help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2006-0888
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/1/258    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Arumugam, R.
Right arrow Articles by Freemark, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Arumugam, R.
Right arrow Articles by Freemark, M.
Endocrinology Vol. 148, No. 1 258-267
Copyright © 2007 by The Endocrine Society

Lactogenic and Somatogenic Hormones Regulate the Expression of Neuropeptide Y and Cocaine- and Amphetamine-Regulated Transcript in Rat Insulinoma (INS-1) Cells: Interactions with Glucose and Glucocorticoids

Ramamani Arumugam, Don Fleenor and Michael Freemark

Departments of Pediatrics and Cell Biology, Duke University Medical Center, Durham, North Carolina 27710

Address all correspondence and requests for reprints to: Drs. Ramamani Arumugam and Michael Freemark, Box 3080, Duke University Medical Center, Durham, North Carolina 27710. E-mail: freem001{at}mc.duke.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lactogenic hormones stimulate food intake in rodents, ungulates, and birds. To test the hypothesis that lactogens regulate expression of neuropeptides that control appetite, we used the prolactin (PRL)-responsive rat insulinoma (INS-1) cell line as an experimental paradigm. INS-1 cells express mRNA for neuropeptide Y (NPY) and cocaine- and amphetamine-regulated transcript (CART) but little or no agouti-related peptide or proopiomelanocortin. As in the hypothalamus in vivo, the levels of NPY mRNA in INS-1 cells were increased by glucose deprivation. Conversely, high media glucose concentrations (11 mM) reduced the levels of NPY mRNA and increased levels of CART mRNA. Rat PRL stimulated a 4- to 7-fold increase in NPY mRNA in INS-1 cells (P < 0.001) and reduced by 50–80% the levels of CART mRNA (P < 0.001). The effects of PRL on NPY mRNA were time and dose dependent and potentiated by glucose deprivation or exogenous dexamethasone (Dex). Hormonal induction of NPY mRNA was accompanied by increased secretion of NPY peptide into cellular conditioned media. PRL stimulated a 1.8- to 3.5-fold increase in expression of AMP-activated protein kinase (AMPK), which mediates in part the effects of hypoglycemia on NPY expression in the hypothalamus in vivo. Pharmacological inhibition of AMPK activity blunted slightly the effects of PRL on NPY and CART but reversed entirely the effects of Dex or of PRL plus Dex on CART mRNA. The effects of PRL on NPY, CART, and AMPK mRNA were mirrored by those of other lactogens and somatogens including placental lactogen and GH. Rat PRL and rat GH in combination had no additive or synergistic effects, suggesting that lactogenic and somatogenic hormones regulate neuropeptide gene expression through similar mechanisms. We conclude that lactogens act in concert with glucose deprivation and glucocorticoids to induce NPY expression and inhibit CART. We speculate that the lactogens facilitate food intake in response to fasting or nutrient deprivation, when glucose levels decline and cortisol levels rise.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MATERNAL FOOD CONSUMPTION increases markedly during late pregnancy and lactation to support the demands of fetal and neonatal development and growth (1, 2, 3, 4). The factors that control maternal food intake are poorly understood; however, emerging evidence implicates a role for the lactogenic hormones produced by the maternal pituitary gland [prolactin (PRL)] and placenta [placental lactogen (PL)]. First, the increases in plasma PL and PRL concentrations during pregnancy and lactation (5) parallel the rise in maternal food consumption. Second, PRL receptors, which bind PL as well as PRL, are detected in hypothalamic neurons that control feeding behavior including the arcuate nucleus of the hypothalamus (ARH), dorsomedial hypothalamus (DMH), paraventricular nucleus (PVN), and ventromedial hypothalamus (VMH) (6, 7, 8, 9, 10). Third, the expression of lactogenic receptors in the hypothalamus increases during late gestation and lactation (8, 9, 10). Finally, PL and PRL stimulate food intake in female rats, ungulates, and birds (11, 12, 13, 14, 15, 16, 17).

