317 leipzig, germany

abstract

the initiation of mammalian puberty requires an increase in pulsatile release of gnrh from the hypothalamus. this increase is brought about by coordinated changes in transsynaptic and glial-neuronal communication. as the neuronal and glial excitatory inputs to the gnrh neuronal network increase, the transsynaptic inhibitory tone decreases, leading to the pubertal activation of gnrh secretion. the excitatory neuronal systems most prevalently involved in this process use glutamate and the peptide kisspeptin for neurotranission/neuromodulation, whereas the most important inhibitory inputs are provided by -aminobutyric acid (gaba)ergic and opiatergic neurons. glial cells, on the other hand, facilitate gnrh secretion via growth factor-dependent cell-cell signaling. coordination of this regulatory neuronal-glial network may require a hierarchical arrangement. one level of coordination appears to be provided by a host of unrelated genes encoding proteins required for cell-cell communication. a second, but overlapping, level might be provided by a second tier of genes engaged in specific cell functions required for productive cell-cell interaction. a third and higher level of control involves the transcriptional regulation of these subordinate genes by a handful of upper echelon genes that, operating within the different neuronal and glial subsets required for the initiation of the pubertal process, sustain the functional integration of the network. the existence of functionally connected genes controlling the pubertal process is consistent with the concept that puberty is under genetic control and that the genetic underpinnings of both normal and deranged puberty are polygenic rather than specified by a single gene. the availability of improved high-throughput techniques and computational methods for global ysis of mrnas and proteins will allow us to not only initiate the systematic identification of the different components of this neuroendocrine network but also to define their functional interactions.

introduction

in mammals, including humans, developmental changes in gonadotropin secretion are controlled by changes in pulsatile release of gnrh. at puberty, pulsatile gonadotropin secretion increases in a diurnal fashion, initially characterized in humans (1), but that also occurs in other species, including the rat (2). this change, necessary for normal gonadal development and function, is determined by activation of a hypothalamic gnrh pulse generator (for review, see refs.2 and 3). the pubertal increase in gnrh secretion is, in turn, prompted by changes in transsynaptic and glial inputs to the gnrh neuronal network. studies conducted by different groups have identified these inputs as being both facilitatory and inhibitory. the former use excitatory amino acids (for review, see refs.2 and 4) and the recently identified neuropeptide metastin/kisspeptin (5, 6) for neurotranission/neuromodulation. -aminobutyric acid (gaba) and opioid peptides provide the inhibitory inputs (7, 8). it is also clear that the pubertal activation of gnrh secretion can no longer be considered as an event driven solely by transsynaptic inputs (2, 9). glial cells produce cell-cell signaling molecules that stimulate gnrh release and that have been shown to be critical for the correct timing of the pubertal process (for review, see ref.10).

this article will briefly review our current understanding of the cell-cell mechanis underlying the neuroendocrine control of puberty. we will also begin to develop the broad (but obviously imperfect) concept that the pubertal activation of gnrh secretion is controlled by a network of genes that, having diverse functions, operate within different cell contexts to coordinate the secretory activity of the gnrh neuronal network at puberty. we will propose the concept that coordination of gnrh release requires the participation of two sets of genes, those that are subordinate and those that govern the pubertal process at a higher hierarchical, transcriptional level of control.

using a systems biology approach, i.e. the coordinated study of a biological system (11), for the understanding of these neuroendocrine regulatory networks, requires by definition (12): 1) identifying the genes, proteins, and other all molecules constituting the pathway of interest; 2) perturbing each pathway component through genetic manipulations and detecting the global cellular response to each perturbation with the help of high-throughput and whole-genomics techniques; 3) integrating the observed mrna and protein responses with existing models of protein-protein, protein-dna, and other interactions, using appropriate computational methods; and 4) formulating new hypotheses to explain observations not predicted by the model. as the reader will readily appreciate from this article, the application of these principles to the understanding of the neuroendocrine control of puberty is, at best, in an early embryonic stage.

subordinate genes required for cell-cell communication

this category include all downstream genes that participate in the excitatory and inhibitory control of gnrh neurons, whether this control is exerted transsynaptically or via glia-to-neuron communication (fig. 1). subordinate genes execute specific cellular functions required for cell-cell signaling, but their expression is hypothetically regulated by a higher order of system network control. for operational purposes they can be considered as the last to be activated.

it was earlier established that the major excitatory transsynaptic event prompting the initiation of puberty is an increase in glutamatergic neurotranission (2, 13), the primary mode of excitatory transsynaptic communication in the hypothalamus (14). activation of glutamatergic inputs increases gnrh secretion (15, 16) and accelerates sexual maturation in both rats and monkeys (17, 18). glutamate acts both directly (19, 20, 21, 22) and via regulatory neuronal subsets (23) to stimulate gnrh secretion.

glutamatergic neurotranission is a complex process controlled by a plethora of genes required for synthesis, transport, and release of the amino acid, as well as for the expression of the various receptors that mediate glutamate actions. puberty-related changes in glutamate receptor expression might be restricted to specific hypothalamic cellular subsets. for instance, the binding capacity of nmda and kainate receptors (which presumably reflect changes in gene expression) does not change in cell membranes derived from whole hypothalami (24). on the other hand, kainate receptor expression measured by in situ hybridization increases in gnrh neurons during sexual development (21). the upstream genes controlling this change are not known. even less is known about the transcriptional control of genes encoding enzymes involved in the synthesis, metaboli, and transport of glutamate. the importance of these homeostatic systems has been made evident by recent studies in which we used a quantitative proteomics approach (25) to identify proteins whose expression is increased in the hypothalamus at the time of rat puberty (26). we observed that the abundance of glutamate dehydrogenase, one of the enzymes that catalyzes the synthesis of glutamate (27), increases in the hypothalamus of female rats undergoing puberty. in contrast, the abundance of glutamine synthase, which catalyzes the metaboli of glutamate into glutamine (27), decreases at this time (fig. 1). these changes were accompanied by an increased capacity of the hypothalamus to release glutamate after blockade of glutamate transport, suggesting that more glutamate is available for both synaptic tranission and glia-to neuron signaling at the time of puberty. because both enzymes are predominantly expressed in glial cells, the results also indicate that an increased glutamate output of glial origin plays a major role in the control of gnrh release at puberty. the upstream genes controlling the transcriptional activity of the glutamate dehydrogenase and glutamine synthase genes remain to be identified. transcriptional regulation of vesicular glutamate transporter expression might represent an even more important control point because vesicular glutamate transporters (28, 29) are critical for the homeostatic minute-to-minute control of glutamate release (30).

much discussion has been centered on the question of the primus movens of puberty: is it the loss of a central restraint (7) or the activation of stimulatory inputs to gnrh neurons (2) the recent finding that mutations of gpr54, the receptor for the kiss1-derived peptide metastin (31, 32, 33), causes hypothalamic hypogonadi (5, 6) suggests that the latter view is correct because gpr54 signaling is coupled to stimulation of gnrh release, instead of inhibition (34, 35). the kiss1-gpr54 signaling complex is a novel, and unsuspected, system involved in the control of gnrh secretion. metastin/kisspeptin is a 53-amino acid peptide encoded by the kiss1 gene (5, 6); proteolytic cleavage of the primary kiss1 protein product originates the decapeptide kisspeptin-10, which is extraordinarily potent in eliciting lh release (36, 37, 38). the comparable effectiveness of intracerebral and systemic administration (36, 39) suggests either a dual hypothalamic-pituitary site of action or a main effect at the median eminence (me) of the hypothalamus, a region of the brain located outside the blood-brain barrier. although kiss1-containing neurons are located in discrete neuronal subsets of the preoptic area (37) and the arcuate nucleus (36, 37), gpr54-containing cells are more diffusely distributed (36, 40), including gnrh neurons (40, 41) and the adenohypophysis (31, 32). this distribution suggests that kiss1 neurons may not only facilitate gnrh secretion by acting on gnrh neuronal perikarya and gnrh nerve terminals at the me but also stimulate gonadotropin secretion directly (39) by releasing metastin into the portal system. kiss1 and gpr54 mrna abundance increases in the nonhuman primate hypothalamus at the time of puberty, indicating that increased gpr54-mediated signaling contributes to the pubertal activation of gnrh secretion (36) (fig. 1). the ability of centrally administered kisspeptin to advance puberty in juvenile female rats (38, 39) supports this concept.

gabaergic neurons acting via gabaa receptors provide a major inhibitory transsynaptic influence controlling gnrh secretion during prepubertal development (42, 43). although this restraining influence has been unambiguously demonstrated in primates (42, 44, 45), an inhibitory role of gabaergic neurotranission in rodent puberty is much less clear because both inhibitory and stimulatory effects have been reported (for review, see ref.2). like glutamate, gaba regulates gnrh secretion by binding to receptors located both on gnrh neurons (46, 47) and on their synaptically connected neuronal partners (for review, see refs.2 and 4). it appears that the main effect of gaba acting via gabaa reception on gnrh neurons is excitation (47), but inhibitory effects have also been reported (46). like glutamate, gaba production requires the participation of different proteins involved in the synthesis, metaboli, transport, and release of the amino acid. because no changes in hypothalamic expression of the mrnas encoding gad-65 and gad-67 (the enzymes responsible for gaba synthesis) have been detected during primate sexual development (48), it does not appear that regulation of their gene expression is an event related to the onset of puberty. by ogy to the glutamatergic system, however, it might be inferred that important control points reside at the level of gaba vesicular transport (30, 49) and/or gaba receptors (fig. 1). again, the upstream genes involved in the transcriptional control of these components at the time of puberty remain to be identified.

the other major inhibitory transsynaptic input to the gnrh neuronal network is provided by opiatergic neuronal systems (such as preproenkephalin-containing neurons). a reduction in opioid input to the gnrh neuronal network at the time of puberty may not be as critical as the loss of gabaergic inhibitory control. however, opioid peptides may provide additional homeostatic counter balance to the cascade of excitatory events leading to the pubertal increase in gnrh output. in rodents, the strength of the prepubertal opioid peptide inhibitory tone (50) diminishes at the time of puberty (51). opioid peptides as a group do not appear to restrain the initiation of puberty (for review, see refs.2 and 52), but it is possible that this inhibitory tone is exerted by a neuronal subset selectively using -endorphin, dynorphin, or met/leu-enkephalin for neurotranission (fig. 1).

gnrh neurons and glial cells share an intimate morphological association (53). in the me, both astroglia (53, 54, 55) and modified ependymoglial cells known as tanycytes (55, 56) appose gnrh terminals. tanycytic end-feet contacting the portal vessels intervene between gnrh nerve endings and endothelial cells of the portal vessels (55, 56) but retract at the time of the preovulatory surge of gonadotropins allowing the terminals to directly contact the endothelial cells (57). it is now clear that glial cells and gnrh neurons also share a functional relationship. this relationship depends upon growth factors acting via serine threonine kinase receptors, such as tgf1, and growth factors signaling through receptors with tyrosine kinase activity, like igf-i, basic fibroblast growth factor, and the members of the epidermal growth factor family, tgf, and neuregulins (nrgs) (fig. 1). we will discuss here only the latter because their role in the control of puberty has been more extensively characterized than the others. a more comprehensive discussion of the roles of igf-1 and basic fibroblast growth factor in the control of gnrh neurons can be found in (2, 58, 59).

tgf binds to erbb1 receptors located on astrocytes and tanycytes, whereas nrgs are recognized by erbb4 receptors expressed only in astrocytes. both receptors recruit the coreceptor erbb2 for signaling, and in both cases, a major outcome is the release of chemical messengers, such as prostaglandin e2 (pge2), that act directly on gnrh neurons to stimulate gnrh secretion (60, 61, 62) (fig. 1). pharmacological and genetic approaches have been used to define the involvement of glial erbb1 receptors in the control of female sexual development. although blockade of erbb1 receptors in the me (63) or a point mutation of the erbb1 gene (64) result in delayed puberty, sexual development is advanced in transgenic mice conditionally overexpressing the tgf gene (65) and rats carrying intrahypothalamic grafts of cells genetically engineered to secrete tgf (66). ligand-dependent activation of erbb1 receptors in tanycytes results in plastic changes that, involving pge2 and tgf1 as downstream effectors, mimic the morphological plasticity displayed by tanycytes during the hours encompassing the preovulatory surge of gnrh (62). erbb1 signaling also has been implicated in the etiology of precocious puberty induced by hypothalamic hamartomas (hhs) in humans (67).

in vivo disruption of hypothalamic erbb2 receptor synthesis using antisense oligodeoxynucleotides resulted in delayed puberty (61). such a delay was also observed in transgenic mice overexpressing, in an astrocyte-specific fashion, a truncated erbb4 protein (dnerbb4) that, lacking the intracellular domain, acts as a dominant negative receptor to block the signaling capability of the intact receptor (68). a combined deficiency achieved by generating dnerbb4 mice carrying a point mutation of the erbb1 receptors accentuated the effect of the single deficiencies (69), indicating that both systems work in a coordinated fashion to facilitate the onset of female puberty.

are there mechanis in place able to coordinate the transsynaptic and glial influences on gnrh neurons one of these mechanis, initiated by excitatory amino acids, has been shown to target the astrocytic erbb signaling system for regulation (70). hypothalamic astrocytes express metabotropic and ionotropic -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. upon concomitant stimulation of both receptor subtypes, astrocytes respond with mobilization of erbb receptors to the cell surface and tgf/nrg-dependent phosphorylation of these receptors, indicating that glutamate stimulation of astrocytes facilitates the interaction of tgf/nrg ligands with their receptors (70). studies in other cell systems have shown that a surface protein with adhesion and protease activity termed tumor necrosis factor--converting enzyme or a disintegrin and metalloproteinase-17 cleaves tgf and nrgs from their tranembrane precursors, allowing the growth factors to bind their erbb receptors (71, 72). this is precisely what activation of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and metabotropic glutamate receptors does in astrocytes, i.e. it enhances tumor necrosis factor--converting enzyme-like activity, which in turn elicits tgf release (73) (fig. 1).

second tier genes controlling cell-cell interactions

the late, but still present, initiation of puberty displayed by animals in which expression of certain candidate genes has been reduced (e.g. tgf, glutamate receptors, etc.) and the very low incidence in the human population of hypothalamic hypogonadi attributed to a single gene defect (for instance, gpr54) suggest that no isolated pathway or cellular subset is solely responsible for the neuroendocrine control of puberty. instead, this control is more likely exerted by functionally interconnected regulatory networks. as indicated in the introductory section, a first step in the implementation of a system biology approach is to identify all the genes, proteins, and other molecules constituting the pathway of interest (12). the conventional single gene/single protein approaches referred to in the previous section do obviously contribute to accomplish this first step. however, a thorough investigation of the diverse constituents of the various cellular networks implicated in the process requires the use of global, high-throughput approaches. using a combination of dna microarrays, proteomics, guilt by association, and retrospective approaches, we have singled out a group of genes that may represent a novel genetic network involved in the neuroendocrine control of female puberty (74). these genes have diverse cellular functions but share the common feature of having been earlier identified as involved in tumor suppression.

with the help of dna microarrays, we queried the hypothalamus of female rhesus monkey at different phases of pubertal development and hhs from human subjects in search of candidate gene transcripts. we investigated hhs because these rare, nonneoplastic congenital malformations of the basal hypothalamus are usually associated with sexual precocity (75). to complement this genomic approach, we used quantitative proteomics (25) to identify hypothalamic proteins that might be down- or up-regulated in dnerbb4 mice, which as indicated above have delayed puberty (68). ysis of the monkey arrays results showed that expression of four tumor-related genes that otherwise participate in normal cell differentiation processes increases selectively in the hypothalamus at the time of monkey puberty (74). the hamartoma arrays identified four additional candidates also implicated in tumor pathology (76). in keeping with these observations, quantitative proteomics using isotope-coded affinity tag labeling revealed that the content of syncam, an immunoglobulin-like adhesion molecule required for synapse formation (77), was decreased in the dnerbb4 mice (78). before its synaptic function was discovered, syncam was known as tumor-suppressor in lung cancer-1 (79). quantitative pcr studies verified the array results (74, 76) and suggested that kiss1 and gpr54 are also part of this neuroendocrine gene network because their mrna expression increases in the hypothalamus at the time of primate puberty (36, 74). before the role of this complex in the control of puberty was discovered, the kiss1 gene was known as a suppressor of tumor metastases (33, 80). it is thus possible that syncam and kiss1 are members of a network of genes that, instead of functioning in the neuroendocrine brain to suppress tumor formation, serves to integrate neuron-to-neuron and glia-to-neuron communication into a functional unit capable of initiating the pubertal process (fig. 2). verification of this broad hypothesis will require implementation of the second step proposed for the application of a system biology strategy (12) to the understanding of the neuroendocrine control of puberty, that is, the controlled perturbation of specific genes followed by the global detection of the attendant cellular responses and the formulation, via repeated iterations, of specific models predicting the functional organization of the network. formidable challenges to be met are the elucidation of the developmental and spatial characteristics of the system.

