This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Belsham, D. D. Articles by Shkreta, L. Search for Related Content PubMed PubMed Citation Articles by Belsham, D. D. Articles by Shkreta, L.

Generation of a Phenotypic Array of Hypothalamic Neuronal Cell Models to Study Complex Neuroendocrine Disorders

Departments of Physiology (D.D.B., F.C., H.C., S.R.S., A.M.F.S., L.S.), Obstetrics and Gyneacology (D.D.B.), and Medicine (D.D.B., A.M.F.S.), University of Toronto, and Division of Cellular and Molecular Biology (D.D.B.), Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada M5S 1A8

Address all correspondence and requests for reprints to: Denise D. Belsham, Ph.D., Department of Physiology, University of Toronto, Medical Sciences Building 3247A, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8. E-mail: d.belsham{at}utoronto.ca.

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Knowledge of how the brain achieves its diverse central control of basic physiology is severely limited by the virtual absence of appropriate cell models. Isolation of clonal populations of unique peptidergic neurons from the hypothalamus will facilitate these studies. Herein we describe the mass immortalization of mouse primary hypothalamic cells in monolayer culture, resulting in the generation of a vast representation of hypothalamic cell types. Subcloning of the heterogeneous cell populations resulted in the establishment of 38 representative clonal neuronal cell lines, of which 16 have been further characterized by analysis of 28 neuroendocrine markers. These cell lines represent the first available models to study the regulation of neuropeptides associated with the control of feeding behavior, including neuropeptide Y, ghrelin, urocortin, proopiomelanocortin, melanin-concentrating hormone, neurotensin, proglucagon, and GHRH. Importantly, a representative cell line responds appropriately to leptin stimulation and results in the repression of neuropeptide Y gene expression. These cell models can be used for detailed molecular analysis of neuropeptide gene regulation and signal transduction events involved in the direct hormonal control of unique hypothalamic neurons, not yet possible in the whole brain. Such studies may contribute information necessary for the strategic design of therapeutic interventions for complex neuroendocrine disorders, such as obesity.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
THE NEUROENDOCRINE HYPOTHALAMUS consists of a complex array of distinct neuronal phenotypes, each expressing a specific complement of neuropeptides, neurotransmitters, and receptors (1). Many of our vital needs, such as those for growth, reproduction, nutrition, sleep, and stress responses, depend on hormonal balance or homeostasis, which is controlled by both external and internal stimuli at the hypothalamic level. Numerous studies have been undertaken to map the afferent connections between distinct hypothalamic neurons using methodology such as double- and, recently, triple-label immunocytochemistry and in situ hybridization (2, 3, 4, 5). These studies are useful to generate an emerging picture of the potential cellular communication within the hypothalamus but are not comprehensive and do not address the mechanisms involved in gene regulation and cellular signaling. Historically it has proven to be difficult to establish immortalized hypothalamic cell lines, due to the lack of naturally occurring central nervous system tumors and the inherent difficulty of transforming or immortalizing highly differentiated neurons from primary culture (6). Cell lines from the peripheral nervous system have been established from neuroblastomas, such as the Neuro2A cell line, and pheochromocytomas, such as the PC12 cell line; however, these models are not truly representative of differentiated central nervous system neurons. Previously, infection of primary cultures of hypothalamic tissue from embryonic d 14 (E14) with SV40 large T-antigen (T-Ag) in the early 1970s produced cell lines that were not considered fully differentiated (7). However, a few cell models from the hypothalamus have been developed, and have proven to be extremely useful toward understanding the cellular biology of specific neuroendocrine cells (7, 8, 9, 10). For instance, the GT1–7 GnRH-secreting neuronal cell model has been used extensively over the past decade to delineate the basic control mechanisms involved in GnRH neuronal function (11), and a cell line from the suprachiasmatic nucleus has allowed the dissection of central clock mechanisms (12). However, the number of cell models from the hypothalamus, and from the entire brain for that matter, consist of a few isolated cell types and represent an infinitesimal percentage of the neuronal phenotypes represented within the brain. For this reason, we have used retroviral transfer of SV40 T-Ag into primary hypothalamic cell culture to generate an array of immortalized cell models from the hypothalamus. The mixed populations were subcloned and defined by expression of specific neuropeptides and receptors. The clonal cell models express neuronal cell markers, exhibit neurosecretory properties, and respond appropriately to hormonal stimulation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Primary cell culture, retroviral infection, and cell culture
Mice (BALB/c females and DC1 males from Charles River Laboratories, Wilmington, MA) were bred and an entire litter was harvested at E15, E17, or E18. All experimental protocols were approved by the Animal Care Committee of the University Health Network, Toronto General Hospital. Three to five hypothalami from embryonic mice were pooled, then dissociated into a single cell suspension by trituration and transferred to a new culture dish containing primary culture medium (DMEM), 10% heat-inactivated defined fetal bovine serum (FBS), 10% heat-inactivated defined horse serum, 1% penicillin-streptomycin, and 20 mM D-glucose (all from Life Technologies/Life Technologies, Inc., Rockville, MD). Osmolarity of the culture medium was adjusted to 320–325 mOsm with glucose. The cell suspension was split into two 75- to 80-cm² tissue culture flasks, coated with 100 µg/ml poly-L-lysine (Sigma Chemical Co., St. Louis, MO). Cells were allowed to attach to the flask for 24 h incubating at 37 C with 5% CO₂ before for infection with retrovirus.