The mechanisms by which lactogenic hormones stimulate food intake are currently unclear, although the induction of feeding after intracerebroventricular hormone administration (12, 15, 16, 17) suggests a central mode of action. A potential locus of activity is the ARH, which, through connections with the median eminence, is exposed to hormones and cytokines in the cerebrospinal fluid, portal blood, and peripheral circulation (18). The ARH integrates feeding behavior through orexigenic neurons that express neuropeptide Y (NPY) and agouti-related peptide (AgRP) and anorexigenic neurons that express proopiomelanocortin (POMC) and cocaine- and amphetamine-related transcript (CART). NPY and AgRP expression in the ARH/VMH are increased during lactation, whereas POMC and CART expression are reduced (12, 19, 20, 21, 22, 23, 24). Systemic administration of PRL is reported to increase signal transducer and activator of transcription 5 (STAT5) activity (25) and AgRP mRNA (17) in the ARH and NPY expression in the DMH (26); conversely, ARH levels of NPY mRNA are decreased in Ames dwarf mice (27), which have deficiencies of PRL, GH, and TSH. These findings suggest that lactogenic hormones may stimulate food intake through effects on hypothalamic gene expression. However, the lack of a PRL-responsive hypothalamic cell line has made it impossible to determine whether such an effect might be mediated directly or indirectly through other hormones, nutrients, or growth factors.

A variety of peptides normally expressed in the hypothalamus are also produced by pancreatic ß-cells from embryonic and perinatal rats and mice and by the PRL-responsive rat insulinoma cell line INS-1. These include NPY, CART, tyrosine hydroxylase, neuron-specific enolase, TRH, orexin A, urocortin III, and the receptors for insulin, leptin, glucagon-like peptide 1 (GLP-1), IL-1, corticotropin-releasing factor, and the glucocorticoids (7, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37). Moreover, INS-1 cells, pancreatic ß-cells, and hypothalamic neurons all express proteins critical for glucose sensing and signaling, including glucose transporters, glucokinase, and ATP-sensitive potassium channels (38). Thus, INS-1 cells and embryonic and perinatal pancreatic ß-cells have certain functional similarities with hypothalamic neurons.

We therefore used INS-1 cells to test the hypothesis that lactogenic hormones regulate directly the expression of neuropeptides in target tissues. We examined the interactions of PRL with glucose and glucocorticoids in the regulation of NPY, CART, and the critical metabolic regulator AMP-activated protein kinase (AMPK) and compared the effects of PRL with those of PL and GH, which also bind to receptors in hypothalamic neurons and pancreatic ß-cells and may stimulate food intake in humans and experimental animals (11, 39, 40, 41, 42, 43, 44).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DMEM, L-glutamine, antibiotic/antimycotic solution, fetal bovine serum (FBS), and Trizol reagent were purchased from Life Technologies (Rockville, MD). Actinomycin D was from Sigma Chemical Co. (St. Louis, MO). Rat GH (lot AFP6284C), rat PRL (lot AFP7545E), mouse PRL (lot AFP306C), mouse GH (lot AFP882), and human PRL (lot AFP9900) were purchased from Dr. Albert Parlow at the National Hormone and Peptide Program. Ovine PRL was purchased from Sigma, and recombinant human PL was obtained from Genentech Corp. (San Francisco, CA). Compound C, a cell-permeable inhibitor of AMPK activity, was kindly provided by Merck Corp. (Rahway, NJ). The High Capacity cDNA Archive kit and SYBR Green PCR Master mix were purchased from Applied Biosystems Inc. (Foster City, CA).