the upper echelon genes

the concept that gnrh neuronal function and, hence, sexual development, is ultimately controlled by a hierarchy of upstream transcriptional regulators acting within functionally linked neuronal and glial subsets (81) is also in its infancy. an underlying premise is that, regardless of the transcriptional process they might control, the net outcome is to establish the conditions required for the productive interaction of neurons and glial cells (fig. 3). importantly, such genes not only reside at the core of any complex regulatory network (for instance, see refs.82 and 83), but they are also required to maintain the hierarchical structure of the network and to ensure that the system contains both redundancy and combinatorial diversity (83). thus far, we have identified three potential candidates for such a role. one of them is oct-2, a transcriptional regulator of the pou-domain family of homeobox-containing genes (84). although the oct-2 gene is expressed throughout the embryonic ventral forebrain, after birth its expression is restricted to a few hypothalamic neuronal subsets (85). oct-2 proteins are more abundant in cultured astrocytes than in neurons (86), suggesting that the oct-2 gene may be important for the trans-regulation of astroglial gene transcription. tgf has been identified as one of such targets (87), but the syncam promoter also contains oct-2 binding sites. the transcriptional activity of the tgf promoter is increased by oct-2. this regulatory mechani is important for the onset of female puberty because: 1) hypothalamic oct-2 mrna levels increase during juvenile development in a gonad-independent manner, 2) blockade of oct-2 synthesis via antisense oligodeoxynucleotides reduced astrocytic tgf synthesis and delayed the age at first ovulation, and 3) lesions of the hypothalamus that induce sexual precocity activate both oct-2 and tgf expression in astrocytes near the lesion site (87).

the second candidate is ttf-1 (thyroid transcription factor-1), another homeobox gene. ttf-1 is required for diencephalic morphogenesis (88); after birth it remains expressed in selected neuronal and glial populations of the hypothalamus, such as gnrh and preproenkephalinergic neurons and tanycytes of the me (89). ttf-1 acts on each of these cell types to promote cell-specific functions. for instance, it enhances gnrh and erbb2 gene transcription but inhibits preproenkephalin promoter activity (89). dna arrays and quantitative pcr ysis of the female rhesus monkey hypothalamus revealed a pubertal increase in ttf-1 expression. employing the cre-loxp system to conditionally delete the ttf-1 gene from those neuronal subsets of the hypothalamus where it is normally expressed, we found that ttf-1 null mutants have delayed puberty, a disruption of initial estrous cyclicity, and decreased reproductive capacity (90). these deficiencies were accompanied by increased preproenkephalin gene expression (90) and by suppressed hypothalamic gnrh and kiss1 mrna levels (mastronardi, c. a., g. iley, t. kusakabe, a. kawagushi, s. heger, r. cabrera, a. e. mungenast, s. kimura, and s. r. ojeda, unpublished data). thus, ttf1 enhances the transcriptional activity of genes required for the facilitatory control of puberty (gnrh, erbb2, kiss1) while repressing the transcription of a gene involved in the inhibition of gnrh secretion.

the third candidate was also discovered using cdna arrays to interrogate the primate hypothalamus at the time of puberty (91). it is a gene earlier known as c14orf4 (92), but that we have now termed eap-1 (91). like ttf-1, eap-1 maps to human chromosome 14. hypothalamic eap-1 mrna levels increase in both monkeys and rats during female puberty (93), suggesting an involvement in the control of the pubertal process. eap-1 encodes a nuclear protein, which is expressed in neuronal subsets involved in the stimulatory and inhibitory control of gnrh secretion, such as glutamatergic, gabaergic, proenkephalinergic, and kiss1 neurons, in addition to gnrh neurons themselves (93). like ttf-1, eap-1 transactivates the promoter of genes involved in facilitating the advent of puberty (e.g. gnrh) while suppressing the expression of genes inhibitory to the pubertal process (such as the preproenkephalin gene). knocking down hypothalamic eap-1 expression via sirna technology delayed puberty and disrupted estrous cyclicity, confirming the importance of eap-1 as an upper echelon gene necessary for the neuron-to neuron regulation of gnrh secretion at puberty (93) (fig. 3).

conclusion

these observations suggest that the neuroendocrine control of puberty is provided by a gene network of hierarchical nature similar in principle to those postulated to exist in less complex cellular systems (see refs.12 , 82 , and 83 and references therein). essential features of such networks are the dominance of a few highly connected upper echelon gene hubs, the partial overlap of second tier gene subnetworks, the large number of less-connected subordinate gene nodes, and the remarkable redundancy of the system (83). although the overall validity of this concept (summarized in figs. 2 and 3) remains to be experimentally tested, the existence of a hypothalamic gene network composed of genes situated at different, but interactive, hierarchical levels is consistent with the idea that the onset of puberty is genetically determined and depends on the contribution of more than one gene (94, 95, 96). further supporting this idea is the recent identification of key quantitative trait loci regulating the abundance of thousands of transcripts in the nervous system in a region-specific manner (97). recent reports have also made clear that, contradicting the current dogma that human central precocious puberty is sporadic in nature, a significant number of cases of this disorder is caused by genetic factors (98). future studies should make clear whether those genes implicated in the control of puberty in animal models are, in fact, required for the normalcy of human puberty.

footnotes

this work was supported in part by national institutes of health grants hd25123, mh65438, hd050798, and rr00163 and through cooperative agreement u-54 hd18185 as part of the specialized cooperative center’s program in reproduction research.

present address for c.r.: department of pediatrics, university of bonn, adenauerallee 119, 53113 bonn, germany.

present address for a.e.m.: department of neuroscience, school of medicine, university of pennsylvania, philadelphia, pennsylvania 19104.

present address for a.-s.p.: departement des sciences cliniques, universite de liege, liege 4000, belgium.

a.l., c.m., s.h., c.r., a.s.p., v.m., and a.e.m. have nothing to declare. s.r.o. received a lecture fee from ferring co. (berkshire, uk).

first published online december 22, 20xx

abbreviations: gaba, -aminobutyric acid; hh, hypothalamic hamartoma; me, median eminence; nrg, neuregulin; pg, prostaglandin; tsg, tumor suppressor gene; ttf-1, thyroid transcription factor-1.

accepted for publication november 21, 20xx.

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第二篇 Both Early and Delayed Treatment with Melanocortin 4 Receptor-Stimulating Melanocortins Produces Neu

【关键词】  melanocortin

department of biomedical sciences (d.g., c.m., s.l., c.b., s.g.), section of pharmacology, department of diagnostic services (m.-m.c., a. bertolini), section of clinical pharmacology

department of anatomy and histology (d.z.), university of modena and reggio emilia, 41100 modena, italy

department of clinical and experimental medicine and pharmacology (d.a., l.m., a. bitto, h.m., f.s.), section of pharmacology, university of messina, 98100 messina, italy

departments of human pathology (a.r.b.) and experimental medicine (r.p., m.s., d.n.), university of pavia, 27100 pavia, italy

department of neuroscience (h.b.s.), university of uppsala, 75124 uppsala, sweden

abstract

ischemic stroke is one of the main causes of death and disability. we investigated whether melanocortin peptides, which have protective effects in severe hypoxic conditions, also produce neuroprotection in a gerbil model of ischemic stroke. a 10-min period of global cerebral ischemia, induced by occluding both common carotid arteries, caused impairment in spatial learning and memory that was associated with activation of inflammatory and apoptotic pathways, including severe dna damage and delayed neuronal death, in the hippocampus. treatment with nanomolar doses of the melanocortin og [nle4, d-phe7] -msh [which activates the melanocortin receptor subtypes (mc) mainly expressed in central nervous system, namely mc3 and mc4] modulated the inflammatory and apoptotic cascades and reduced hippocampus injuries even when delayed up to 9 h after ischemia, with consequent dose-dependent improvement in subsequent functional recovery. the selective mc3 receptor agonist 2-msh had no protective effects. pharmacological blockade of mc4 receptors prevented the neuroprotective effects of [nle4, d-phe7] -msh and worsened some ischemia outcomes. together, our findings suggest that mc4 receptor-stimulating melanocortins might provide potential to develop a class of drugs with a broad treatment window for a novel approach to neuroprotection in ischemic stroke.

introduction

transient or prolonged severe decrease in cerebral blood flow may eventually lead to delayed neuronal death (1), and ischemic stroke is one of the main causes of death and the leading cause of disability for impairment in several functions, including learning and memory (2). within minutes to days after a cerebrovascular accident, several pathological pathways are triggered, which may potentially damage brain cells. this is caused by several mechanis, including excitotoxicity (due to marked release of glutamate and aspartate), inflammatory response, and apoptosis (1, 3).

several melanocortins, which are endogenous peptides of the acth/msh group and include acth-(124), -msh, shorter fragments, and synthetic ogs such as [nle4, d-phe7] -msh (ndp-msh), have a life-saving effect in animal and human conditions of circulatory shock (4, 5, 6), as well as in other severe hypoxic conditions, including prolonged respiratory arrest (7) and myocardial ischemia (8). this life-saving effect is associated with blunted nuclear factor b (nf-b) activation (9) and a marked reduction in the levels of tnf- and free radicals and intercellular adhesion molecule expression by vascular endothelium (5, 9, 10, 11). this is in agreement with the notion that melanocortins have a peculiar antiinflammatory activity (12, 13, 14). moreover, it has been shown that preventive or early-initiated treatment with -msh increases brain stem auditory-evoked potentials in dogs subjected to transient basilar artery occlusion (15), decreases cerebrocortical tnf- and il-1 expression in mice subjected to transient middle cerebral artery/carotid artery occlusion (16), and decreases local and circulating tnf- in lipopolysaccharide-induced brain inflammation in mice (17).

here we provide the first clear evidence that melanocortins cause a strong protection, with a broad therapeutic treatment window, against inflammatory, apoptotic (including dna damage), and histopathological and behavioral consequences of brain ischemia by activating central nervous system (cns) melanocortin 4 (mc4) receptors.

materials and methods

transient global brain ischemia in gerbils

for these studies, we used male mongolian gerbils (charles river laboratories, calco, lecco, italy) weighing 7080 g. housing conditions and experimental procedures were in strict accordance with the european community regulations of the use and care of animals for scientific purposes (european economic community council 89/609; italian d.l. 22-1-92 no. 116) and were approved by the committee on animal health and care of modena and reggio emilia university. transient global brain ischemia was induced, under general anesthesia with chloral hydrate (400 mg/kg ip; sigma, st. louis, mo), by occluding with atraumatic clips both common carotid arteries for 10 min (18). this experimental model represents human stroke conditions due, for example, to atherosclerotic involvement of the common carotid arteries, respiratory arrest, and cardiac arrest (19, 20). rectal and cranial (left temporalis muscle) temperatures were monitored from the induction of anesthesia until death with temperature probes and were maintained close to 37 c by means of heating lamps. animals used in behavioral studies were allowed to recover for 4 d before starting testing. sham ischemic gerbils received the same surgical procedure except that the carotid arteries were not occluded.

drugs and treatment schedules

ndp-msh (sigma), 2-msh (kindly provided by prof. paolo grieco, department of pharmaceutical and toxicological chemistry, university of naples federico ii, naples, italy), and hs024 (neosystem, straourg, france) were dissolved in saline (1 ml/kg) and administered ip. control animals (ischemic or sham ischemic) received equal volume of saline by the same route. for behavioral studies, ndp-msh, 2-msh, or saline was administered every 12 h (for 11 d) starting 2 min before carotid occlusion; in two other groups of animals, ndp-msh treatment started 3 or 9 h after the ischemic episode. for the assay of mapk activity and cytokine expression, gerbils were treated with a single dose of ndp-msh, 2-msh, or saline 2 min before carotid occlusion (for the assay of mapk activity: in some animals, ndp-msh was given 3 or 9 h after the ischemic episode). for the assay of caspase-3 activity, gerbils were treated with two doses of ndp-msh, 2-msh, or saline: the first dose was given 2 min before carotid occlusion (in some animals, the first dose of ndp-msh was given 3 or 9 h after the ischemic episode) and the second, 12 h after the first treatment. for the assay of dna damage, treatment schedule (first dose 2 min before carotid occlusion, then treatment every 12 h) lasted 3 d. pretreatment with hs024 (cyclic msh og, potent and highly selective mc4 receptor antagonist) (21) or saline, when done, was performed ip 20 min before each administration of ndp-msh or saline (that is, 22 min before carotid occlusion). drug doses were chosen on the basis of previous studies (8, 9, 12, 14, 21). the different treatment protocols were chosen to perform the assays of cytokines, mapk and caspase-3 activities, and dna damage at times (see below) considered of prominent expression (1, 22, 23, 24).

assesent of spatial learning and memory

we used the morris water-maze test (25, 26) in a double-blinded manner. this test measures the ability of the gerbil to learn, remember, and go to a place in space defined only by its position relative to distal extramaze cues. the apparatus consisted of a circular white pool (80 cm in diameter and 55 cm in height) filled to a depth of 15 cm with water (27 c) rendered opaque with milk powder (18). gerbils were trained to find the spatial location of a platform of clear perspex hidden by arranging for its top surface (7 cm in diameter) to be 1 cm below the water level. latency to escape onto the hidden platform was recorded. gerbils were subjected to a 5-d training sequence (to assay learning) starting 4 d after the ischemic episode, then, 2 d after the end of learning assay, to a 1-d training (to assay memory).

histology

at the end of behavioral studies, in 56 gerbils (early or delayed treated), the brains were removed and processed as previously described (8, 26). hippocampus morphology was studied on hematoxylin-eosin stained sections (7 μm thick). glial fibrillary acidic protein (gfap) and antiapoptotic activity of cells were yzed on slides immunocytochemically treated with monoclonal anti-gfap (zymed laboratories, san francisco, ca) and monoclonal anti-bcl-2 (dako, glostrup,denmark), respectively. the slides were incubated overnight at 4 c with the antibodies, in a moist and darkened chamber. the slides were then incubated with 1:200 streptavidin biotinylated complex (dako) for 60 min and developed in diaminobenzidine (fluka, buchs, switzerland), and counterstained in harris hematoxylin. morphological yses were performed using an axiophot photomicroscope (carl zeiss, jena, germany). histometric yses were performed at the magnification factor on the monitor screen of x50 (length) and x800 (thickness and cell number) using an image system (vidas, carl zeiss). thickness of the pyramidal cell layer (at level of the ca1 subfield), ischemic extent [percentage of the linear size of the hippocampus ca1-ca4 subfields containing (after hematoxylin-eosin stain) neurons having red cytopla and pyknotic or shrunk nuclei], viable neurons in the ca1 subfield (neurons having, after hematoxylin-eosin stain, granular cytopla and euchromatic nucleus with large nucleoli), number of astrocytes in the ca1 subfield (cells positive to gfap), and cells positive to bcl-2 (in the ca1 subfield) were evaluated on three different slides of serial sections for each hippocampal sample. the density of neurons, astrocytes, and cells positive to bcl-2 was estimated in a 100-μm-thick band overlapping the pyramidal cell layer.

isolation of cytoplaic proteins

at different times after the ischemic episode, whole hippocampi were dissected from gerbil brains and immediately frozen into liquid nitrogen and stored at 80 c to permit adequate preservation of the phosphorylation state. the hippocampi were used to measure mapk activity (0.5, 3.5, and 9.5 h after the ischemic episode), tnf- and il-6 amount (2 h after), caspase-3 activity (24 h after), and concentrations of bcl-2 family proteins (bcl-2, bcl-xl, and bax: 11 d after). after homogenization in lysis buffer [10 mm tris (ph 7.4); 1% sds; protease inhibitor cocktail; phosphatase inhibitors: 100 mm na3vo4, 10 mm naf, 10 mm na4p2o7] and centrifugation, the supernatant was collected and employed for protein determination using the bio-rad protein assay kit (bio-rad, richmond, va), as previously described (27).

western blot ysis of erks, c-jun n-terminal kinases, p38 mapk, tnf-, caspase-3, il-6, bcl-2, bcl-xl, and bax

as previously described (27), cytoplaic proteins (40 μg for each sample) were denatured, electrophoretically separated, and transferred onto nitrocellulose membranes. staining of the blots with ponceau’s solution showed that total protein amount was equal in each lane. the blots were then blocked and incubated overnight at 4 c with a primary antibody for phosphorylated erks, c-jun n-terminal kinases (jnks), and p38 mapk (cell signaling, charlottesville, va); tnf-, il-6, caspase-3, and bax (chemicon international, temecula, ca); and bcl-2 and bcl-xl (biovision, mountain view, ca). the day after, the membranes were incubated with a specific secondary antibody peroxidase conjugated (pierce, rockford, il) for 1 h at room temperature. the membranes were yzed by the enhanced chemiluminescence system according to the protocol of the manufacturer (amersham, buckinghamshire, uk). the protein signals were quantified by scanning densitometry using a bioimage ysis system (bio-profil, celbio, italy). tnf-, il-6, bcl-2, bcl-xl, and bax values, as well as the activities of erks, jnks, p38 mapk, and caspase-3, were expressed as arbitrary units (27). the change was calculated vs. the respective sham value.