The primary cells were incubated for 1 h, twice successively, with fresh virus-containing medium harvested from confluent culture of 2 cells (psitex cells) (13) producing a replication-defective, recombinant murine retrovirus. This virus, constructed by using the pZIPNeo SV(X) 1 vector, harbors the intact cDNA sequence for simian virus (SV40) large T-Ag and neomycin resistance gene. The producer psitex cells harboring the oncogene were cocultured with NIH3T3 cells at a 1:4 ratio. The viral supernatant was prepared at a titer between 10⁵ and 10⁶ colony-forming units/ml. The supernatant was stored at-80 C until primary cell culture infection. Retrovirus-infected cells (after 48 h in culture medium with retrovirus) were incubated with medium containing geneticin (G418) in selective concentration (400–600 µg/ml for initial selection, 250 µg/ml for cell maintenance).

Resistant colonies, appearing after 2–3 wk, were picked using cloning cylinders and further expanded. The growth curves of cloned cell lines, still representing a mixed population of hypothalamic cells, displayed a doubling time of approximately 24–48 h. The new clones were selected if they demonstrated predominantly neuronal lineage morphology, as small, rounded, or ovoid perikarya and long neuritic processes. Generally, the cloned lines form monolayers, grow rapidly, and retain growth contact inhibition. Further expansion was performed only after evidence for expression of SV40 T-Ag and neuron-specific enolase (NSE) and lack of glial fibrillary acidic protein. Mixed populations of hypothalamic cells were further subcloned through successive dilutions of the trypsinized cells into 96-well tissue culture plates coated with poly-L-lysine. The optimal dilution allowed only one or two cells per well. The cells were incubated in conditioned medium, i.e. medium taken from the mixed cultures, at a 1:1 ratio with culture medium, DMEM with 15% FBS (Life Technologies). Cell colonies were allowed to grow and then successively split into 24-well plates, and finally 60-mm plates, for RNA analysis and cryopreservation. Immortalized cell lines were grown in DMEM supplemented with 10% FBS, 20 mM glucose, and penicillin/streptomycin and maintained at 37 C with 5% CO₂.

Cell line screening
Each cell line was analyzed for the expression of specific markers by RT-PCR. First-strand cDNA was synthesized from 10 µg of DNase I-treated RNA, using SuperScript II RT (Life Technologies). The RT reaction was primed with random primers. cDNA synthesis was followed by RNase H (180 U/ml) digestion of RNA in a total volume of 20 µl. Control reactions were performed where amplification was carried out on samples in which the RT was omitted (RT–). Whenever possible, primer sequences flanked an intron, as an extra control for DNA contamination (Table 1). In most cases, the agarose gels were transferred for Southern blot analysis with an internal primer sequence as a probe. All products were sequenced to confirm identity.