Cell culture
INS-1 cells (originally provided by M. Asfari, INSERM, Paris, France) were grown in RPMI 1640 (11.1 mM glucose) supplemented with 10% FBS, 50 µM 2-mercaptoethanol, 1 mM sodium pyruvate, 10 mM HEPES, and 1% antibiotic/antimycotic solution in 5% CO2 at 37 C. When the cells were approximately 80% confluent, the medium was changed to basal medium DMEM containing 5.5 mM glucose, 0.1% human serum albumin, 10 µg/ml human transferrin, 50 µM ethanolamine, 0.1 nM T3 , 50 µM phosphoethanolamine, and 1% antibiotic/antimycotic solution. Additional hormones were supplemented daily. To test the effects of glucose deprivation on gene expression, we performed some experiments using basal medium containing 2 mM, rather than 5.5 mM, glucose.

Quantification of mRNA expression
Total RNA from the INS-1 cells was extracted using Trizol reagent according to the manufacturer’s protocol. cDNA was synthesized from 2.5 µg RNA using the High Capacity cDNA Archive kit (Applied Biosystems) according to the manufacturer’s protocol. Quantitative real-time PCR was performed using an ABI 7900 Real Time PCR System. Oligonucleotide primers were designed using the Primer Express program from Applied Biosystems. For measurements of mature mRNA, all primer pairs spanned introns; amplicon lengths ranged from 90–150 bp. Thermal cycling conditions were 10 min at 95 C followed by 35–40 cycles for 15 sec at 95 C and 1 min at 57 C; SYBR green incorporation into a single peak was monitored using a dissociation curve. Negative controls were processed without reverse transcriptase. All samples from a single experiment were run using a single PCR mixture. Expression levels were normalized against the levels of acidic ribosomal phoshoprotein PO (riboprotein), a housekeeping gene that shows little change during cellular growth or differentiation (45).

The levels of mRNA were quantified using the comparative threshold cycle (CT) method. CT was determined from a log-linear plot of the PCR signal vs. cycle number. The amount of mRNA was normalized to that of the housekeeping gene (riboprotein) and expressed relative to controls by the formula 2–{Delta}{Delta}CT, where {Delta}CT = CT (riboprotein) – CT (target) and {Delta}{Delta}CT = {Delta}CT (treated sample) – {Delta}CT (untreated sample). The levels of riboprotein mRNA were not affected by any of the hormonal treatments.

The oligonucleotide primer pairs, all of which encode rat genes, and CT values obtained in cells at 80% confluence (before the change to basal medium) are shown in Table 1Go.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Analysis of gene expression in INS-1 cells by quantitative real-time PCR

 
NPY RIA
NPY peptide levels in serum-free conditioned media were determined by RIA using a kit from ALPCO Diagnostics (Windham, NH). The cells were not lysed before collection of conditioned media; thus, media levels of NPY reflect NPY secretion. The sensitivity of the RIA is 6 pM. The intraassay and interassay variations approximate 3 and 10%, respectively. The assay does not cross-react with either peptide YY (PYY) or pancreatic polypeptide. Preliminary experiments demonstrated that the slopes of the dose-response curves produced by serial dilutions of conditioned media were statistically identical to that produced by NPY standards and that recovery of NPY in conditioned medium exceeded 80%.

Statistical analysis
All assays were performed in duplicate or triplicate and were repeated at least three times. Data are expressed as mean ± SEM. Differences among sample means were tested by ANOVA, followed by the Neuman-Keuls or Bonferroni tests of multiple comparisons. A P value of <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NPY and CART expression in INS-1 cells
INS-1 cells express PRL and GH receptors in abundance and have been used extensively to study the regulation of ß-cell proliferation and insulin production by lactogenic and somatogenic hormones (46, 47). However, previous investigations showed that INS-1 cells, like normal pancreatic ß-cells, also express a variety of neural genes and hypothalamic neuropeptides including tyrosine hydroxylase, neurogenin-3, neuro-D, TRH, orexin-A, NPY, CART, and the melanin-concentrating hormone receptors (28, 29, 30, 32, 33, 34, 35, 36, 37). In initial studies, we confirmed that mRNAs encoding NPY and CART are readily detected in INS-1 cells (Table 1Go); in contrast, there is only very low level expression of AgRP or POMC mRNAs.