dna damage

we measured dna single-strand breaks using the comet assay, as previously described (28, 29), and dna fragmentation by means of the alkaline halo/diffusion assay (30). seventy-two hours after the ischemic episode (or sham ischemia), whole hippocampi were dissected from gerbil brains, and then the cells were lysed to obtain nucleoids. in the comet assay, after electrophoresis and staining, nucleoids were visually detected using a fluorescence microscope (olympus, tokyo, japan). one hundred nucleoids (comets) randomly selected on each of two slides/gerbil were scored and classified according to the relative intensity of fluorescence in the tail and given an arbitrary value from 0 (undamaged nucleus) to 4 (severely damaged nucleus), and then the total score was divided in two; therefore, the score in each gerbil ranged from 0400. in the alkaline halo/diffusion assay, after incubation in an alkaline hypotonic buffer and staining, the images of nucleoids were detected by means of a digital camera operating on a fluorescence microscope and yzed on a computer by using the public domain national institutes of health image program (r.info.nih.gov/nih-image). in all ischemic gerbils, the level of dna fragmentation was quantified, as percentage of increase of nuclear diameter compared with that of sham ischemic animals, from 25 randomly selected cells/gerbil.

statistical ysis

the values obtained in the comet assay were yzed using kruskal-wallis test followed by mann-whitney u test. mapk activities in gerbils treated 3 and 9 h after the ischemic episode were compared with those of the corresponding controls by means of student’s t test. all other data were yzed by means of anova followed by student-newman-keuls test.

results

ndp-msh improves learning and memory performance after brain ischemia

the hippocampus, particularly the ca1 subfield, is an area of the brain that plays a critical role in learning and memory. a brief, transient period of global cerebral ischemia causes selective loss of ca1 pyramidal cells 23 d after the ischemic episode. in the mongolian gerbil, this phenomenon, termed delayed neuronal death, represents a useful model for exploring the mechanis of neuronal death consequent to cerebral ischemia and the effects on spatial learning and memory, as well as for evaluating neuroprotective drugs in ischemic stroke (18, 31, 32, 33). therefore, we studied the ability of gerbils subjected to a 10-min period of global cerebral ischemia to learn, remember, and go to a place in space defined only by its position relative to distal extramaze cues (25, 26). in control gerbils (treated with saline), such a period of ischemia caused a significant impairment (as compared with sham ischemic) in place finding both during the first training session (assay of learning) and during the second session (assay of memory) (fig. 1, a and b). on the other hand, in gerbils treated ip (starting immediately before or 3 or 9 h after the ischemic episode) with the melanocortin ndp-msh, there was a dose-related improvement in learning and memory performance compared with ischemic control animals (fig. 1, a and b). interestingly, at the highest dose, ndp-msh accelerated learning compared with sham ischemic gerbils (fig. 1, a and c). melanocortins are known to reach the cns after systemic injection (34, 35) and much experimental evidence points in this direction (12, 13, 14, 36). five melanocortin receptor subtypes (mc1mc5) are known so far, and mc3 and mc4 are the predominant subtypes expressed in the cns (12, 13, 14, 16, 36). therefore, we investigated the role of these receptors. the protective effect of the maximally effective dose of ndp-msh [which activates mc1, mc3, mc4, and mc5 receptor subtypes (12)] on the behavioral performance of gerbils was completely prevented by a pretreatment with the selective mc4 receptor antagonist hs024 (21) (fig. 1, c and d). in these animals, as well as in those treated only with the mc4 receptor antagonist hs024, there was a worsening of memory, as compared with ischemic gerbils treated with saline. moreover, the selective mc3 receptor agonist 2-msh (12) (treatment equimolar to ndp-msh) had no protective effect (fig. 1, c and d).

ndp-msh inhibits mapk activation and proinflammatory cytokine overexpression

some members of mapks, such as jnks, p38 mapk, and erks, are early activated after transient forebrain ischemia in the gerbil hippocampus (24). the activation of some members of mapks has been implicated in the transcription of several genes involved in inflammatory processes, including genes encoding for proinflammatory cytokines (37). indeed, cytokines such as tnf- and il-6 increase early (within 13 h) in the hippocampus after brain ischemia (1, 38). in our study, immunoblot ysis showed that the activities of jnks, p38 mapk, and erks in whole hippocampus were increased in saline-treated gerbils subjected to a 10-min period of global brain ischemia, as detected 0.5, 3.5, and 9.5 h after reperfusion (40, 30, and 30 min after treatment, respectively) (fig. 2, ac). in gerbils treated with ndp-msh (also late), but not in those treated with the mc3 agonist 2-msh (at equimolar dose), there was a significant reduction of such increase (more markedly in jnks and p38 mapk); this protective effect of ndp-msh was counteracted by gerbil pretreatment with the mc4 receptor antagonist hs024 (fig. 2, ac). accordingly, tnf- and il-6 hippocampal levels were increased in saline-treated gerbils, as detected 2 h after the ischemic episode, and treatment with ndp-msh, but not with 2-msh, significantly prevented such increase (fig. 3, a and b). also this effect of ndp-msh was counteracted by pretreatment with the mc4 receptor antagonist hs024 (fig. 3, a and b).

ndp-msh reduces histological damage and delayed neuronal death

at the end of the second session of behavioral study, we processed the hippocampus for histology and histometry. in saline-treated gerbils subjected to transient brain ischemia, we observed loss of hippocampal neurons mostly inside ca1 subfield and partially replaced by glial cell hyperplasia (astrocytes), as indicated by gfap positivity (fig. 4, b and d); astrocytes spread from the pyramidal cell layer to the adjacent layers, oriens, and radiatum (fig. 4f). moreover, we found a great number of dead neurons showing pyknosis, nuclear dust, swollen perikaryon, cellular shrinkage, and absence of nissl substance (fig. 4g); accordingly, we detected scant expression of antiapoptotic activity (number of cells expressing the antiapoptotic protein bcl-2; fig. 4, e and h). in the hippocampus (from ca1 to ca4 subfield) of gerbils treated (early or late) with doses of ndp-msh maximally effective in improving the behavioral performance, we detected an ischemic extent quite similar to that of saline-treated ones (fig. 4a) but with a significantly larger thickness of the pyramidal cell layer in the ca1 subfield (fig. 4c). in this subfield, we also recorded a significantly reduced number of ischemic neurons and a higher number of viable neurons (also when the first treatment was delayed up to 3 or 9 h), when compared with the corresponding areas of saline-treated gerbils (fig. 4, d and g). in the ca1 subfield, astrocyte immunoreaction was quite similar to that observed in saline-treated gerbils, but without oriens and radiatum spread (fig. 4, b and f), whereas the antiapoptotic activity (number of bcl-2 positive cells) was more expressed (fig. 4, e and h). treatment with the selective mc3 agonist 2-msh (equimolar to ndp-msh), as expected, had no protective effects (fig. 4, ce). the effect of ndp-msh on thickness of the pyramidal cell layer, number of viable neurons, and bcl-2 immunoreaction was prevented by pretreatment of gerbils with the selective mc4 receptor antagonist hs024 (fig. 4, ce).

ndp-msh modulates the apoptotic process

the activation of mapks has been implicated in the apoptotic processes occurring after brain ischemia (1, 24, 39); in our experimental model of brain ischemia, ndp-msh blunted the activation of mapk members jnks, p38 mapk, and erks (fig 2, ac). the interaction between bcl-2 family members that promote (bax) and suppress (bcl-2 and bcl-xl) apoptosis determines whether cells undergo programmed death (40, 41, 42); in our conditions, ndp-msh caused overexpression of bcl-2 immunoreactivity in the cells of hippocampus pyramidal layer (fig. 4, e and h). this would indicate that melanocortins also affect apoptosis. to further investigate the role of melanocortins in the apoptotic process after transient global brain ischemia, at the end of the second session of behavioral study, we detected bcl-2, bcl-xl, and bax by means of immunoblot ysis. hippocampal levels of both bcl-2 and bcl-xl increased in ischemic saline-treated gerbils (fig. 5, a and b). in gerbils treated with ndp-msh (only early treatment was tested), but not in those treated with the selective mc3 agonist 2-msh (at equimolar dose), there was a further significant increase in bcl-2 and bcl-xl levels; this effect of ndp-msh was counteracted by pretreatment with the mc4 receptor antagonist hs024 (fig. 5, a and b). ndp-msh did not affect bcl-2 and bcl-xl levels in sham ischemic gerbils. similar high levels of bax protein were found in all experimental groups, including sham ischemic animals treated with ndp-msh (fig. 5c).

moreover, we explored the caspase pathway and the possible influence of ndp-msh. among the known 14 caspases, we chose to study the activity of the downstream executioner caspase-3 for its specific involvement in dna damage, with consequent cell death (1, 22, 23). in the hippocampi of saline-treated control gerbils, caspase-3 activity was significantly higher than that of sham ischemic animals, as detected 24 h after the ischemic episode [time of prominent expression (maintained at high levels for at least 72 h) of caspase-3 mrna in ca1 pyramidal neurons] (22) (fig. 6). in gerbils treated with ndp-msh, but not in those treated with the selective mc3 agonist 2-msh (at equimolar doses), caspase-3 activity was very reduced also when the first administration was delayed up to 3 or 9 h, and such effect of ndp-msh was again counteracted by gerbil pretreatment with the mc4 receptor antagonist hs024 (fig. 6).

the ultimate feature of apoptosis is dna fragmentation (1, 22, 23, 40). we, therefore, evaluated the degree of dna damage in the hippocampus by means of both the comet test (to assay dna single strand breaks, sign of initial lesions) (28, 29) and the alkaline halo/diffusion assay (to quantify dna fragmentation) (30). seventy-two hours after the ischemic episode, we found a significantly greater number of nuclei with long tail (high comet visual score) and with wide halo (high nuclear diameter due to radial diffusion of dna fragments) in the hippocampus of saline-treated control gerbils, when compared with those of sham ischemic animals (fig. 7, ad). in gerbils treated with ndp-msh, but not in those treated with 2-msh (at equimolar doses), dna damage was significantly lower when compared with that of control animals (fig. 7, ad). the selective mc4 receptor antagonist hs024 prevented such protective effect of ndp-msh against dna damage and even increased dna fragmentation as compared with that of ischemic gerbils treated with saline (fig. 7, a and b).

discussion

the third main cause of death in the united states and europe is ischemic stroke, and the only approved therapy for this condition is early thrombolysis (within 3 h) (43). early reperfusion with thrombolytic agents may reverse ischemic lesions, but recent investigations have shown that such reversible lesions may recur after 24 h (1). lesion recurrence may be related to occurrence of inflammation and delayed apoptotic death, which emphasizes the need for neuroprotective therapies targeting the mechanis involved in brain cell damage (1, 3).

here we demonstrate that the melanocortin peptide ndp-msh significantly protects against impairment in learning and memory caused by transient global brain ischemia. this clear neuroprotective effect is dose dependent, occurs at nanomolar doses, and is seemingly mediated by brain melanocortin mc4 receptors. the ndp-msh-induced improvement in behavioral performance, which is greater than that of sham ischemic animals, is associated with a suppression of the inflammatory cascade, as indicated by the significant decrease in tnf- and il-6 levels in the hippocampus, as well as by the blunted activation of the mapk members jnks, p38 mapk, and erks. moreover, our data show that, despite a quite similar extent of ischemia in the hippocampus of all experimental groups, this protective effect is associated with a reduction of the morphological damage in the ca1 subfield, including a reduction of neuronal death and a larger thickness of the pyramidal cell layer. overexpression of bcl-2 and bcl-xl (but not bax) proteins is also induced by ndp-msh, as detected 11 d after ischemia (but not after sham ischemia), although an influence on bcl-2 family members, including bax, 12 d after ischemia cannot be ruled out. blunted activation of jnks, p38 mapk, and erks, as well as increased expression of bcl-2 and bcl-xl, indicate that ndp-msh also suppresses apoptosis. this notion is likewise supported by the data showing that ndp-msh causes inhibition of caspase-3 activity and decrease of dna fragmentation. it is, therefore, likely that melanocortins modulate (directly or indirectly) the apoptotic process in brain ischemia. these protective events seem to be triggered by activation of central mc4 receptors, because ndp-msh fails to protect the ischemic gerbils pretreated with the selective mc4 receptor antagonist hs024 (21), and 2-msh [selective agonist at mc3 receptors, the other mc subtype mainly expressed in the cns (12, 36)] has no protective effect.

our present data show, for the first time, the antiapoptotic effect of melanocortins in conditions of brain ischemia and give further evidence of the antiinflammatory activity of such neuropeptides (9, 12, 13, 14, 16, 17). our data confirm that melanocortins are able to reduce cerebral tnf- and il-1 levels in brain ischemia and are in agreement with the proposed important role of this mechani in neuroprotection (16). it has been repeatedly reported that the antiinflammatory effects of melanocortins are adrenal independent, and ndp-msh does not stimulate adrenal glands, because it does not bind mc2 receptors (expressed in adrenal cortex and mediating glucocorticoid release) (12, 14, 36). in recent years, it has been also reported that melanocortins, including ndp-msh, stimulate the hypothalamic-pituitary-adrenal axis through central mc receptors (44, 45, 46). but, more recently, it has been found that -msh (at low doses) and -msh inhibit il-1-induced activation of the hypothalamic-pituitary-adrenal axis, also through central mc receptors (47). the possibility that the antiinflammatory effects of ndp-msh that we saw in this study may be an indirect consequence of glucocorticoid release from adrenal glands, therefore, cannot be completely ruled out.

although several innovative neuroprotective approaches have been shown to reduce brain lesions in animal models of stroke, clinical trials have failed to confirm these animal data so far, often because of toxic side effects and short therapeutic treatment window and because almost all studies aimed to block just one of the mechanis leading to neuronal damage (1, 48). rather, effective neuroprotection may require rational polytherapy or identification of single agents active against more than just one mechani of brain damage. recently, it has been suggested that among jnk isoforms (also activated owing to the excitatory amino acid glutamate receptor activation), jnk3 may be a potential target for neuroprotection therapies in stroke (49, 50). our data show for the first time that ndp-msh, an og of endogenous melanocortins, which lack of appreciable toxicity (14), has a therapeutic treatment window of at least 9 h (figs. 1, 4, and 6) and affects several ischemia-related mechanis of damage, including jnk3 activity (which contributes to both p46jnk and p54jnk) (49) (figs. 27). indeed, there seems to be a good correlation between behavioral and biomolecular changes; however, performing a statistical correlation between these parameters is beyond the scope of this investigation, and it could be the object of an additional study.

in brain ischemic areas, excitotoxicity directly and/or indirectly generates large amounts of radical species (1, 40). melanocortins, albeit lacking in scavenger properties, reduce free radical blood levels, including nitric oxide, in other hypoxic conditions, such as circulatory shock and myocardial ischemia (8, 10, 11). in brain ischemia, therefore, it is possible that, after signal transduction of ndp-msh, free radical-triggered activation of mapk and bcl-2 pathways be modulated (1, 51, 52), thus leading to antiinflammatory and antiapoptotic effects. an influence on nf-b activity―also involved in ischemic brain injury (53)―may also be hypothesized, in view of the sequential link between mapk and nf-b activation in other hypoxic conditions (54), and because of the fact that melanocortins blunt nf-b activation in the inflammatory response (9, 12, 14).

recently, it has been suggested that -msh-induced hypothermia might contribute to neuroprotection during global cerebral ischemia (55). we can, however, rule out a role of hypothermia for the protective effects observed in our study, because we maintained rectal and cranial temperatures of gerbils close to 37 c from the induction of anesthesia until death.

melanocortins are largely distributed in the cns, and mc4 receptors have been found in various brain areas including the hippocampus (12, 13, 14, 16, 36), and recently they have also been found outside the cns in the rat (56). it is known that melanocortins improve learning and memory in normal animals (57) and increase production of the antiinflammatory cytokine il-10 in modulating the inflammatory cascade (12, 14). on the other hand, low plaa concentrations of il-10 are associated with early worsening of neurological symptoms in patients with acute ischemic stroke (58), and -msh plaa levels are markedly decreased in patients with an unfavorable outcome after acute traumatic brain injury (14). accordingly, our data show an apparently faster learning in ndp-msh-treated ischemic animals, when compared with saline-treated sham ischemic ones, and a postischemic worsening of memory―as well as an increase in dna fragmentation―after the blockade of brain mc4 receptors, when compared with that of ischemic gerbils treated with saline. the suggestion that melanocortins might be physiologically involved in neuroprotection in conditions of brain ischemia, and that mc4 receptors might play an eminent role, is, for the moment, still speculative.

in conclusion, considering the broad time window for successful drug treatment (several hours), the activity against several ischemia-related mechanis of damage, the high pharmacological potency (they are effective at nanomolar doses), and the lack of apparent toxicity (14), melanocortins, in particular selective agonists at mc4 receptors, in our opinion are worth evaluating in other animal models of brain ischemia. such studies might aid in further development of novel approaches to neuroprotection in ischemic stroke.

footnotes

this work was supported in part by grants from ministero dell’istruzione, dell’universita e della ricerca, roma, and fondazione cassa di risparmio di modena, modena, italy.

first published online october 27, 20xx

abbreviations: cns, central nervous system; gfap, glial fibrillary acidic protein; jnk, c-jun n-terminal kinase; mc, melanocortin receptor subtype; ndp-msh, [nle4, d-phe7] -msh; nf-b, nuclear factor b.

accepted for publication october 20, 20xx.