Calcium imaging and microscopy
Ca²⁺ imaging experiments were performed with Fura2 (Molecular Probes, Eugene, OR) using an Olympus IX70 inverted epifluorescence microscope, an Ultrapix camera with an EEV CCD37-10-1-019 chip, a monochromator, and a PC with Merlin imaging software (Life Science Resources, Cambridge, UK) using standard Fura2 optics and imaging techniques. The extracellular solution used for imaging experiments consisted of (in mM): 140 NaCl, 4 KCl, 2 CaCl₂, 1 MgCl₂, 10 glucose, and 10 HEPES at pH 7.3. Cells were loaded with 4 µM Fura2-AM for 40 min at 37 C in standard extracellular solution. Intracellular free Ca²⁺ ([Ca²⁺]_i) concentrations were calculated using the Grynkiewicz equation (14), where K_d is the dissociation constant. Transmission electron microscopy was performed by the Advanced Bioimaging Centre, Mount Sinai Hospital (Toronto, Ontario, Canada), using a Hitachi H-7000 TEM, equipped with a digital image acquisition system.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Generation of immortalized hypothalamic cell lines
We have established a series of clonal, immortalized hypothalamic neuronal cell lines that express the potent oncogene SV40 large T-Ag and specific neuronal markers. To obtain these cell lines we have used primary cell culture from fetal mouse hypothalamus at E15, E17, and E18, representing a period of recognized neurogenesis in the mouse (15). The pZIPNeo SV(X)1 vector, containing the intact cDNA sequence for SV40 T-Ag and a neomycin resistance gene, was used as an immortalization factor during retroviral infection of the primary hypothalamic cultures (6, 13). The cells generated by this method are unique, compared with other immortalized cell lines, because they did not originate as a tumor but were transformed only by the expression of SV40 T-Ag in monolayer primary culture. The mixed cell populations were isolated using geneticin (G418) at a selective concentration (400–600 µg/ml) (Fig. 1A). The mixed cell populations are considered to be neuron-like due to the expression of NSE, a specific neuronal marker (6), and neurofilament, but not glial fibrillary acidic protein (GFAP) (Fig. 1, B and C). Incorporation of the oncogene was also determined by analysis of SV40 T-Ag expression. The heterogeneous cell populations potentially represent a source of any cell of interest from the hypothalamus. To test this, the cell lines were then serially diluted until clonal cell populations were generated. Thirty-eight representative clonal cell lines have been isolated from the initial screenings of the heterogeneous cell populations, and 16 of these have been further characterized to date (Table 2). Each cell line has a distinct cell morphology, indicating that they represent unique cell types demonstrating the potential diversity from the heterogeneous cells, although all of the cells have some common characteristics, such as overall neuronal phenotype and the appearance of neurites (Fig. 2A). Using electron microscopy, we have found that the cells exhibit dense core granules, indicative of secretory neurons, and form cell-cell contacts, with what appears to be the presence of dense material at the contact point, indicative of what has been seen previously at a synaptic cleft (16) (Fig. 2B). The lines are stable and maintain expression of SV40 T-Ag and NSE but do not express GFAP (Fig. 2B). For instance, the N-38 cell line has been passaged to P-44 and maintains SV40 T-Ag expression after 2 yr of continuous growth. Furthermore, all cell lines express syntaxin-1, a SNARE protein complex member localized to the presynaptic plasma membrane that has an indispensable role in neurosecretion (17). These immortalized, clonal cell lines provide valid model systems for molecular and biochemical investigations on the regulation of specific hormones, characteristics of their respective secretory neuronal population and an unlimited source of homogenous cell material and of the neuropeptide itself.

View larger version (95K): [in this window] [in a new window]   FIG. 1. Mixed populations of immortalized neuroendocrine cells exhibit neuronal morphology. A, Representative phase contrast micrographs of the mixed populations of cells after G418 selection. Immortalized cells from E15 and E17 express neuronal cell markers, as analyzed by RT-PCR of NSE (B) and neurofilament (C) from mixed cell populations. GT1–7 cell and hypothalamic RNA are used as positive controls; reactions without RT (RT–) were also included.