Effect of glucose on NPY and CART expression
In the hypothalamus in vivo, NPY mRNA is induced by hypoglycemia and suppressed by hyperglycemia (48, 49, 50). We found (Fig. 1Go) that glucose deprivation (2 mM) stimulated an increase in NPY mRNA levels in INS-1 cells. Conversely, glucose excess (11 mM) inhibited expression of NPY mRNA but increased transiently the expression of CART. Thus, the response of INS-1 cells to varying glucose concentrations in vitro mirrors the response of the hypothalamus to varying glucose concentrations in vivo (48, 49, 50), suggesting that INS-1 cells may serve as a model of neuropeptide gene regulation.


Figure 1
View larger version (10K):
[in this window]
[in a new window]

  		FIG. 1. Effects of glucose on NPY and CART mRNAs. INS-1 cells were grown to 80% confluence in RPMI 1640 (11.1 mM glucose) containing 10% FBS (time 0). The medium was then changed to serum-free basal medium containing 2 mM, 5.5 mM, or 11 mM glucose. Cellular RNA was processed at time 0, 24 h (d 1), or 48 h (d 2). NPY and CART mRNA levels were analyzed by real-time PCR. Control values at time 0, normalized to riboprotein mRNA levels, were arbitrarily set at 1.0. Values show the mean ± SE of three samples; similar results were obtained in three separate experiments.

 
Effect of PRL on NPY and CART mRNAs and interactions with glucose and glucocorticoids
Figures 2Go and 3Go show the effects of PRL on NPY and CART mRNA levels in INS-1 cells. Rat PRL stimulated a 4- to 7-fold increase in NPY mRNA (Fig. 2Go) and reduced by 50–80% the levels of CART mRNA (Fig. 3Go). In contrast, PRL had no effects on the levels of pancreatic polypeptide or PYY mRNAs (not shown). The effects of PRL on NPY and CART were dose dependent and observed at concentrations within the physiological range (0.5–2 nM).


Figure 2
View larger version (12K):
[in this window]
[in a new window]

  		FIG. 2. Effect of rat PRL on NPY mRNA levels. INS-1 cells were grown to 80% confluence in RPMI 1640 (11.1 mM glucose) containing 10% FBS (time 0). The medium was then changed to serum-free basal medium DMEM containing various concentrations of rat PRL or diluent. The left panel shows the time course of induction of NPY mRNA by rat PRL (500 ng/ml). Control values at time 0, normalized to riboprotein mRNA levels, were arbitrarily set at 1.0. The right panel shows the dose dependence of the PRL effect at 48 h of incubation. Control (diluent) values were arbitrarily set at 1.0. Values show the mean ± SE of three samples. Similar results were obtained in four separate experiments.

 

Figure 3
View larger version (12K):
[in this window]
[in a new window]

  		FIG. 3. Effect of rat PRL on CART mRNA levels. INS-1 cells were grown to 80% confluence in RPMI 1640 (11.1 mM glucose) containing 10% FBS (time 0). The medium was then changed to serum-free basal medium (5.5 mM glucose) containing various concentrations of rat PRL or diluent. The left panel shows the time course of inhibition of CART mRNA by rat PRL (500 ng/ml). The right panel shows the dose dependence of the PRL effect at 48 h of incubation. Control values at time 0, normalized to riboprotein mRNA levels, were arbitrarily set at 1.0. Values show the mean ± SE of three samples. Similar results were obtained in four separate experiments.