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第三篇 Cyclic Adenosine 5-Monophosphate-Dependent Sphingosine-1-Phosphate Biosynthesis Induces Human CYP17

【关键词】  sphingosine

school of biology, parker h. petit institute for bioengineering and biosciences, georgia institute of technology, atlanta, georgia 30332-0230

abstract

in the human adrenal cortex, acth activates steroid hormone biosynthesis by acutely increasing cholesterol delivery to the mitochondrion and chronically increasing the transcription of steroidogenic genes (including cyp17) via a camp-dependent pathway. in the present study, we characterized the role of sphingolipids in acth-dependent steroidogenesis. h295r human adrenocortical cells were treated with acth or dibutyryl camp (bt2camp) and the content of several sphingolipid species quantified by mass spectrometry. both acth and bt2camp decreased cellular amounts of several sphingolipids, including sphingomyelin, ceramides, and sphingosine and stimulating the activity of sphingosine kinase and increasing the release of sphingosine-1-phosphate (s1p) into the media. s1p increased cyp17 mrna expression by promoting the cleavage and nuclear localization of sterol regulatory element binding protein (srebp) 1. chromatin immunoprecipitation assays revealed that bt2camp and s1p increased acetylation of histone h3 and promoted binding of srebp1 to the 520/331 region of the cyp17 promoter. in summary, our studies demonstrate a role for sphingolipid metaboli and srebp1 in acth-dependent cyp17 regulation and steroidogenesis.

introduction

steroid hormone biosynthesis in adrenal cortex involves the coordinate action of several cytochrome p450 enzymes, whose genes (cyp) are transcriptionally activated by the peptide hormone acth (1). in the zona fasciculata and zona reticularis, acth directs increased steroid hydroxylase gene transcription via the activation of adenylyl cyclase and subsequent increase in intracellular camp. this second messenger then activates protein kinase a (pka), which induces gene transcription by phosphorylating transcription factors, coactivators, and/or other proteins in the acth signaling pathway.

we have previously shown that the acth/camp increases human cyp17 gene expression by promoting the binding of a protein complex containing steroidogenic factor-1 (sf-1), p54nrb, and polypyrimidine-tract binding protein-associated splicing factor (psf) (2) to a 20-bp region between 57 and 37 of the cyp17 promoter. the affinity of this sf-1/p54nrb/psf complex for region 57/37 of the cyp17 promoter is induced by camp and is dependent on phosphatase activity (3, 4). although acth/camp plays a central role in regulating cyp17 gene expression, other signaling cascades, and second-messenger systems have been shown to modulate cyp17 gene expression (5). moreover, it is likely that acth/camp activates other signal transduction cascades that result in the induction of cyp17 transcription.

sphingolipids are a diverse family of phospholipids and glycolipids that serve as structural components of the cell membrane and key mediators of numerous cellular processes (6, 7, 8, 9). ceramide has been shown to act as a second messenger for events as diverse as differentiation, senescence, proliferation, cell cycle arrest, and apoptosis (7, 8, 9). sphingosine-1-phosphate (s1p) also modulates a wide variety of physiological functions, including cell proliferation and survival (10, 11, 12, 13), chemotaxis (14), and protection against ceramide-mediated apoptosis (15).

over the past few years, several studies have examined the role of sphingolipids on steroid hormone biosynthesis in both gonadal and adrenal cell lines (16, 17, 18, 19, 20, 21, 22, 23). ceramide, bacterial sphingomyelinase [(ase), which converts sphingomyelin () to ceramide], and dihydroceramide all increase basal and human chorionic gonadotropin-stimulated progesterone synthesis in ma-10 murine leydig cells (16). exposure of glutaraldehyde-fixed ma-10 cells to recombinant ase increases degradation, cholesterol movement from the cell surface into the mitochondria, and progesterone secretion (18). ase also enhances camp-stimulated steroidogenesis, suggesting that activating sphingolipid turnover may promote steroid hormone biosynthesis by increasing cholesterol movement to the inner mitochondrial membrane (18). similar stimulatory effects of ceramide on steroid hormone production have also been found in jeg-3 human choriocarcinoma cells (17). finally, s1p was found to stimulate cortisol secretion in zona fasciculata bovine adrenal cells in a protein kinase c- and ca2+-dependent manner (19).

studies have also been published demonstrating an inhibitory role of sphingolipids on steroid hormone production (20, 21, 22, 23). ceramide ogs have no effect on rat leydig cell steroidogenesis, whereas ase inhibits human chorionic gonadotropin-stimulated production (22). budnick et al. (23) reported that tnf inhibits production by inducing ceramide accumulation, thereby resulting in decreased steroidogenic acute regulatory (star) protein expression levels. the star protein plays an essential role in steroidogenesis by enabling the transport of cholesterol to inner mitochondrial membrane in which p450 side chain cleavage enzyme (p450scc; encoded by cyp11a1) converts cholesterol to pregnenolone in the first enzymatic step of steroid hormone biosynthesis. in rat granulosa cells, both ase and ceramide inhibit fsh-stimulated progesterone biosynthesis and the mrna expression levels of cyp11a1 and 3-hydroxysteroid dehydrogenase (20).

interestingly, sphingolipids have been shown to induce sterol regulatory element binding protein (srebp) 1 cleavage, leading to increase in intracellular cholesterol (24). srebp1a has been shown to activate transcription of the star protein (25). srebps are a family of transcription factors that regulate the genes that encode for more than 30 enzymes that are involved in cholesterol, triacylglyceride, phospholipid biosynthesis, fatty acid desaturation, and cholesterol uptake (26).

the aim of this study was to examine the relationship between acth/camp-dependent steroidogenesis and the sphingolipid metabolic pathways in the h295r human adrenocortical cell line. we used mass spectrometric ysis to determine the effects of acth and camp on the cellular content of several sphingolipid species. we also characterized the effect of s1p on cyp17 gene expression. we show that acth and camp rapidly alter the cellular sphingolipid profile by stimulating sphingolipid cataboli. acth and dibutyryl camp (bt2camp) increased the activity of sphingosine kinase (sk). moreover, the s1p produced in response to bt2camp increased the transcription of cyp17 by stimulating binding of srebp1 to the promoter. in summary, our studies establish a link between acth/camp-dependent steroidogenesis and sphingolipid metaboli in the human adrenal cortex and demonstrate that s1p can serve as a signaling mediator in acth/camp-stimulated cyp17 transcription by activating srebp1.

materials and methods

reagents

bt2camp and n-acetyl-leu-leu-norleucine (alln) were obtained from sigma (st. louis, mo). sphingolipids were obtained from avanti polar lipids inc. (alabaster, al). n-acetyl-d-erythro-sphingosine (c2-ceramide) was freshly prepared before each experiment by dissolving in ethanol. d-erythro-sphingosine (sph) was prepared by dissolving in ethanol, followed by dilution in fatty acid-free bsa (calbiochem, la jolla, ca). d-erythro-s1p and dihydro-s1p (dhs1p) were prepared by dissolving in ethanol and dimethylamine, followed by evaporation and solubilization in 2 mm fatty acid-free bsa. acth was obtained from calbiochem. all interfering rna (sirna) oligonucleotides directed against srebp1, srebp2, sk1, and sk2 were obtained from dharmacon (lafayette, co).

cell culture

h295r adrenocortical cells (27, 28) were generously donated by dr. william e. rainey (medical college of georgia, augusta, ga) and cultured in dulbecco’s modified eagle’s/f12 medium (invitrogen, carlad, ca) supplemented with 10% -serum i (bd biosciences, palo alto, ca), 0.5% its plus (bd biosciences), antibiotics, and antimycotics.

ysis of sphingolipid molecular species

for sphingolipid measurements, cells were treated for 30 min or 2 h with 50 nm acth or 1 mm bt2camp and sphingolipids in the cells, and media were yzed by liquid chromatography, electrospray ionization, and tandem mass spectrometry (lc-esi-ms/ms) as described previously (29, 30). the internal standards for quantification of the sphingolipids were obtained from avanti polar lipids.

sk assay

sk activity was assayed as described by olivera et al. (31). briefly, cells were plated onto 100-mm dishes and treated with 50 nm acth or 1 mm bt2camp for 560 min. alternatively, cells transfected with sirna oligonucleotides directed against sk1 and sk2 were treated with 1 mm bt2camp for 15 min. after the desired treatment period, the cells were washed with ice-cold pbs and then harvested into 200 μl of lysis buffer [20 mm tris-hcl (ph 7.4), 20% glycerol, 1 mm dithiothreitol, 1 mm edta, 1 mm sodium orthovanadate, 10 mm mgcl2, 15 mm sodium fluoride, 1 mm phenylmethylsulfonyl fluoride, 0.5 mm 4-deoxypyridoxine, and protease inhibitors (calbiochem)]. the cytosolic fraction was incubated with 5 mm d-erythro-sphingosine and [-32p]atp for 30 min at 37 c. sphingosine used in the reactions was solubilized as a sphingosine-bsa complex as described above. reactions were terminated by placing the tubes on ice and adding 20 μl of 1 m hcl and 800 μl of chloroform/methanol/hcl (100:200:1, vol/vol). the samples were vortexed for 10 min, followed by the addition of 200 μl chloroform and 200 μl of 2 m kcl, vortexing again for 10 min, and phase separation by centrifugation. an aliquot (50 μl) of the organic phase was spotted onto a silica gel 60 tlc plate and developed in 1-butanol/methanol/acetic acid/water (80:20:10:20, vol/vol). unlabeled s1p was also spotted onto each plate and visualized by spraying with ninhydrin. the dried plates were exposed to a phosphor imager screen and the amount of radioactivity measured normalized to the total protein concentration of each sample.

rna isolation and northern blotting

cells were cultured onto 12-well plates and treated with 1 mm bt2camp, 0.110 μm sph or 0.110 μm s1p for 112 h. total rna was prepared from treated cells by acid-phenol extraction (32). rna was fractionated by agarose (1%) gel electrophoresis in the presence of 5% formaldehyde and transferred onto nylon transfer membrane filters (millipore, bedford, ma). a 1.2-kb cdna fragment of cyp17 was used to detect cyp17 mrna expression. cdna fragments were radiolabeled with [a-32p]dctp using a random primer labeling kit (takara, shiga, japan). blots were hybridized overnight at 42 c in 50% formamide, 5x sodium chloride/sodium phosphate/edta, 1% sodium dodecyl sulfate (sds), 5x denhardt’s, 50 μg/ml trna, and 32p-labeled cdna fragments. the hybridized membranes were sequentially washed for 2 x 10 min in 2x saline sodium citrate, 0.2% sds, and 2 x 5 min in 0.2x saline sodium citrate, 0.2% sds at 42 c. the amount of probe bound to the filter was quantified using a fluorescence/phosphor imager (fuji film, tokyo, japan). results were normalized to the content of glyceraldehyde-3-phosphate dehydrogenase mrna.

rna interference (rnai) and real-time rt-pcr

for rnai experiments, cells were subcultured onto 12-well plates and 24 h later media replaced by optimem (invitrogen). cells were transfected with 150 nm of srebp1, srebp2, sk1, sk2 or nonspecific sirna oligonucleotides using siimporter (upstate biotechnology, lake placid, ny). seventy-two hours (srebp1 and srebp2) or 48 h (sk1 and sk2) after transfection, cells were treated with 1 mm bt2camp or 1 μm s1p for 6 h. total rna was extracted using trizol (invitrogen) and amplified using the iscript one-step rt-pcr kit with sybr green (bio-rad, hercules, ca) and an icycler real-time thermocycler (bio-rad). the following pcr primers were used: cyp17 (forward, 5'-ctcttgctgcttcaccta, and reverse, 5'-tcaaggagatgacattggtt); actin (forward, 5'-acggctccggcatgtgcaag-3', and reverse, 5'-tgacgatgccgtgctgcatg-3'), sk1 (forward, 5'-ctggcagcttccttgaaccat-3', and reverse, 5'-tgtgcagagacagcaggttca-3'); and sk2 (forward, 5'-ccagtgttggagagctgaaggt-3', and reverse, 5'-gtccattcatctgctggtcctc-3'). cyp17 expression is normalized to actin and calculated using the - cycle threshold method. s1p receptor expression was determined using taqman gene expression probes and the iscript one-step rt-pcr kit for probes (bio-rad).

transient transfection and reporter gene ysis

cells were subcultured onto 12-well plates and 24 h later transfected with 500 ng of reporter plaids containing 1100, 700, 300, or 57 bp of the cyp17 promoter upstream of the start site (2) using genejuice (novagen, madison, wi). the cyp17 1100-, 700-, and 300-pgl3 constructs were generated by pcr using a plaid containing a 1.8-kb fragment of the cyp17 promoter fused to the luciferase gene in the pgl3 vector [generously donated by dr. janette m. mcallister (pennsylvania state university, hershey, pa)]. the cyp17 57-pgl3 plaid was constructed by ligating double stranded oligonucleotides corresponding to the region 57/2 of the cyp17 5' flank upstream of the luciferase gene in the pgl3 vector (promega, madison, wi). cells were cotransfected with 10 ng of the renilla luciferase plaid (prl cmv, promega) for normalization. cells were then treated with 1 mm bt2camp, 1 μm sph, or 1 μm s1p for 6 h and harvested for dual luciferase assays (promega).

chromatin immunoprecipitation (chip)

for chip assays (33, 34), h295r cells (150-mm dishes) were stimulated with 50 nm acth, 1 mm bt2camp, 1 μm c2-ceramide, 1 μm sph, 1 μm s1p, or 500 nm trichostatin a (tsa) for 1 or 4 h. cross-linking was performed by incubation in 1% formaldehyde (in pbs) for 10 min. the reaction was stopped by the addition of glycine (0.125 m final concentration), the cells washed, the nuclei harvested, and the lysates sonicated to obtain optimal dna fragment lengths of 100-1000 bp. the purified chromatin solutions were immunoprecipitated using antiacetyl histone h3 (upstate, charlottesville, va) or anti-srebp1 (santa cruz biotechnology, santa cruz, ca) and protein a/g plus (santa cruz biotechnology). the reaction was centrifuged at 4000 rpm for 5 min and 100 μl of the supernatant (input) retained. the antibody/protein/dna bound beads were subjected to a series of 5-min washes: three times in radioimmunoprecipitation assay (ripa) buffer [50 mm tris, cl (ph 7.4), 1% nonidet p-40, 0.25% sodium deoxycholate, 150 mm nacl, 1 mm edta, 150 nm aprotinin, 1 mm leupeptin, 1 mm e-64, 500 mm 4-(2-aminoethyl)benzenesulfonylfluoride], three times in ripa buffer plus 500 mm nacl, three times in washing buffer [10 mm tris-cl (ph 8), 0.25 m licl, 1 mm edta, 1 mm egta, 1% nonidet p-40, 1% sodium deoxycholate, 10 mm sodium butyrate, 20 mm -glycerophosphate, and protease inhibitors], and three times in tris/edta buffer. the cross-links were reversed and protein digested using proteinase k (100 μg/ml). dna was purified by phenol-chloroform extraction and ethanol precipitation. precipitated dna was amplified by pcr using the following primer pairs: forward 5'-ggggaccattaacccgacagcccttatcgc-3' (520/491 cyp17), reverse 5'-gacagatgacagattcaggagggtcacaag-3' (331/360 cyp17). pcrs were as follows: 1) 1 x 94 c, 5 min; 2) 35 x 95 c, 1 min, 55 c, 1 min, 72 c, 2 min; 3) 1 x 72 c, 10 min; 4) cool to 4 c. pcr products were then subjected to agarose (2%) gel electrophoresis.

sds-page and western blot ysis

to confirm that rnai of sk1, sk2, srebp1, and srebp2 was effective, cells were transfected with sirna oligonucleotides for 48 h (sk1 and sk2) or 72 h (srebp1 and srebp2) and cell lysates harvested for sds-page and western blotting. twenty-five micrograms of each sample were run on 10% sds-page gels, transferred to polyvinylidene difluoride membranes (pvdf, pall corp., pensacola, fl) and probed with anti-sk1 (1:250), anti-sk2 (1:250), anti-srebp1(k10) (1:500), or anti-srebp2(h164) (1:500). antibodies to sk1 and sk2 were obtained from exalpha biologicals inc. (watertown, ma), and antibodies to srebp1 and srebp2 were obtained from santa cruz biotechnology. protein expression was detected using an ecf western blotting kit (amersham biosciences, piscataway, nj) and a fluor/phosphor imager (fuji film).

to determine the effect of s1p on srebp processing, cells were subcultured onto six-well plates and treated with 1 μm s1p for periods ranging from 1 to 4 h. two hours before harvesting, 25 μg/ml alln were added to inhibit proteosomal degradation of mature srebp. for the 1 h time point, cells were pretreated with alln before stimulation with s1p. cells were washed twice in pbs, harvested into ripa buffer, and lysed by passing 10 times through a 22-gauge needle. lysates were centrifuged for 15 min at 4 c and the supernatant collected for ysis by sds-page. aliquots of each sample (25 μg of protein) were run on 10% sds-page gels, transferred to pvdf membranes, and probed with anti-srebp1. srebp1 protein expression was detected and imaged as described above. for ysis of membrane and nuclear srebp1, cells were plated onto 60-mm dishes and treated with 1 μm s1p or dhs1p. cells were isolated and membrane and nuclear extracts prepared using ne-per (pierce, rockford, il) containing protease inhibitors. twenty-five micrograms of nuclear or membrane extracts were run on 10% sds-page gels, transferred to pvdf membranes, probed with anti-srebp1, and expression detected as described above.