View larger version (76K): [in this window] [in a new window]   FIG. 2. Immortalized, clonal cell lines express neuronal cell markers but exhibit unique cellular morphologies and neuroendocrine markers and respond to hormonal stimulation. A, Representative phase contrast micrographs of clonal cell lines N-1, N-4, N-6, N-20, N-36, and N-38. B, Electron micrographs of N-38 exhibiting dense core material (i and iii; indicated by open arrows) and cell contact regions (ii, iii, and iv; indicated by closed arrows), whereas iv is an enlarged version of the boxed region in iii. C and D, RT-PCR of NSE, T-Ag, and GFAP (C) or NPY, AgRP, and Ob-Rb (D) in N-38 cells. GT1–7 cell and hypothalamic RNA are used as positive controls, whereas reactions without RT (RT–) were also included, as indicated. E, N-38 cells were exposed to 10 nM leptin, and real-time RT-PCR was performed over a 12-h time course. *, P < 0.05 (n = 3).

The successful generation of cell models from the hypothalamus that are not yet available allows the study of the transcriptional regulation of many genes associated with complex neuroendocrine pathways. Specifically, very little is known of the control of ghrelin, melanin-concentrating hormone (MCH), urocortin, neurotensin, proglucagon, and GHRH in the hypothalamus, and to our knowledge, our cell lines are the first available to study the regulation of these genes. Importantly, many of the lines expressing peptides associated with energy homeostasis also express the long form of the leptin receptor, ObR_b (4), and suppressor of cytokine signaling, a downstream effector molecule of ObR_b (21). As an example, the N-38 cell line expresses NPY, AgRP, and ObR_b, but not POMC (Fig. 2D). To determine whether the cell lines respond appropriately to hormone stimulation, we used the N-38 cell line as a model for leptin responsiveness. Stimulation of the N-38 cell line with 10 nM leptin over a 12-h time course resulted in a significant down-regulation of NPY gene expression at 2 and 4 h (Fig. 2E). These results conform to previous reports in vivo suggesting that leptin is able to directly effect NPY-expressing neurons in the hypothalamus, specifically by decreasing NPY gene expression (22, 23, 24). However, because N-38 is a clonal cell model of NPY-expressing neurons, it is difficult to directly compare the temporal down-regulation of NPY mRNA levels to the whole animal. Although the N-38 cells were continuously exposed to leptin, the metabolism of the peptide may be altered due to the presence or absence of specific binding proteins, enzymes, and afferent neuronal connections that may contribute to the sustained repression of NPY in the animal. Nonetheless, the N-38 cell model will prove useful to understand the direct regulation of the NPY neuron by leptin vs. indirect or afferent control of NPY expression and to dissect the molecular events involved in this process.

A number of the neuronal cell models reported herein have the potential to produce major neurotransmitters, as many of the lines expressed tyrosine hydroxylase, a marker of catecholaminergic neurons. Cells expressing tyrosine hydroxylase have the potential to produce dopamine, norepinephrine, and epinephrine, depending upon the complement of downstream catalytic enzymes. Other cell lines expressed tryptophan hydroxylase, the rate-limiting enzyme of serotonin production and an important component of melatonin biosynthesis. Due to the involvement of these neurotransmitters in the development of neurological disorders, such as depression, the use of the cell lines to study their regulation is fundamental for the development of satisfactory treatment options. Cell lines were also generated expressing specific releasing peptides, such as GHRH, GnRH, or secreted peptides, such as proglucagon-derived peptides, oxytocin, or arginine vasopressin. Because many of the peptides and enzymes present in the individual cell lines are secretory products, one representative cell line, N-38, was studied for its response to depolarization by addition of 60 mM KCl. Intracellular calcium levels ([Ca²⁺]_i) were monitored, and a typical single-spike [Ca²⁺]_i response was observed, consistent with a functional neurosecretory response (Fig. 3). Although each cell line will be investigated in terms of the secretory properties of specific neuropeptides, these preliminary studies indicate that these cells are capable of neurosecretion, as expected.

View larger version (44K): [in this window] [in a new window]   FIG. 3. Depolarization of N-38 neuroendocrine cells causes a significant increase in intracellular calcium concentration. Graph demonstrates the effect of the addition of KCl (60 mM, indicated by arrow) on intracellular calcium levels ([Ca2+]i) for the cells pictured. Images are pseudocolored according to the bar on the right. Shown is a representative experiment from six experiments; n = 116 cells in total.