results

acth stimulates sphingolipid metaboli in h295r cells

several studies have characterized the role of sphingolipids in steroid hormone biosynthesis and found both stimulatory and inhibitory roles for ceramide, sph, and s1p on hormone secretion (16, 17, 18, 19, 20, 21, 22, 23). however, the effect of acth on cellular sphingolipid content is unknown. thus, we treated h295r human adrenocortical cells for 2 h with 50 nm acth or 1 mm bt2camp and quantified the amounts of several sphingolipids by lc-esi-ms/ms. as shown in fig. 1a, both acth and bt2camp decreased cellular . of the different molecules in h295r cells, both acth and bt2camp decreased c16 and c18 (fig. 1b).

activation of sphingolipid turnover of complex sphingolipid molecules like is linked to the increased production of bioactive species such as ceramide, sph, and s1p. unexpectedly, however, we also observed acth/camp-stimulated reduction of cellular ceramides and sph (fig. 1c). bt2camp treatment also decreased the amounts of multiple chain-length subspecies (fig. 1d).

acth/camp activate sk activity

because acth and bt2camp evoked decreases in cellular ceramides and sph, we hypothesized that acth and bt2camp were activating sk. increased sk activity would explain decreases in cellular sph due to phosphorylation to form s1p. thus, we carried out enzyme assays to determine whether acth/camp was activating sphingosine kinase activity. as shown in fig. 2, exposure of h295r cells to acth and bt2camp resulted in rapid increases in sk activity. maximal increases in enzyme activity were observed at 15 min for both agents and returned to basal levels after 30 min. incubation of h295r cells for 15 min with acth increased sk activity 3.2-fold, whereas bt2camp resulted in a 4.8-fold increase in catalytic activity (fig. 2).

acth/camp promote s1p secretion into the media

the increase in sk activity after exposure to acth or bt2camp suggested that acth/camp may activate sphingolipid turnover and increase the cellular concentration of s1p. however, mass spectrometric ysis of cells treated for 30 min or 2 h with 50 nm acth or 1 mm bt2camp revealed decreases in the cellular amounts of s1p (fig. 3). because some cell types secrete s1p, the amounts in the medium were also yzed by tandem mass spectrometry. as shown in fig. 3, s1p in the medium increased in a manner that mirrored the decreases in the cells; therefore, it appears that acth and camp are stimulating cataboli of , resulting in decreases in the cellular content of ceramide, sph, and s1p and secretion of s1p into the media. unexpectedly, no significant difference was observed in the amount of s1p in the media at the 30-min and 2-h time points. the cellular levels of most sphingolipids returned back to control levels after 6 h (data not shown).

s1p induces steroidogenic gene expression

given that acth alters the sphingolipid profile in h295r cells (fig. 1), we carried out studies to further explore the relationship between sphingolipid metaboli and a steroidogenic enzyme. cyp17 mrna expression in bt2camp-, s1p-, or sph-treated cells was yzed by northern blotting (fig. 4a) and real-time rt-pcr (fig. 4, b and c). densitometric ysis of northern blots revealed that sph and s1p increased cyp17 mrna expression by 1.3- and 3.6-fold, respectively, whereas bt2camp induced hcyp17 mrna expression 5.2-fold (fig. 4a). as shown in fig. 4b, both s1p and bt2camp increased cyp17 mrna expression in a time-dependent manner; however, maximal stimulatory effects of s1p were observed at 6 h, whereas bt2camp maximally increased cyp17 mrna at the 12-h time point.

camp-dependent cyp17 mrna expression requires sk1

because sk activity was increased by bt2camp treatment (fig. 3) and s1p increased cyp17 expression (fig. 4), we used rnai to determine whether camp-stimulated cyp17 transcription required sk. h295r cells were transfected with sk1 and sk2 sirnas and then treated with 1 mm bt2camp for 12 h. real-time rt-pcr revealed that silencing both sk1 and sk2 decreased the stimulatory effect of bt2camp on cyp17 mrna expression (fig. 5a). we confirmed that rnai suppressed sk1 and sk2 protein expression by western blotting (fig. 5b) and sk activity ysis (fig. 5c).

sphingolipids stimulate cyp17 transcriptional activity

to determine whether the stimulatory effects of sphingolipids on cyp17 mrna (fig. 4) were mediated at the level of transcription, we performed transient transfection assays using plaids containing varying lengths of the cyp17 promoter fused to the firefly luciferase gene. sph and s1p increased the transcriptional activity of the 1100- and 700-pgl3 cyp17 reporter constructs, whereas having no significant effect on the cyp17 57- and 300-pgl3 plaids (fig. 6a). these findings suggest that the region of the cyp17 promoter required for sphingolipid-dependent gene transcription lies between 700 and 300 bp. in contrast to the effect of sph and s1p, bt2camp significantly stimulated the luciferase activity of all plaids tested (fig. 6a) as seen previously (2). in silico ysis (35) revealed a putative binding site for srebps (fig. 6b). thus, we determined the effect of mutating this site on the ability of s1p to stimulate cyp17 reporter gene activity. as shown in fig. 6c, mutation of the putative sre abolished the stimulatory effects of s1p.

sphingolipids increase acetylation of histone h3 at the cyp17 promoter

next we examined the effect of s1p on the acetylation state of histone h3 at the cyp17 promoter. cells were treated with bt2camp sphingolipids or the histone deacetylase inhibitor, tsa, for 4 h, followed by chip. pcr was performed using primers that amplified regions 142/+45 and 520/360. although we have previously shown that acth/camp-dependent gene transcription occurs on binding of a complex containing sf-1/p54nrb/psf to region 57/37 of the cyp17 promoter (2), studies described herein show that s1p activates cyp17 gene transcription by stimulating the binding of a trans-acting factor(s) to a more distal region of promoter (436/448).

as shown in fig. 7a, acetylation of histone h3 at the 520/360 region of the cyp17 gene is increased by bt2camp, sph, and s1p. tsa also increased the acetylation of histone h3 at the cyp17 promoter (fig. 7a). these findings are in contrast to the results of pcr using primers designed to amplify the 142/+45 region of the cyp17 promoter, in which only bt2camp and tsa significantly increased the acetylation of histone h3 (data not shown). finally, we performed chip assays using antibodies to srebp1. as shown in fig. 7b, acth, bt2camp, and s1p increased binding of srebp1 to the 520/60 region of the cyp17 gene.

s1p induces cyp17 transcription by activating srebp1

because sphingolipid-stimulated srebp1a cleavage (24) mediates increased transcription of star (25), we investigated the role of srebps in s1p-evoked cyp17 gene expression. h295r cells were transfected with srebp1 and srebp2 sirnas and treated with 1 mm s1p for 6 h followed by total rna extraction and quantitative rt-pcr as described in materials and methods. as shown in fig. 8a, srebp1 sirna abolished s1p-stimulated transcription of cyp17, whereas sirnas targeted against srebp2 decreased s1p-stimulated cyp17 mrna expression by 31%. all interfering oligonucleotides specifically decreased the protein expression srebp1 and srebp2 (fig. 8b).

s1p stimulates srebp1 cleavage and nuclear translocation

nascent srebps contain two tranembrane domains that are integrally inserted into the endoplaic reticulum. processing of srebp to generate the transcriptionally active n-terminal fragment involves the transport of the full-length protein to the golgi apparatus in which it is cleaved sequentially by two membrane-associated proteases. the transcriptionally active fragment of srebp is then translocated to the nucleus in which it binds to the promoters of srebp target genes. to determine whether s1p stimulates the processing of srebp1, we treated h295r cells with s1p for varying time points and measured the levels of mature and precursor forms by western blotting. because the mature form of srebp is subjected to ubiquitination and subsequent proteasomal degradation (36), h295r cells were treated with the proteasome inhibitor alln as previously described by others (24, 37). as shown in fig. 9a, s1p stimulates srebp1 maturation in a time-dependent manner. to determine whether s1p activates srebp cleavage by binding to a s1p receptor or acting intracellularly, we treated h295r cells with dhs1p. like s1p, dhs1p binds to and activates s1p receptors (38, 39); however, because of its increased hydrophilic nature, dhs1p is unable to permeate the plaa membrane (40, 41). both s1p and dhs1p stimulated an increase in the mature form of srebp1 in the nucleus (fig. 9b), suggesting that s1p acts extracellularly to induce srebp1 maturation and induce cyp17 transcription.

our observation that camp stimulates the secretion of s1p into the media (fig. 3) coupled with the finding that dhs1p promotes srebp1 translocation to the nucleus (fig. 9b) suggests that s1p may act in a paracrine or autocrine manner to increase cyp17 transcription by binding to s1p receptors on surface of h295r cells. s1p regulates biological processes by serving as a ligand for the s1p family of g protein-coupled receptors (42, 43) and acting intracellularly (42). to date, five s1p receptors have been identified (44, 45, 46). these g protein-coupled receptors were initially called endothelial differentiation gene (edg) receptors but have been renamed as s1p1 (edg-1), s1p2 (edg-5), s1p3 (edg-3), s1p4 (edg-6), and s1p5 (edg-8) (44). as shown in fig. 9c, all five receptors are expressed in h295r cells; however, s1p4 is not as highly expressed as the other four receptor subtypes. finally, like bt2camp and s1p, dhs1p is able to induce cyp17 mrna expression (fig. 9d), further supporting our hypothesis that s1p acts in a paracrine manner to stimulate srebp1 binding to the cyp17 promoter by binding to a g protein-coupled receptor.

discussion

acth exerts its stimulatory actions on steroid hormone biosynthesis via two temporally distinct camp/pka-dependent pathways. a rapid, acute response results in the transport of cholesterol into the inner mitochondrial membrane for conversion to pregnenolone by p450 11a1 (p450scc). during the acute response, an essential site of phosphorylation by pka is cholesterol ester hydrolase, which on activation catalyzes the conversion of cholesterol esters to free cholesterol (47). key regulators of the acute response, star protein (48, 49) and the peripheral benzodiazapene receptor (50, 51), facilitate cholesterol movement in the mitochondria. the chronic effect of acth is to increase the transcription of steroidogenic enzymes.

in this report, we provide evidence for a novel mechani by which acth induces cyp17 transcription. we show herein that both acth and camp decrease the cellular amounts of several sphingolipid species, including and ceramides (fig. 1). in addition, acth and camp rapidly and transiently activate sk catalytic activity (fig. 2). previous studies have demonstrated the relationship between and p450scc (52). p450scc catalyzes the first step in steroid hormone biosynthesis: conversion of cholesterol to pregnenolone. inhibits the ability of cholesterol to bind to p450scc (52). furthermore, the interaction between cholesterol and is cooperative (52), indicating that interactions between cholesterol and lipids can play a role in steroidogenesis. in light of these previous findings, the studies presented herein suggest that in addition to increasing availability of cholesterol for steroidogenesis, acth activates sphingolipid metaboli to maintain optimal cholesterol to lipid ratios in the membranes of adrenocortical cells.

because decreases in cellular levels of s1p were paralleled by increases in this bioactive molecule in the medium (fig. 3) and s1p receptors are expressed in h295r cells, we speculated that s1p acts in a paracrine manner to increase steroidogenesis. our findings that the s1p receptor agonist dhs1p mimicked the stimulatory effect of s1p on cyp17 gene expression suggest that s1p acts extracellularly by binding to one of the s1p receptors. studies are underway to determine which one of the five s1p receptors mediates cyp17 transcription in response to s1p. the differences in signaling through these receptors are primarily a result of differential coupling to g proteins. s1p1 couples to gi (53, 54), whereas s1p2 and s1p3 couple to gi, gq, and g13 (54). s1p4 has been shown to associate with gi (39, 55) and g12/13 (56) and s1p5 couple to gi/o and g12 (57). interestingly, s1p2 activates adenylyl cyclase (58); however, because the receptor does not couple to gs, the activation of adenylyl cyclase may occur through an indirect mechani. it has also been shown that s1p induces mapk phosphatase-1 (mkp-1) in mouse fibroblast ch10t1/2 cells (59). we have previously shown that induction of cyp17 gene expression in response to camp requires activation of mkp-1 (1). thus, it is possible that camp-stimulated release of s1p into the media may activate cyp17 gene expression via mkp-1. although our data demonstrating that s1p is secreted into the media (fig. 3) and that dhs1p induces cyp17 transcription (fig. 9c) suggest that s1p acts extracellularly, it is possible that s1p may also be acting intracellularly. the generation of fatty acids for prostaglandin synthesis by phospholipase a2 has been shown to have both extracellular and intracellular components (60, 61, 62, 63, 64).

srebps regulate genes involved in the sterol synthesis and uptake as well as fatty acid biosynthesis and desaturation (26, 65). when the levels of free cholesterol in the cell are low, srebps undergo proteolytic processing, resulting in the release of a mature transcription factor that translocates to the nucleus for increased expression of genes involved in cholesterol biosynthesis and fatty acid metaboli (26, 65). the activity of these transcription factors is controlled by transport from endoplaic reticulum to golgi by an escort protein called srebp-cleavage activating protein (scap), which senses the absence of cholesterol (26). recently sphingolipids have been shown to stimulate srebp-1 cleavage by causing the cholesterol to be sequestered in endosomes or lysosomes, leading to a compartmentalization of cellular cholesterol (24). this redistribution of cholesterol is sensed by scap, which leads to the translocation of srebps to the golgi for maturation (24). in agreement with these studies, we found that s1p promotes srebp1 maturation (fig. 9a) and nuclear expression (fig. 9b). moreover, transcription of star is increased by srebp-1 (25). chip data presented in fig. 7 showing increased acetylation of histone h3 and recruitment of srebp1 to the 700/300 region of the cyp17 promoter suggest that s1p stimulates chromatin remodeling, resulting in increased accessibility to this region of the promoter and srebp binding. of note, hyperacetylation of histone h3, but not histone h4, has been found to occur in chromatin at the promoters for the low-density lipoprotein receptor gene and hydroxymethyl glutaryl coa reductase (66). our findings agree with these findings and suggest that in addition to increasing the acetylation of histone h3 at the promoters of sterol-regulated genes, srebp also promotes hyperacetylation of a steroidogenic gene. additionally, recent studies by hughes et al. (67), demonstrate that orthologs of srebp and scap, sre1+, and scp1+, respectively, function as an oxygen sensor in fission yeast.

in these studies, microarray ysis carried out to identify genes activated by srebp1 revealed that two genes required for the hydroxylation of sphingolipids were highly up-regulated (67). previously it was proposed by lawler et al. (68) that stimulation of human hepatocytes by tnf leads to the activation of neutral ase, which in turn induces the maturation of srebp1. this induction of srebp1 cleavage occurred independent of cholesterol depletion. furthermore, studies have suggested that whereas sre-mediated gene transcription is decreased by the inhibition of ceramide synthesis, it is stimulated by increasing sph levels (69).

based on the findings presented herein, we propose a model (fig. 10) for the role of s1p and srebp1 in acth/camp-dependent cyp17 transcription and cortisol biosynthesis. binding of acth to its receptor on cell surface activates adenylyl cyclase, which leads to the production of camp and activation of pka. pka promotes the turnover of , which leads increased sphingolipid turnover due to the activation of enzymes (such as sk) in the sphingolipid pathway, causing decreased levels of several sphingolipid species. the s1p released into extracellular space binds to an s1p receptor, thereby activating a signal transduction cascade that activates srebp1 cleavage and maturation. although we found that total cellular amounts of s1p decreased in camp-treated cells (fig. 1), it is also possible that s1p concentrations are increased in distinct subcellular organelles, such as the endoplaic reticulum or golgi and that this organelle-specific increase may result in an intracellular pool of s1p that may contribute to the increase in cyp17 transcription. cellular fractionation studies are ongoing to determine the effects of acth and camp on organellar concentrations of various sphingolipid moieties. moreover, it is probable that acth/camp may affect de novo sphingolipid biosynthesis. studies are underway to determine whether the decreases in cellular sphingolipid content are due to increased cataboli, decreased de novo synthesis, or both. in our model (fig. 10), we propose that the s1p produced in response to camp promotes the migration of srebp1 to the golgi in which it is processed to the mature form. mature srebp1 then translocates to the nucleus, dimerizes, and binds to the sre site on the cyp17 promoter causing the activation of transcription of cyp17 gene. in summary, our studies demonstrate that acth/camp rapidly activates sphingolipid cataboli and s1p production, which ultimately leads to induction of cyp17. these findings provide evidence for a role for sphingolipids in acth/camp-dependent cortisol biosynthesis and establish a novel mechani by which s1p, acting as a paracrine or autocrine factor, can increase cyp17 gene transcription by activating srebp1.

acknowledgments

we thank samuel kelly, elaine wang, and dr. alfred h. merrill, jr. for mass spectrophotometric ysis of sphingolipid molecular species.

footnotes

this work was supported by the national science foundation (mcb-0347682), the national institutes of health (gm073241), and the georgia cancer coalition. mass spectrometric ysis of cellular lipids was supported by the national institutes of health (pa-02-132 to a.h.m.).

the authors have nothing to declare.

first published online november 23, 20xx

abbreviations: alln, n-acetyl-leu-leu-norleucine; bt2camp, dibutyryl camp; c2-ceramide, n-acetyl-d-erythro-sphingosine; chip, chromatin immunoprecipitation; dhs1p, dihydro-s1p; edg, endothelial differentiation gene; lc-esi-ms/ms, liquid chromatography, electrospray ionization, and tandem mass spectrometry; mkp-1, mapk phosphatase-1; pka, protein kinase a; p450scc, p450 side chain cleavage enzyme; psf, protein-associated splicing factor; pvdf, polyvinylidene difluoride; ripa, radioimmunoprecipitation assay; rnai, rna interference; scap, srebp-cleavage activating protein; sds, sodium dodecyl sulfate; sf-1, steroidogenic factor-1; sirna, all interfering rna; sk, sphingosine kinase; , sphingomyelin; ase, sphingomyelinase; s1p, sphingosine-1-phosphate; sph, d-erythro-sphingosine; sre, sterol regulatory element; srebp, sterol regulatory element binding protein; star, steroidogenic acute regulatory; tsa, trichostatin a.

accepted for publication november 14, 20xx.

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第四篇 Tracing from Fat Tissue, Liver, and Pancreas: A Neuroanatomical Framework for the Role of the Brain

netherlands institute for brain research (f.k., y.s.k., c.v.h., j.v.d.v., a.k., r.m.b.), 1105 az amsterdam, the netherlands

department of endocrinology and andmetaboli (f.k., h.p.s., e.f.), academic medical center, university of amsterdam, 1100 dd amsterdam, the netherlands

department of endocrinology and metaboli (f.k., j.a.r.), leiden university medical center, 2300 rc leiden, the netherlands

friedrich-loeffler-institute (t.c.m.), federal research institute for animal health, d-17493 insel riems, germany

abstract

the hypothalamus uses hormones and the autonomic nervous system to balance energy fluxes in the body. here we show that the autonomic nervous system has a distinct organization in different body compartments. the same neurons control intraabdominal organs (intraabdominal fat, liver, and pancreas), whereas sc adipose tissue located outside the abdominal compartment receives input from another set of autonomic neurons. this differentiation persists up to preautonomic neurons in the hypothalamus, including the biological clock, that have a distinct organization depending on the body compartment they command. moreover, we demonstrate a neuronal feedback from adipose tissue that reaches the brainstem. we propose that this compartment-specific organization offers a neuroanatomical perspective for the regional malfunction of organs in type 2 diabetes, where increased insulin secretion by the pancreas and disturbed glucose metaboli in the liver coincide with an augmented metabolic activity of visceral compared with sc adipose tissue.

introduction

to balance energy fluxes, the brain needs a precise and clear view on the metabolic state of the body. circulating humoral factors, in fact, are averaged whole body signals, whereas sensory neurons add detailed information from specific regions of the body to this global view. an early report indicates that information from sc fat tissue to the brain is being transported by the sympathetic nervous system (1). hence, the first part of this study addressed the question of to which extent adipose tissue acts as any other organ and provides neuronal feedback to the brain. we investigated the presence of sensory feedback to the central nervous system by injection of the neuronal tracer cholera toxin b (ctb) into intraabdominal adipose tissue.

the hypothalamus integrates peripheral signals delivered by the blood (fatty acids, glucose, and hormones) and by neuronal input from peripheral organs (2). this hypothalamic integration of peripheral information results in a modulated metabolic state. for instance, fatty acids and insulin are sensed in the hypothalamus and inhibit endogenous glucose production by the liver, stimulate glycogen synthesis in muscle, and reduce food intake (3, 4, 5, 6, 7, 8, 9, 10). the biological clock, located in the suprachiaatic nucleus (scn), modulates the set point of the hypothalamus by oscillating its sensitivity to metabolic signals (11, 12, 13). the scn induces diurnal metabolic variations such as the rise of glucose and glucocorticoids in the early morning, known as the "dawn-phenomenon" (8, 9).

in general, in a chain of four events, the brain receives input, integrates it, and generates a hormonal and autonomic output that finally affects the peripheral organs. two steps in this chain are far less established than the others, namely the direct neuronal sensory information from the organs and the neuronal output of the hypothalamus to the organs. the exact mechani by which the hypothalamus directs the organs into the desired metabolic state is far from evident. adipose tissue, liver, and pancreas have been shown to receive sympathetic and parasympathetic innervation, but the organization of this autonomic outflow from the hypothalamus to metabolic organs has not been investigated (14, 15, 16, 17, 18).

to clarify the output of the brain to the abdominal cavity we yzed the vagal output to the abdomen by means of injecting ctb with different fluorescent labels in intraabdominal fat, liver, and pancreas. injecting the retrograde neuronal tracer pseudorabies virus (prv) into retroperitoneal and sc fat allowed us to determine the projections from hypothalamus and brainstem to these fat compartments. thus, we uncovered from the biological clock separate sets of neurons controlling sc and visceral adipose tissues. in contrast, located in the same body region, visceral adipose tissue, liver, and pancreas share the same vagal motor neurons. we propose that this region-specific organization contributes to the concord in malfunction of organs in type 2 diabetes mellitus (t2 diabetes), in which condition an increased insulin secretion by the pancreas and hepatic insulin resistance coincides with an augmented metabolic activity of visceral compared with sc adipose tissue (19, 20, 21, 22).

materials and methods

materials

all experiments were performed in male wistar rats (250350 g; harlan, zeist, the netherlands) according to the netherlands institute for brain research guidelines for animal experiments and with approval of the animal care committee of the royal netherlands academy of arts and sciences.

fat denervation

the sympathetic or parasympathetic fibers entering the retroperitoneal fat pad were cut, as described earlier by our group (14). for a detailed description including perioperative photos, please refer to the supplemental data published on the endocrine society’s journals online web site at endo.endojournals.org.

liver denervation

the liver was sympathetically denervated by a technique described earlier by buijs et al. (23). the bile duct was isolated from the portal vein complex. at the level of the hepatic portal vein, the hepatic artery, a branch of the celiac artery, branches into the hepatic artery proper and the gastroduodenal artery. this division occurs on the ventral surface of the portal vein. at this point, the arteries were separated from the portal vein via blunt dissection. the nerve bundles running along the hepatic artery proper were removed using microsurgical techniques. then that part of the hepatic artery was closed by surgical thread on two sides and cut in between the knots in such a way that all sympathetic nerves were sectioned.

ctb tracing

two microliters of ctb (2%, sigma-aldrich, st. louis, mo; no. c167) or ctb-alexa fluor 488/555/647 (1%, molecular probes, eugene, or; no. c22841/c22843/c22844) were injected in retroperitoneal fat, liver, or pancreas using a 30-gauge needle connected to a hamilton syringe at a single spot. in a control experiment, ctb was applied on top of the intact or totally denervated organ.

three, 4, or 5 d after tracer injection, the animals were first perfused with saline and then with a solution of 4% paraformaldehyde and 0.15% glutaraldehyde in pbs (ph 7.4). they were postfixed overnight and cryoprotected by immersion with 30% sucrose in 0.2 m pbs (ph 7.4) for a further 24 h. brains were frozen and coronal sections (40 μm) were cut. after rinsing in 0.05 m tris-buffered saline (ph 7.4), brains with ctb sections were incubated overnight at 4 c with polyclonal rabbit anti-ctb (sigma-aldrich; no. c3062), then incubated for 60 min in the secondary antibody, biotinylated goat antirabbit (vector laboratories inc., burlingame, ca), followed by incubation in abc complex (vector laboratories inc.). finally, the sections were reacted with 0.025% 3,3-diaminobenzidine tetrahydrochloride-nickel in tris-buffered saline containing 0.5% h2o2. the light microscopy color figures were imported using a zeiss axioplan 2 microscope (zeiss, jena, germany) fitted with a progress camera 3012 (jenoptik, jena, germany). the figures were of 1488 x 1120 pixel size in rgb 24-bit true color. contrast and color were adapted using adobe photoshop (adobe systems inc., mountain view, ca) without any other image manipulation. brain sections with ctb-alexa fluor were rinsed extensively in pbs (ph 7.2) and coverslipped in 50% pbs glycerol for examination under a philips (eindhoven, the netherlands) confocal laser-scanning microscope (l410/510). digital images of the fluorescent sections were obtained using filters that prevented cross talk of the fluorophores. figures were contrast-enhanced but not otherwise manipulated in adobe photoshop.

prv tracing

five microliters prv-bartha (5 x 106 plaque-forming units; a generous gift of c. e. jacobs from the institute for animal science and health, lelystad, the netherlands), prv b80 (5 x 107 plaque-forming units prv -galactosidase b80; institute for molecular biology, insel riems, germany), or prv green fluorescent protein (gfp) (5 x 107 plaque-forming units prv gfp; institute for molecular biology) were injected into liver, sc inguinal or retroperitoneal fat using a 30-gauge needle connected to a hamilton syringe at a single spot. in a control experiment, prv was applied on top of the intact or the totally denervated organ.

three, 4, or 5 d after tracer injection, the animals were first perfused with saline and then with a solution of 4% paraformaldehyde and 0.15% glutaraldehyde in pbs (ph 7.4). [for a discussion on survival times, see buijs and colleagues (16)]. they were postfixed overnight and cryoprotected by immersion with 30% sucrose in 0.2 m pbs (ph 7.4) for a further 24 h. brains were frozen and coronal sections (40 μm) were cut. sections were incubated overnight at 4 c with a polyclonal mouse anti-prv bartha (a generous donation of c. e. jacobs), rabbit-anti gfp (molecular probes), or mouse-anti galactosidase (sigma-aldrich), depending on the tracers used, and then with a secondary antibody for 60 min for ysis under a confocal laser-scanning microscope (see ctb tracing).

results

adipose tissue feeds back to nociception-related central structures

two microliters of 2% ctb solution was either injected into (n = 15) or, as control, applied onto retroperitoneal fat in rats (n = 5). consequently, spinal cord, brainstem, and hypothalamus were stained for ctb. the absence of ctb-label in the central nervous system of rats that received an injection into a completely denervated fat pad or a topical application of ctb onto fat tissue served as a control and excluded false-positive results due to leakage. in six animals, the neurons in vagal motor nuclei and nerve endings in the gracile nucleus were positive, but no other areas, whereas nine did not show any central ctb (fig. 1).

liver, pancreas, and intraabdominal fat share one set of vagal motor neurons, intraabdominal or sc fat do not

two microliters of 1% ctb-488 (fluor-conjugate), ctb-555, or ctb-647 was injected alternately into liver and pancreas (n = 8). in the control group, rats received ctb-fluor-conjugate into the vagal denervated liver or pancreas (n = 6). because the fluorescence signal in the cns was absent in the control groups, false-positive results due to leakage could be excluded. in the dorsal motor nucleus of vagus (dmv), labeled vagal motor neurons contained both fluorescent labels from liver and pancreas. in a second set of experiments, the liver was sympathetically denervated as described earlier and injected with 5 μl of the retrograde tracer prv, and 2 μl of 2% ctb was injected into retroperitoneal fat (n = 12) (23). in the control groups, injection of prv or ctb into the denervated liver or fat pad or application onto liver or fat pad did not result in labeling of the cns (n = 7). in the intervention group, all vagal motor neurons projecting to retroperitoneal fat labeled with ctb contained also prv from the liver (fig. 2; five animals with tracing of ctb and prv in the dmv). some vagal motor neurons were filled with prv only, indicating that more neurons might control the liver than retroperitoneal adipose tissue (fig. 2). earlier, we reported that sc and intraabdominal fat pads are controlled by separate sets of vagal motor neurons (14).

distinct sets of hypothalamic and amygdalar neurons project to either intraabdominal or sc adipose tissue

five microliters of prv b80 or prv gfp was injected into intraabdominal or sc adipose tissue. injecting prv into completely denervated intraabdominal fat or applying the tracer on top of intraabdominal or sc adipose tissue did not result in labeling of the cns (n = 6). after parasympathetic denervation of both the right and left retroperitoneal fat pad, prv b80 and prv gfp were injected alternately (n = 32). in animals with a comparable infection rate of both tracers (n = 9), neuronal colocalization of both tracers exceeded 95%, both in second order infection of prv in paraventricular nucleus (pvn) and medial preoptic area (mpo) (n = 5) as well as in third order infection in scn and amygdala (n = 4). this demonstrates that the used viruses have the capacity to infect, simultaneously or shortly after each other, the same neuron; in addition, it indicates a shared control of both intraabdominal fat pads. parasympathetic (n = 37) denervation of the left retroperitoneal fat pad and alternate injection of prvb80 or gfp in this fat pad and sc fat, forced the virus to infect the brain via the sympathetic motor neurons only and allowed us to investigate whether these different sympathetic neurons receive input from different or the same preautonomic neurons. now, instead of major overlap as found with infection via functionally the same fat pads, none or only sparse overlap of both tracers (a maximum of one neuron per section) could be observed in animals with comparable infection (fig. 3) (pvn/mpo, n = 6; scn/amygdala, n = 5). earlier we showed that sympathetic motor neurons are specialized in intraabdominal or sc fat pads (14). thus, the projections of the pvn, mpo, scn, and amygdala are specialized by body region.

discussion

neuronal feedback from adipose tissue

several fat-derived humoral factors have been demonstrated to affect the brain. for instance, the hormone leptin acts on the hypothalamus and other brain regions, inhibits food intake, and stimulates sympathetic nerve activity (24). other studies have shown that free fatty acids inhibit endogenous glucose production by the liver via the hypothalamus (5, 6, 7). sympathetic feedback from sc fat tissue has been demonstrated earlier by labeling of a retrograde tracer in the dorsal root ganglia. however, in our tracing study from intraabdominal fat tissue, no nerve endings were found in the dorsal horn of the spinal cord (1). the presence of primary afferent projections from adipose tissue to the gracile nucleus of the brainstem not only presents evidence of neuronal feedback of fat tissue but also opens the question of the functional role of this feedback. the gracile nucleus receives afferent signals from the whole body and has a role in nociception (25, 26, 27, 28, 29, 30, 31). earlier studies demonstrated primary afferents from various organs to the gracile nucleus, for instance from sc tissue of primates, as well as from the hind limb, splanchnic nerve, pelvic nerve, and pudendal nerve in rats (27, 30, 31, 32, 33). in view of this pain-related feedback, the anatomical position of adipose tissue within the body suggests a function in monitoring skin and visceral organs. earlier, dermatologists had suggested a role of sc fat tissue in the perception of pain (34).

the afferents could sense mechanical, temperature, or hormonal stimuli such as cytokines not only under the skin, but also from the viscera (35). few studies have addressed nociception in brown and white fat tissue. it has been shown that capsaicin-sensitive fibers are present in brown adipose tissue (36). the nociceptive function of the afferents is supported by experiments in rats where capsaicin was injected into white sc fat tissue on the back. as a consequence, skin lesions appeared 10 d later on the back but also in the neck, suggesting a reaction mediated by the autonomic nervous system (ans) (37). fat pads in the knee joint and around spine ligaments contain nociceptive substance p fibers (38, 39, 40). recently, a study demonstrated the induction of local and referred pain by injection of saline into the infrapatellar knee fat pad (41).

thus, nociceptive fibers from adipose tissue to the gracile nucleus might sense mechanical stress or paracrine factors. consequently, the present study shows that adipose tissue has equal hormonal and neuronal access to the brain just as other metabolic organs.

shared (pre-) autonomic output links intraabdominal obesity to diabetes

recently, several studies reported early dysfunction of the ans in the development of t2 diabetes (42, 43, 44, 45, 46). other publications demonstrate a link between cardiovascular disease or insulin resistance in muscle and sympathetic overweight (47, 48). in contrast, hyperinsulinemia, obesity, and fatty liver are connected to parasympathetic overweight (49, 50, 51, 52). thus, the ans might have a different tone in different parts of the body at the same time. however, when the local status of the ans in a certain region is understood as an indicator of autonomic balance of the whole body, the picture becomes confusing; some authors find a high sympathetic tone, others find a high parasympathetic tone, and a third group finds low sympathetic and parasympathetic tone in patients with t2 diabetes (53, 54, 55, 56).

our experiments show a neuronal network that might control the body per compartment. these observations indicate that the brain may group the organs by anatomic location. we reveal a shared parasympathetic control of the abdomen that might connect one single neuron to visceral fat growth, hyperinsulinemia, and a fatty liver by a parasympathetic overweight (57). using the first order tracer ctb, we demonstrate that liver, pancreas, and intraabdominal fat indeed share the same vagal motor neurons. in contrast, distinct sets of vagal motor neurons project either to intraabdominal or to sc fat (14).

moreover, we describe the output of hypothalamus and limbic system to the intraabdominal and sc compartment using two different labels of the transneuronal retrograde tracer prv. tracing from sc fat tissue results in a strong (pre-) sympathetic picture, with much slower development of parasympathetic labeling, and in the controls with prv tracers injected into sc and sympathetically denervated retroperitoneal fat, no colocalization was found (14). in consecutive groups with increasing survival times, we yzed sequentially first order neurons in the sympathetic motor nuclei, then upstream second order neurons in the hypothalamus, and third order neurons in the hypothalamus and amygdala, and found them separated on all levels.