	Acknowledgments

	Footnotes

 
This work was supported by the Canadian Institutes of Health Research, Natural Sciences and Engineering Research Council of Canada, Premier’s Research Excellence Award, and the Canada Foundation for Innovation.

Abbreviations: AgRP, Agouti-related protein; CART, cocaine- and amphetamine-regulated transcript; E14, embryonic d 14; FBS, fetal bovine serum; GFAP, glial fibrillary acidic protein; MCH, melanin-concentrating hormone; NPY, neuropeptide Y; NSE, neuron-specific enolase; POMC, proopiomelanocortin; SV, simian virus; T-Ag, T-antigen.

Received July 25, 2003.

Accepted for publication September 30, 2003.

	References

Top Abstract Introduction Materials and Methods Results and Discussion References

 

1. Everitt BJ, Hokfelt T 1990 Neuroendocrine anatomy of the hypothalamus. Acta Neurochir Suppl (Wien) 47:1–15
2. Hoffman GE, Smith MS, Verbalis JG 1993 c-Fos and related immediate early gene products as markers of activity in neuroendocrine systems. Front Neuroendocrinol 14:173–213[CrossRef][Medline]
3. Dufourny L, Warembourg M, Jolivet A 1999 Quantitative studies of progesterone receptor and nitric oxide synthase colocalization with somatostatin, or neurotensin, or substance P in neurons of the guinea pig ventrolateral hypothalamic nucleus: an immunocytochemical triple-label analysis. J Chem Neuroanat 17:33–43[CrossRef][Medline]
4. Elmquist JK, Elias CF, Saper CB 1999 From lesions to leptin: hypothalamic control of food intake and body weight. Neuron 22:221–232[CrossRef][Medline]
5. Smith MS, Grove KL 2002 Integration of the regulation of reproductive function and energy balance: lactation as a model. Front Neuroendocrinol 23: 225–256
6. Cepko CL 1989 Immortalization of neural cells via retrovirus-mediated oncogene transduction. Annu Rev Neurosci 12:47–65[CrossRef][Medline]
7. de Vitry F, Camier M, Czernichow P, Benda P, Cohen P, Tixier-Vidal A 1974 Establishment of a clone of mouse hypothalamic neurosecretory cells synthesizing neurophysin and vasopressin. Proc Natl Acad Sci USA 71:3575–3579[Abstract/Free Full Text]
8. Mellon PL, Windle JJ, Goldsmith P, Pedula C, Roberts J, Weiner RI 1990 Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis. Neuron 5:1–10[CrossRef][Medline]
9. Zakaria M, Dunn IC, Zhen S, Su E, Smith E, Patriquin E, Radovick S 1996 Phorbol ester regulation of the gonadotropin-releasing hormone (GnRH) gene in GnRH-secreting cell lines: a molecular basis for species differences. Mol Endocrinol 10:1282–1291[Abstract]
10. Earnest DJ, Liang FQ, DiGiorgio S, Gallagher M, Harvey B, Earnest B, Seigel GM 1999 Establishment and characterization of adenoviral E1A immortalized cell lines derived from the rat suprachiasmatic nucleus. J Neurobiol 39:1–13[CrossRef][Medline]
11. Wetsel WC 1995 Immortalized hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons: a new tool for dissecting the molecular and cellular basis of LHRH physiology. Cell Mol Neurobiol 15:43–78[CrossRef][Medline]
12. Earnest DJ, Liang FQ, Ratcliff M, Cassone VM 1999 Immortal time: circadian clock properties of rat suprachiasmatic cell lines. Science 283:693–695[Abstract/Free Full Text]
13. Brown M, McCormack M, Zinn KG, Farrell MP, Bikel I, Livingston DM 1986 A recombinant murine retrovirus for simian virus 40 large T cDNA transforms mouse fibroblasts to anchorage-independent growth. J Virol 60:290–293[Abstract/Free Full Text]
14. Grynkiewicz G, Poenie M, Tsien RY 1985 A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450[Abstract/Free Full Text]
15. Gardette R, Courtois M, Bisconte JC 1982 Prenatal development of mouse central nervous structures: time of neuron origin and gradients of neuronal production. A radioautographic study. J Hirnforsch 23:415–431[Medline]
16. Tixier-Vidal A, De Vitry F 1979 Hypothalamic neurons in cell culture. Int Rev Cytol 58:291–331[Medline]
17. Rettig J, Neher E 2002 Emerging roles of presynaptic proteins in Ca++-triggered exocytosis. Science 298:781–785[Abstract/Free Full Text]
18. Sawchenko PE, Imaki T, Vale W 1992 Co-localization of neuroactive substances in the endocrine hypothalamus. Ciba Found Symp 168:16–30[Medline]
19. Broberger C, De Lecea L, Sutcliffe JG, Hokfelt T 1998 Hypocretin/orexin- and melanin-concentrating hormone-expressing cells form distinct populations in the rodent lateral hypothalamus: relationship to the neuropeptide Y and agouti gene-related protein systems. J Comp Neurol 402:460–474[CrossRef][Medline]
20. Hahn TM, Breininger JF, Baskin DG, Schwartz MW 1998 Coexpression of AgRP and NPY in fasting-activated hypothalamic neurons. Nat Neurosci 1:271–272[CrossRef][Medline]
21. Bjorbaek C, El-Haschimi K, Frantz JD, Flier JS 1999 The role of SOCS-3 in leptin signaling and leptin resistance. J Biol Chem 274:30059–30065[Abstract/Free Full Text]
22. Sahu A 1998 Evidence suggesting that galanin (GAL), melanin-concentrating hormone (MCH), neurotensin (NT), proopiomelanocortin (POMC), and neuropeptide Y (NPY) are targets of leptin signaling in the hypothalamus. Endocrinology 139:795–798[Abstract/Free Full Text]
23. Elias CF, Aschkenasi C, Lee C, Kelly J, Ahima RS, Bjorbaek C, Flier JS, Saper CB, Elmquist JK 1999 Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area. Neuron 23:775–786[CrossRef][Medline]
24. Baskin DG, Hahn TM, Schwartz MW 1999 Leptin sensitive neurons in the hypothalamus. Horm Metab Res 31:345–350[Medline]
25. Hokfelt T, Meister B, Villar MJ, Ceccatelli S, Corts R, Schalling M, Everitt B 1990 Colocalization of messenger substances with special reference to the hypothalamic arcuate and paraventricular nuclei. Prog Clin Biol Res 342: 257–264
26. Ahima RS, Osei SY 2001 Molecular regulation of eating behavior: new insights and prospects for therapeutic strategies. Trends Mol Med 7:205–213[CrossRef][Medline]

This article has been cited by other articles:

				D. Titolo, C. M. Mayer, S. S. Dhillon, F. Cai, and D. D. Belsham Estrogen Facilitates both Phosphatidylinositol 3-Kinase/Akt and ERK1/2 Mitogen-Activated Protein Kinase Membrane Signaling Required for Long-Term Neuropeptide Y Transcriptional Regulation in Clonal, Immortalized Neurons J. Neurosci., June 18, 2008; 28(25): 6473 - 6482. [Abstract] [Full Text] [PDF]

				D. L. Fox and D. J. Good Nescient Helix-Loop-Helix 2 Interacts with Signal Transducer and Activator of Transcription 3 to Regulate Transcription of Prohormone Convertase 1/3 Mol. Endocrinol., June 1, 2008; 22(6): 1438 - 1448. [Abstract] [Full Text] [PDF]

				J. Wauman, A.-S. De Smet, D. Catteeuw, D. Belsham, and J. Tavernier Insulin Receptor Substrate 4 Couples the Leptin Receptor to Multiple Signaling Pathways Mol. Endocrinol., April 1, 2008; 22(4): 965 - 977. [Abstract] [Full Text] [PDF]

				H. Cheng, F. Isoda, D. D. Belsham, and C. V. Mobbs Inhibition of Agouti-Related Peptide Expression by Glucose in a Clonal Hypothalamic Neuronal Cell Line Is Mediated by Glycolysis, Not Oxidative Phosphorylation Endocrinology, February 1, 2008; 149(2): 703 - 710. [Abstract] [Full Text] [PDF]