these findings are in agreement with earlier studies that suggest that higher brain regions such as the hypothalamus affect body fat distribution. lesions rostral from the autonomic motor neurons in the midbrain and lh lead to a different body fat distribution than lesions in the ventromedial hypothalamus (58). as to the function of such differentiation, it has been proposed that the hypothalamic temperature center, the mpo, might selectively activate the projections to the intraabdominal compartment to mobilize energy in times of low food and low temperature by burning specifically visceral fat which means that the isolation layer of the body, the sc fat, can then be spared (59, 60, 61, 62, 63).

in the amygdala, we revealed separated groups of neurons projecting either to the intraabdominal or to the sc body compartment. earlier studies showed that amygdala lesions lead to a change in body composition in favor of fat, hyperinsulinemia, and impaired skin conduction (64, 65). the direct connections to brain regions that process ell and taste suggest that the amygdala prepare the body for upcoming food. when food is detected by the nose, a specific parasympathetic activation of the intraabdominal compartment by the amygdala might induce secretion of insulin and enhance the uptake of substrate in fat tissue and liver.

our group proposed a role for the biological clock in the metabolic syndrome, where disturbed circadian rhythms play a prominent role, such as hormonal rhythms, a less pulsatile insulin secretion or a reduced dipping of the heart rate at night (49, 57, 66, 67, 68). here, we show that indeed a somatotopic organization exists up to the biological clock (scn) in the hypothalamus. possibly, this neuronal network might coordinate the dawn-phenomenon, where enhanced glucose production by the liver and high insulin levels coincide with enhanced glucose uptake of the target organs at the beginning of the active phase of the day (69, 70, 71). inappropriate timing and protracted activation of the shared vagal input to intraabdominal compartment (intraabdominal fat, pancreas, and liver) might lead to intraabdominal obesity, hyperinsulinemia, and a fatty liver. at the same time, an enhanced sympathetic activation in the thorax compartment and to the vasculature of the muscles might induce cardiovascular disease and insulin resistance. in the current thinking, the proposed failure of the brain in t2 diabetes might be caused by a genetic or developmental defect (72). however, probably in a majority of obese patients, the main cause of the system failure is a huge "environmental mutation" of our lifestyle. overeating without compensating for this by physical activity might induce confusing feedback to the brain (73, 74, 75).

because energy homeostasis is warranted by countless mechanis in our body, it is unlikely that one single cause of t2 diabetes will be identified. unbalanced food intake is not an endocrinological disease that could be cured by a replacement therapy of one single hormone. the step from a physiological buildup of energy stores to a metabolic derailment and t2 diabetes might occur at many points of the system. in our opinion, future experiments that address the physiological relevance of the neuroanatomical network established in the present paper should incorporate the cross talk between blood-borne factors and neurons in their experimental design.

footnotes

first published online december 8, 20xx

abbreviations: ans, autonomic nervous system; ctb, cholera toxin b; dmv, dorsal motor nucleus of vagus; gfp, green fluorescent protein; mpo, medial preoptic area; prv, pseudorabies virus; pvn, paraventricular nucleus; scn, suprachiaatic nucleus; t2 diabetes, type 2 diabetes.

accepted for publication november 17, 20xx.

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第五篇 Liver Triglyceride Secretion and Lipid Oxidative Metabolism Are Rapidly Altered by Leptin in Vivo_内科

【关键词】  metaboli

department of medicine, division of endocrinology (w.h., n.d., a.b., r.m.o.)

department of molecular genetics and biochemistry (r.m.o.), university of pitturgh, pitturgh, pennsylvania 15261

cardiovascular research group, university of alberta (g.d.l.), edmonton, alberta, canada t6g 2s2

abstract

leptin has potent lipid-lowering effects in peripheral tissues and plaa that are proposed to be important for the prevention of cellular lipotoxicity and insulin resistance. the current study addressed in vivo the effects of acute leptin delivery on liver triglyceride (tg) metaboli, the consequence of hepatic leptin action on whole-body tg homeostasis, and the mechanis of leptin action. a 120-min iv leptin infusion (plaa leptin, 14 ng/ml) decreased liver tg levels (53 ± 3%; p = 0.001), but not skeletal muscle tg levels, and increased liver phosphatidylinositol 3-kinase activity (341 ± 95%; p = 0.01) in lean rats. leptin had no effect on liver tg levels or phosphatidylinositol 3-kinase activity in diet-induced obese rats. in lean animals, leptin decreased the plaa tg concentration (20 ± 7%; p = 0.017), the rate of tg accumulation in plaa after tyloxapol administration (26 ± 6%; p = 0.003), and tg secretion from isolated liver (51 ± 8%; p = 0.004). to determine possible metabolic fates of depleted hepatic tg, we assessed leptin effects on liver oxidative metaboli. leptin increased hepatic acetyl-coenzyme a carboxylase phosphorylation (85 ± 13%; p = 0.006), fatty acid oxidation (49 ± 7%; p = 0.001) and ketogenesis (69 ± 15%; p = 0.004). finally, intracerebroventricular delivery of leptin for 120 min had no effect on liver tg levels, but did increase signal transducer and activator of transcription 3 phosphorylation (162 ± 40%; p = 0.02). these data present in vivo evidence for a role for leptin in the acute regulation of hepatic tg metaboli, and whole body tg homeostasis. a likely contributing mechani for these effects is leptin-induced partitioning of tg into oxidative pathways.

introduction

leptin-deficient states resulting from leptin gene mutations or lipodystrophy in rodents and humans are characterized by hyperlipidemia, excessive storage of lipid in tissues such as liver and skeletal muscle, and insulin resistance. these defects are markedly improved by the administration of leptin in humans and rodents (1, 2, 3, 4, 5, 6), establishing leptin as an important regulator of lipid metaboli and insulin sensitivity. in a previous study (7) we demonstrated that a 90-min perfusion of isolated rat liver with leptin was sufficient to decrease triglyceride levels by approximately 50%. these effects were absent in diet-induced obese (dio) rats, a situation of elevated liver and plaa triglyceride (tg) concentrations, establishing hepatic leptin resistance as a potential mechani contributing to dyslipidemia in obesity.

a number of questions arose from these observations. first, because our previous observations were made in a perfused liver model, the acute in vivo effects of leptin on liver tg levels are unclear. this is an important issue, because the regulation of liver tg metaboli in vivo occurs in a neural, nutrient, and hormonal milieu that is disrupted in the isolated liver. second, the rapid depleting effects of leptin on liver tg levels suggest that leptin may play a role in the acute regulation of hepatic lipoprotein metaboli. tg export to peripheral tissues from the liver as a component of very low-density lipoprotein (vldl) is important for the maintenance of normal lipid homeostasis. however, dysregulation of lipoprotein metaboli is common in obesity, resulting in a so-called atherogenic lipid profile [large vldl and all, dense low-density lipoprotein (ldl)]. interestingly, the plaa tg concentration is markedly decreased by chronic leptin treatment in leptin-deficient humans and in rodents (1, 2, 8, 9, 10). however, it is unclear what role, if any, leptin-induced alterations in tg secretion may play in reducing plaa tg. finally, the mechanis responsible for the depleting effects of leptin on liver tg levels were not addressed. in skeletal muscle and heart, leptin acutely increases fatty acid oxidation (11, 12, 13, 14, 15, 16, 17), an effect that in skeletal muscle is a consequence of decreased acetyl-coenzyme a carboxylase (acc) activity mediated by increased ampk activity (16). leptin-induced increases in oxidative metaboli have also been demonstrated (18, 19) or implied (20) in other tissues, but the effects of leptin in liver are unknown. the current study was undertaken to address each of these issues. specifically, we determined in vivo the effects of acute leptin delivery on liver tg metaboli, the consequence of hepatic leptin action on whole-body triglyceride homeostasis, and the potential mechanis of leptin action on liver tg levels. the data demonstrate that iv delivery of leptin for 120 min substantially decreases liver tg levels and activates phosphatidylinositol 3-kinase (pi3-kinase) in lean, but not dio, rats; that leptin decreases liver tg secretion and increases hepatic fatty acid oxidation and ketogenesis (most likely by inhibition of acc activity), and that 120 min of intracerebroventricular (icv) leptin administration does not decrease liver tg levels.

materials and methods

animal care and maintenance

male wister rats were purchased from charles river laboratories (madison, wi) at a weight of 175200 g. after arrival, rats were maintained on a constant 12-h light, 12-h dark cycle with free access to water and were fed ad libitum a standard rat chow diet (11% of calories from fat) or a high-fat diet (td 96001, harlan teklad, madison wi; 45% of calories from fat) for 56 wk. all materials and methods were approved by the institutional animal care and use committee of university of pitturgh and were performed in accordance with the national research council’s guide for the care and use of laboratory animals.

implantation of chronic indwelling catheters

animals were anesthetized with a :xylazine:acepromazine mix (60 mg/kg , 5 mg/kg, xylazine, and 1 mg/kg acepromazine). catheters (pe-50, intramedic, bd biosciences, sparks, md) were introduced into the left carotid artery (advanced to the aortic arch) and the right jugular vein (advanced to the right atrium) as previously described (9, 21). the catheters were exteriorized at the back of the neck, filled with a sterile glycerol:heparin mix (3:1), and flame-sealed. the wounds were closed with sutures and treated with betadine. the animals were treated with ketoprofen (2 mg/kg, sc), and their recovery was monitored with special attention given to food intake, weight gain, and healing of wounds. animals were allowed at least 4 d to recover, and only those that had achieved more than 90% of presurgery weights were used in the studies.

implantation of third cerebral ventricle cannula

anesthetized animals were placed on a stereotactic apparatus. the incisor bar was set at 3.3 mm. the skull was exposed so that cranial sutures could be viewed. a steel guide cannula was inserted at coordinates from bregma of 2.8 mm anterior/posterior, 0.0 mm medial/lateral from the midsagittal suture, and 8.4 mm ventral to dura according to the brain atlas (22, 23). three screws placed approximately 5 mm from the cannula insertion site and reinforced with dental cement secured the cannula in place. after checking for cerebrospinal fluid return, a dummy cannula was inserted to prevent clogging of the guide cannula. the skin was closed and sutured over the mound of the dental cement. antibiotic treatments and recovery procedures were the same as described above.

liver perfusions

the perfusion equipment and procedures were described previously (7) with modifications. briefly, livers isolated from lean postprandial rats were perfused with krebs-henseleit buffer, which contained 15 mm d-(+)-glucose, 1.7 mm fructose, 2 mm l-lactate, 0.2% bsa, and 0.2 mm palmitate in a recirculating system at a rate of approximately 2.7 ml/min/g liver. additions of recombinant rat leptin (r&d systems, inc., minneapolis, mn) or vehicle together with human insulin (eli lilly & co., indianapolis, in) were accomplished by admixing into the portal vein perfusate using a syringe pump as described previously (7). this administration regimen gave constant perfusate concentrations of leptin (14 ng/ml) and insulin (1 mu/ml).

experimental design

effects of iv leptin delivery on liver tg levels.

conscious 18-h fasted rats received a continuous iv infusion of either vehicle or leptin using a syringe pump (model 11, harvard apparatus, natick, ma) at a rate of 0.2 μg/kg・min for 120 min (preceded by a 2-min priming dose of 2 μg/kg・min) or for 15 min in the experiments designed for measuring liver pi3-kinase activity and akt phosphorylation. volume delivery was 5 ml/kg・h for both vehicle and leptin infusions. blood samples were taken before infusions and at 120 min, the rats were then anesthetized, and liver and skeletal muscle (soleus) were promptly removed, snap-frozen in liquid nitrogen, and stored at 80 c until ysis.

effects of leptin delivery on liver tg secretion.

three experiments were preformed to address this question. in the first experiment, the plaa tg concentration was measured at the end of a 120-min leptin infusion. a second experiment assessed the rate of tg accumulation in blood after the administration of tyloxapol (triton wr1339, sigma-aldrich corp., st. louis, mo), an inhibitor of tg clearance (24, 25). specifically, leptin or vehicle was infused into lean rats for 120 min, then all animals received a bolus injection of tyloxapol (300 mg/kg). immediately before tyloxapol administration and 45 and 90 min after tyloxapol administration and a continuing leptin infusion, blood samples were taken and apolipoprotein b (apob)-containing lipoproteins were precipitated as described below. subsequently, the tg content was measured. because the vast majority of tg is associated with the apob-containing vldl particle, we refer to these tg measurements as vldl-tg. preliminary experiments determined that the rate of tg appearance in blood was linear for at least 90 min after tyloxapol administration (data not shown). vldl-tg secretion rates were calculated from the slope of the line after tyloxapol injection, based on the assumption of a plaa volume of 3.5% of body weight. in a third experiment, the secretion of tg from isolated livers was assessed directly. livers were perfused as described above for 90 min in the absence or presence of leptin. subsequently, aliquots of perfusate were taken, and vldl-tg was measured after precipitation of apob-containing lipoproteins as described below.

effects of leptin on hepatic fatty acid oxidation and ketogenesis.

leptin or vehicle was infused iv for 120 min into 18-h fasted animals. subsequently, four pieces of liver weighing approximately 200 mg each were isolated; each piece of tissue was then further divided into four pieces of approximately 50 mg and immediately incubated with 9,10-[3h]palmitate for the measurement of fatty acid oxidation, as described below. to assess ketogenesis, isolated livers were perfused for 90 min in the absence or presence of leptin and 0.2 mm palmitate. subsequently, aliquots of the perfusate were taken, and ketone bodies were measured.

effects of acute icv delivery of leptin on liver tg levels.

conscious 18-h fasted rats received a continuous icv infusion of leptin (1 μg/kg・h) or artificial cerebrospinal fluid at a rate of 2 μl/h. at the end of the 120-min infusion, the rats were anesthetized, and the hypothalamus and liver were promptly removed, snap-frozen in liquid nitrogen, and stored at 80 c until ysis.

tissue, plaa, and perfusate measurements

tissue tg levels were determined as described previously (7, 9). briefly, approximately 50 mg frozen liver tissue was extracted in 1 ml of a chloroform-methanol mix (2:1). after redissolving the lipid pellet in 60 μl tert-butanol and 40 μl of a triton x-114-methanol (2:1) mix, tg were measured spectrophotometrically (du 530, beckman coulter, fullerton, ca) using the glycerol phosphate oxidase-tg kit and lintrol lipids as standard (sigma-aldrich corp.). fatty acid oxidation was assessed in liver sections previously exposed to leptin in vivo for 120 min as described above. liver sections (200 mg) were incubated in 2 ml of a culture medium containing 10 mm hepes, 5 mm glucose, and 0.2 mm 9,10-[3h]palmitate in a shaking water bath at 37 c for 2 h. palmitate oxidation was assessed by measuring the quantity of tritiated water released into the medium, as previously described (19, 26). briefly, at the end of the 2-h incubation, 200 μl medium was transferred to an open 1.5-ml tube, the tube was placed into a capped scintillation vial containing 0.5 ml unlabeled water, and the vial was kept at 50 c for 18 h. during this period, tritiated water in the tube equilibrated with unlabeled water in the vial. to determine the equilibration coefficient, 200 μl medium containing a known quantity of tritiated water (10 μci) was incubated similarly to other samples. after the 18-h incubation, 10 ml scintillation fluid was added to the vials, and the samples were counted in duplicate in a beckman scintillation counter. the rate of palmitate oxidation was calculated from the specific activity of the starting fatty acid solution.

apob-containing lipoproteins in perfusate (8 ml) or plaa (200 μl; 1:3 diluted with saline) were precipitated by adding 5% 2 g/liter dextran sulfate and 10% 2.25 m magnesium sulfate (27). after gentle mixing, a 10-min incubation at room temperature, and centrifugation at 2000 x g for 10 min, the supernatant was removed, and the pellet was dissolved in 200 μl of a high-salt buffer (containing 2 m nacl, 2 mm edta, and 50 mm napi), 200 μl tert-butanol, and 100 μl of a methanol-triton x-100 mixture (1:1), each added separately followed by a 30-sec vortex. after centrifugation at 4000 x g, tg in the upper phase of the supernatant was yzed spectrophotometrically as described above. -hydroxybutyrate in perfusate samples was measured enzymatically using the -hydroxybutyrate liquicolor kit (stanbio, boerne, tx).

leptin was measured using a rat-specific ria kit (linco research, inc., st. charles, mo). pi3-kinase activity and insulin receptor substrate-1 (irs-1) tyrosine phosphorylation were assayed as previously described (7). hypothalamic phospho-signal transducer and activator of transcription 3 (phospho-stat3) and total stat3, hepatic phospho-akt (ser473), akt, and hepatic phospho-acc (ser79) were measured using standard immunoblotting techniques [-phospho-stat3 catalogue no. 9131, -stat3 catalog no. 9132; -phospho-akt catalog no. 9271, and -akt catalog no. 9272 (cell signaling technology, beverly, ma); and -phospho-acc catalog no. 07-303 (upstate biotechnology, inc., lake placid, ny); all used at a 1:1000 dilution]. immunoblots were quantified by densitometry.