				A.-S. Carlo, M. Pyrski, C. Loudes, A. Faivre-Baumann, J. Epelbaum, L. M. Williams, and W. Meyerhof Leptin Sensitivity in the Developing Rat Hypothalamus Endocrinology, December 1, 2007; 148(12): 6073 - 6082. [Abstract] [Full Text] [PDF]

				R. M. Luque, R. D. Kineman, and M. Tena-Sempere Regulation of Hypothalamic Expression of KiSS-1 and GPR54 Genes by Metabolic Factors: Analyses Using Mouse Models and a Cell Line Endocrinology, October 1, 2007; 148(10): 4601 - 4611. [Abstract] [Full Text] [PDF]

				R. D Kineman, M. D Gahete, and R. M Luque Identification of a mouse ghrelin gene transcript that contains intron 2 and is regulated in the pituitary and hypothalamus in response to metabolic stress J. Mol. Endocrinol., May 1, 2007; 38(5): 511 - 521. [Abstract] [Full Text] [PDF]

				F. Cai, A. V Gyulkhandanyan, M. B Wheeler, and D. D Belsham Glucose regulates AMP-activated protein kinase activity and gene expression in clonal, hypothalamic neurons expressing proopiomelanocortin: additive effects of leptin or insulin J. Endocrinol., March 1, 2007; 192(3): 605 - 614. [Abstract] [Full Text] [PDF]

				H. Cui, F. Cai, and D. D. Belsham Leptin signaling in neurotensin neurons involves STAT, MAP kinases ERK1/2, and p38 through c-Fos and ATF1 FASEB J, December 1, 2006; 20(14): 2654 - 2656. [Abstract] [Full Text] [PDF]

				J. Piessevaux, D. Lavens, T. Montoye, J. Wauman, D. Catteeuw, J. Vandekerckhove, D. Belsham, F. Peelman, and J. Tavernier Functional Cross-modulation between SOCS Proteins Can Stimulate Cytokine Signaling J. Biol. Chem., November 3, 2006; 281(44): 32953 - 32966. [Abstract] [Full Text] [PDF]

				D. Titolo, F. Cai, and D. D. Belsham Coordinate Regulation of Neuropeptide Y and Agouti-Related Peptide Gene Expression by Estrogen Depends on the Ratio of Estrogen Receptor (ER) {alpha} to ER{beta} in Clonal Hypothalamic Neurons Mol. Endocrinol., September 1, 2006; 20(9): 2080 - 2092. [Abstract] [Full Text] [PDF]

				S. Ezzat, R. Mader, S. Fischer, S. Yu, C. Ackerley, and S. L. Asa An essential role for the hematopoietic transcription factor Ikaros in hypothalamic-pituitary-mediated somatic growth PNAS, February 14, 2006; 103(7): 2214 - 2219. [Abstract] [Full Text] [PDF]

				H. Cui, F. Cai, and D. D. Belsham Anorexigenic Hormones Leptin, Insulin, and {alpha}-Melanocyte-Stimulating Hormone Directly Induce Neurotensin (NT) Gene Expression in Novel NT-Expressing Cell Models J. Neurosci., October 12, 2005; 25(41): 9497 - 9506. [Abstract] [Full Text] [PDF]

				V. C. Russo, S. Metaxas, K. Kobayashi, M. Harris, and G. A. Werther Antiapoptotic Effects of Leptin in Human Neuroblastoma Cells Endocrinology, September 1, 2004; 145(9): 4103 - 4112. [Abstract] [Full Text] [PDF]

				F Bai, T Rankinen, C Charbonneau, D D Belsham, D C Rao, C Bouchard, and G Argyropoulos Functional dimorphism of two hAgRP promoter SNPs in linkage disequilibrium J. Med. Genet., May 1, 2004; 41(5): 350 - 353. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Belsham, D. D. Articles by Shkreta, L. Search for Related Content PubMed PubMed Citation Articles by Belsham, D. D. Articles by Shkreta, L.