5'-amp-activated protein kinase activity (ampk) was measured by quantifying the incorporation of 32p into a synthetic substrate peptide amaraasaaalarrr as previously described (28). briefly, ampk was extracted from a liver homogenate using the polyethylene glycol precipitation method (28). the protein concentration was measured colorimetrically. subsequently, ampk activity was measured in a 25-μl total volume in the presence of a buffer containing 40 mm hepes-naoh (ph 7.0), 80 mm nacl, 8% glycerol, 0.8 mm edta, 0.8 mm dithiothreitol, and [-32p]atp/mg at 30 c for 10 min.

statistical methods

all results are expressed as the mean ± se. statistical significance was determined by unpaired t test using the systat statistical program (evanston, il). statistical significance was assumed at p < 0.05.

results

acute iv leptin delivery substantially decreases liver tg levels and activates pi3-kinase in lean, but not dio, rats

in a previous study (7) we demonstrated that leptin rapidly decreases liver tg levels in a perfused liver model and that these effects are absent in diet-induced obesity. however, it is unknown whether similar effects or defects of leptin action on liver tg levels are present in vivo. furthermore, it is unclear whether these rapid effects on liver tg levels are replicated in other peripheral tissues, such as skeletal muscle. to address these issues, leptin was infused into lean animals (body weight, 286 ± 5 g) at a rate that increased the plaa leptin concentration from 0.4 ± 0.1 to 14.2 ± 1.2 ng/ml (fig. 1), values within the physiological range of leptin concentrations in rodents. this plaa leptin concentration decreased liver tg levels by 53 ± 3% (p = 0.001) compared with vehicle-infused controls (fig. 2a). furthermore, leptin increased liver pi3-kinase activity (fig. 2b) by 341 ± 95% (p = 0.01) and irs-1 tyrosine phosphorylation by 393 ± 136% (p = 0.03), consistent with our previous observations in isolated perfused liver (7). interestingly, leptin did not decrease skeletal muscle tg levels (fig. 2a). in dio rats (body weight, 462 ± 10 g), hepatic tg levels were increased by 129 ± 6% compared with standard chow-fed controls [p < 0.0001; fig. 2 (leptin ) vs. fig. 3 (leptin )], and the fasting plaa leptin concentration was increased by approximately 10-fold compared with lean animals (fig. 1, 0 min point). however, a leptin infusion that resulted in a plaa concentration of 22.9 ng/ml had no effect on liver tg levels, the activity of liver pi3-kinase, or irs-1 tyrosine phosphorylation in dio rats (fig. 3).

leptin decreases liver tg secretion

whole body tg homeostasis is critically dependent on the secretion of tg as components of the apob-containing vldl particle. the demonstration that leptin decreases liver tg levels in lean animals raises the possibility that leptin may alter tg secretion from liver. thus, we next undertook three experiments to address this hypothesis (fig. 4). first, the plaa tg concentration was determined subsequent to a 120-min leptin infusion (fig. 4a). tg concentrations in leptin-infused animals were reduced by 20 ± 7% compared with vehicle-infused animals (p = 0.017). to determine whether leptin was decreasing tg appearance or disposal, we next inhibited the clearance of plaa tg using tyloxapol and subsequently measured the appearance of tg in plaa in the absence or presence of a leptin infusion. leptin infusion in the presence of tyloxapol reduced the rate of vldl-tg appearance in plaa from 58 ± 3 to 43 ± 4 mg/kg・min (p = 0.003, leptin vs. vehicle; fig. 4b). finally, to rule out changes in the tg content of other apob-containing lipoproteins and other possible confounding in vivo variables, we performed an experiment that assessed the effects of leptin on tg secretion from isolated perfused liver (fig. 4c). as suggested by our in vivo experiment, leptin decreased perfusate vldl-tg by 51 ± 8% (p = 0.004, leptin vs. vehicle).

leptin increases lipid oxidative metaboli in liver

we have demonstrated that leptin decreases total liver tg and tg secretion. however, these observations give no indication of the metabolic fate of the depleted liver tg. it is well established that leptin increases fatty acid oxidation. thus, one potential mechani to decrease liver tg would be to increase the rate of fatty acid oxidation subsequent to tg hydrolysis. therefore, we next examined the effects of leptin on the activity of lipid oxidative pathways in liver. livers taken from animals infused with leptin for 120 min oxidized exogenous palmitate at a rate 49 ± 7% (p = 0.001) greater than the rate of fatty acid oxidation in livers taken from vehicle-infused animals (fig. 5a). furthermore, leptin increased ketogenesis, another important oxidative pathway in liver, in perfused liver by 69 ± 15% (p = 0.004) compared with vehicle-perfused control livers (fig. 5b). in the same experiment we evaluated palmitate incorporation into lipids and found no differences between leptin- and vehicle-treated livers, suggesting that esterification of fatty acids into the tg pool was not altered by leptin (data not shown). increased rates of oxidation induced by leptin were matched by decreases in the activity (as measured by phosphorylation) of acc, demonstrating a biochemical mechani for leptin action on fatty acid oxidation (fig. 6c). we also examined potential biochemical mechanis of leptin action by assessing the responses of akt and ampk to a leptin stimulus. leptin increased phosphorylation of the pi3-kinase downstream target akt by 73 ± 15% (fig. 6a; p = 0.01). however, similar to our previous observations in isolated liver (7), leptin had no effect on ampk activity after a 120-min leptin stimulus (fig. 6b) or a 15-min leptin infusion (data not shown).

acute icv leptin delivery does not replicate the effects of iv leptin delivery on liver tg levels

a number of studies have demonstrated the effects of icv delivery of leptin on carbohydrate (29) and lipid (24, 30) metaboli. to determine the role of central mechanis in mediating the acute effects of leptin on liver tg levels in vivo, we delivered leptin by the icv route for 120 min. icv leptin delivery at a rate of 1 μg/kg・h increased hypothalamic stat3 phosphorylation by 162 ± 40%; p = 0.005) (fig. 7a), demonstrating activation of central pathways of leptin action, but did not decrease liver tg levels (fig. 7b). similar to lean rats, icv infusion of leptin in obese rats did not alter liver tg levels (data not shown).

discussion

the primary goal of the current study was to establish in vivo the role of leptin in the acute regulation of liver tg metaboli. a number of novel observations arise from these studies. we demonstrate, first, that iv delivery of leptin for 120 min substantially decreases liver tg levels and activates pi3-kinase in lean, but not dio, rats; second, that leptin decreases tg secretion from liver, implicating a role for leptin in the regulation of vldl metaboli; third, that leptin elevates hepatic fatty acid oxidation and ketogenesis, most likely by inhibition of acc activity; and finally, that icv leptin administration does not replicate the iv effects of leptin on liver tg levels.

liver tg metaboli in vivo is altered by hormonal, neural, and nutrient signals that regulate the metabolic pathways involved in the synthesis, degradation, and/or secretion of tg. in a previous study (7), we demonstrated that leptin rapidly decreases liver tg levels in the isolated perfused liver. the current study addressed whether there were similar effects of leptin in vivo when a physiological hormonal and metabolic milieu was present. we demonstrate that the extent of leptin effects on tg levels is the same in vivo and in the perfused liver (50% depletion in both models). furthermore, skeletal muscle tg were not reduced by leptin, suggesting that the effects of leptin were specific to liver. a note of caution should be attached to this conclusion, because acute stimulation of tg hydrolysis by leptin in isolated skeletal muscle has been reported (11, 13). however, we would point out that these studies were performed in an in vitro system (isolated muscle), and the dose of leptin used was 1000 times higher then that used in our study (10 μg/ml vs. 10 ng/ml). either of these issues may explain the lack of an effect observed in the current study on muscle tg. finally, mechanis similar to those responsible for the actions of leptin in the isolated liver are implicated in the action of leptin in vivo. thus, we previously demonstrated that the effects of leptin on liver tg levels are dependent on the activation of pi3-kinase. furthermore, muoio et al. (14) reported that the pi3-kinase inhibitor, wortmannin, attenuates the effects of leptin on skeletal muscle lipid metaboli. in the present study leptin substantially increased hepatic pi3-kinase activity and irs-1 tyrosine phosphorylation, and also increased the serine phosphorylation of akt, a major metabolic target of pi3-kinase activity. indeed, the activation of pi3-kinase occurs at a relatively early time point (15 min) in response to a leptin stimulus and is present at even earlier times (5 and 10 min; data not shown). these data confirm previous reports of leptin activation of pi3-kinase in liver (7, 31) and in primary hepatocytes (32). however, although this combination of observations is suggestive, a definitive demonstration that leptin acts through pi3-kinase to regulate liver tg metaboli in vivo is still required.

despite substantial progress, the effects of leptin resistance on the metabolic actions of leptin in vivo remain ill defined. a previous study (33) demonstrated decreased leptin regulation of hepatic glucose metaboli in rats on a high-caloric diet. the current study expands these observations to demonstrate that the acute effect of leptin on liver tg levels is absent in dio rats. these observations are in agreement with our previous study of isolated livers from dio rats and rats fed the high-caloric diet described above. the mechani of hepatic leptin resistance in dio is most likely post receptor, because hepatic leptin receptor expression is not decreased in dio rats (7). furthermore, in both isolated liver (7) and in vivo (current study), leptin does not activate pi3-kinase in dio rats. in the broader context of leptin resistance, our observations are consistent with the loss of the effects of leptin on fatty acid oxidation in skeletal muscle isolated from obese humans and dio rats (11, 34). taken together, these studies indicate that there is a substantial loss of leptin’s ability to regulate lipid and carbohydrate metaboli in obesity. furthermore, the data support the hypothesis that leptin resistance may contribute to the atherogenic plaa lipid profile (large vldl and all, dense ldl) and the inappropriate accumulation of lipid in peripheral tissues commonly found in obesity.

studies in normal animals (8, 9), leptin-deficient humans (1, 2), and transgenic mice (10) demonstrated substantial leptin-induced decreases in plaa tg. increased uptake of tg into peripheral tissues or decreased synthesis/export of tg from liver are two possible mechanis that could result in a decrease in the circulating concentration. the current study addressed the acute effects of leptin on the export of tg from liver, based on the hypothesis that a decrease in total liver tg levels may also decrease hepatic tg secretion. we offer three independent lines of evidence to support a role for leptin in the regulation of whole body tg and, by extension, vldl metaboli. we demonstrate, first, that plaa tg are decreased by approximately 20% in response to a 120-min leptin infusion; second, that the accumulation of vldl-tg after tyloxapol administration is reduced by approximately 26%, suggesting that it is liver export, rather than peripheral uptake, of tg that is altered by leptin; and third that leptin decreases tg secretion from the perfused liver by approximately 50%. we also addressed the effects of leptin on plaa apob levels and associated cholesterol, and found no differences between leptin- and vehicle-infused animals (huang, w., n. dedousis, and r. o’doherty, unpublished observations). however, these measurements included cholesterol and apob associated with ldl; for that reason, these data are not reported in the current study. additional studies are planned that will address the acute effects of leptin on intrahepatic vldl metaboli.

the current study clearly demonstrates that leptin increases the activity of oxidative pathways in the liver. thus, exogenous fatty acid oxidation by the liver was increased after a 2-h leptin infusion in vivo, and leptin increased ketogenesis in the isolated, perfused liver. these data raise a number of discussion points. first, our data are in good agreement with previous observations (7) demonstrating increased oxygen consumption in leptin-perfused livers and increases in fatty acid oxidation in skeletal muscle and cardiac muscle in response to leptin (11, 12, 13, 14, 15, 16, 17). second, one plausible explanation for the metabolic fate of the reduced liver tg level is the partitioning of fatty acids derived from tg into oxidative pathways. this hypothesis is supported by previous studies in skeletal muscle that demonstrate leptin-induced increases in tg hydrolysis and partitioning of fatty acids toward oxidation (12). also, in the current study the incorporation of palmitate into the total lipid pool, a large portion of which is fatty acid esterification into tg, was unaltered by leptin in the perfused liver (data not shown), suggesting that leptin stimulates the hydrolysis of tg in liver. third, despite the observation that leptin increased acc phosphorylation, ampk activity was unaltered. the current data are in agreement with our previous observations in perfused livers (7). in skeletal muscle there appears to be a clear relationship between leptin-induced increases in ampk activity and fatty acid oxidation (16). however, this is not the case in cardiac muscle (17). furthermore, leptin increased akt activity, as measured by elevated phosphorylation, an event that may be expected to decrease ampk activity, as previously reported in mouse heart (35). thus, the role of ampk in mediating the effects of leptin on fatty acid oxidation in the liver remains unclear. finally, the combined observations of leptin-induced decreases in liver tg levels and secretion, and increased oxidative metaboli suggest that tg partitioning to oxidative pathways contributes to the effects of leptin on tg homeostasis. this may have implications for a role for leptin in the regulation of the plaa lipoprotein profile and, perhaps more generally, the capacity of increased intrahepatic fatty acid oxidation to improve vldl profiles in hyperlipidemic states. in support of a role for leptin in the regulation of lipoprotein metaboli are the reports of acute and chronic effects of leptin on liver cholesterol (24, 36), bile acid (37), and high-density lipoprotein cataboli (38).

the role of the hypothalamus in mediating leptin action is well established. the most relevant observations in the context of the current study are the demonstrations that acute (5 h) icv delivery of leptin elevates hepatic gluconeogenesis and decreases glycogenolysis (29), that a single low dose of leptin delivered icv decreases the activity of 3-hydroxy-3-methyl-glutaryl-coenzyme a reductase and cholesterol 7-hydroxylase, enzymes that play important roles in the regulation of hepatic cholesterol metaboli (24), and that a 2-wk icv delivery of leptin substantially reduces hepatic steatosis in a mouse model of lipodystropy (30). based on these observations, it was somewhat surprising that icv delivery of leptin in the current study did not decrease liver tg levels. hypothalamic activation by leptin was apparent, because leptin increased stat3 phosphorylation. the lack of an effect of leptin could not be explained by the time of infusion, because this was the same for both icv and iv infusions (120-min), or by the leptin dose, because the icv infusion rate directly into the third ventricle (1 μg/kg・h) most likely resulted in a higher concentration of leptin in the hypothalamus than that obtained with an iv infusion (plaa leptin, 14 ng/ml), although direct measurements of leptin concentration in the hypothalamus were not possible. it should also be noted that the results obtained in the current study are consistent with those obtained in isolated perfused liver (7) and with the study by vanpatten et al. (24) that demonstrated unaltered liver tg levels in rats 12 h after receiving an icv leptin stimulus. finally, our data are in accord with a number of studies demonstrating metabolic effects of leptin in isolated peripheral tissues and in vivo, including observations that the expression of wild-type leptin receptors in zucker diabetic fatty rat liver decreases liver tg levels (39), and that administration of leptin to gold thioglucose obese mice that lack hypothalamic leptin function decreases hepatic lipogenesis (40).

in conclusion, the current study clearly establishes a role for leptin in the acute regulation of liver tg levels in vivo. leptin-induced decreases in tg secretion from the liver also suggests a role for leptin in determining the tg content of vldl, a critical determinant of the plaa lipoprotein profile. the mechanis responsible for decreased intrahepatic tg stores and tg secretion probably involve nutrient partitioning away from storage, because leptin increases the activity of fat oxidation pathways in liver.

acknowledgments

we thank cory wagg for his technical assistance.

footnotes

this work was supported by an american diabetes association career development award and national institutes of health grant ro1-dk-58855-01 (both to r.o.d.) and a university of pitturgh obesity and nutrition research center (p30dk462) pilot and feasibility award (to w.h.). w.h. is supported by national institutes of health grant t32-dk-07052 (research training in diabetes and endocrinology).

first published online december 8, 20xx

abbreviations: acc, acetyl-coenzyme a carboxylase; apob, apolipoprotein b; ampk, 5'-amp-activated protein kinase; dio, diet-induced obesity; icv, intracerebroventricular; irs-1, insulin receptor substrate-1; ldl, low-density lipoprotein; pi3-kinase, phosphatidylinositol 3-kinase; stat, signal transducer and activator of transcription; tg, triglyceride; vldl, very low-density lipoprotein.

accepted for publication november 28, 20xx.

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steinberg gr, dyck dj 2000 development of leptin resistance in rat soleus muscle in response to high-fat diets. am j physiol 279:e1374e1382

kovacic s, soltys cl, barr aj, shiojima i, walsh k, dyck jr 20xx akt activity negatively regulates phosphorylation of amp-activated protein kinase in the heart. j biol chem 278:3942239427

hyogo h, roy s, paigen b, cohen de 20xx leptin promotes biliary cholesterol elimination during weight loss in ob/ob mice by regulating the enterohepatic circulation of bile salts. j biol chem 277:3411734124

vanpatten s, ranginani n, shefer s, nguyen lb, rossetti l, cohen de 20xx impaired biliary lipid secretion in obese zucker rats: leptin promotes hepatic cholesterol clearance. am j physiol 281:g393g404

silver dl, jiang xc, tall ar 1999 increased high density lipoprotein (hdl), defective hepatic cataboli of apoa-i and apoa-ii, and decreased apoa-i mrna in ob/ob mice. possible role of leptin in stimulation of hdl turnover. j biol chem 274:41404146

lee y, wang my, kakuma t, wang zw, babcock e, mccorkle k, higa m, zhou yt, unger rh 20xx liporegulation in diet-induced obesity. the antisteatotic role of hyperleptinemia. j biol chem 276:56295635

bryson jm, phuyal jl, swan v, caterson id 1999 leptin has acute effects on glucose and lipid metaboli in both lean and gold thioglucose-obese mice. am j physiol 277:e417e422

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