 
The effect of PRL on NPY mRNA levels was facilitated by glucose deprivation (2 mM). In the experiment shown in Fig. 4Go, left panel, PRL stimulated a 5.5-fold increase in NPY mRNA, whereas 2 mM glucose stimulated a 3.2-fold increase in NPY mRNA. The combination of PRL plus 2 mM glucose increased NPY mRNA 8.3-fold. Thus, the effects of PRL exposure and glucose deprivation appear to be additive, rather than synergistic, in nature. In contrast, glucose deprivation did not facilitate the effect of PRL on CART mRNA levels (Fig. 4Go, right panel).


Figure 4
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 4. Glucose deprivation potentiates the effect of PRL on NPY mRNA but not CART mRNA. INS-1 cells were grown to 80% confluence in RPMI 1640 (11.1 mM glucose) containing 10% FBS. The medium was then changed to serum-free basal medium containing 2 or 5.5 mM glucose in the presence or absence of rat PRL. Cellular RNA was processed at 48 h of incubation; NPY and CART mRNA levels were analyzed by real-time PCR. The labels 5.5 and 2 mM signify 5.5 and 2 mM glucose, respectively. PRL means that the cells were incubated with rat PRL, 500 ng/ml. Control values, normalized to riboprotein mRNA levels, were arbitrarily set at 1.0. Values show the mean ± SE of three samples; similar results were obtained in two separate experiments.

 
The effects of PRL on NPY mRNA were also potentiated by glucocorticoids (Fig. 5Go, left panel); at 48 h of incubation, PRL alone stimulated a 4.9-fold increase in NPY mRNA whereas dexamethasone (DEX) stimulated a 5.2-fold increase. The combination of PRL and DEX, however, stimulated a 16- to 26-fold increase in NPY mRNA. Thus, PRL and DEX act synergistically to enhance NPY expression.


Figure 5
View larger version (17K):
[in this window]
[in a new window]

  		FIG. 5. The effect of PRL on NPY and CART mRNAs is potentiated by DEX. INS-1 cells were grown to 80% confluence in RPMI 1640 (11.1 mM glucose) containing 10% FBS. The medium was then changed to serum-free basal medium (5.5 mM glucose), and the cells were incubated in the presence or absence of rat PRL (500 ng/ml), DEX (1 µM), a combination of the two, or diluent (control). Cellular RNA was processed at 48 h of incubation. Control values, normalized to riboprotein mRNA levels, were arbitrarily set at 1.0. Values show the mean ± SE of three samples; similar results were obtained in three separate experiments.

 
The effects of PRL and Dex on CART mRNA are shown in Fig. 5Go, right panel. PRL alone caused a 70–80% reduction in the levels of CART mRNA (P < 0.001), whereas DEX alone reduced the levels of CART mRNA by 65% (P < 0.001). The combination of PRL and DEX reduced by 88% the levels of CART mRNA. The inhibitory effect of PRL and DEX in combination exceeded (P < 0.05) that of DEX alone but was not statistically different (P = 0.06) from that of PRL alone.

Effects of PRL and DEX on NPY peptide levels in cellular conditioned media
That the PRL-dependent effect on NPY mRNA translates to induction of NPY peptide was demonstrated by measuring the NPY content of cellular conditioned media. The cells were not lysed before collection of conditioned media; thus, media levels of NPY reflect peptide secretion. The results are shown in Table 2Go. PRL alone stimulated a 57.6% increase in media NPY levels, whereas DEX alone stimulated a 119.8% increase. The combination of PRL plus DEX stimulated a striking 11-fold increase in NPY levels, confirming the synergism of the two hormones on NPY expression.


View this table:
[in this window]
[in a new window]

  		TABLE 2. Effects of PRL and DEX on NPY levels in cellular conditioned media

 
Regulation of INS-1 cell AMPK mRNA
In the arcuate hypothalamus in vivo, the effects of hypoglycemia and fasting on NPY expression and food intake are mediated in part by activation of AMPK, which serves as a cellular sensor of nutrient deficiency (49, 50). As shown in Fig. 6Go (bottom), AMPK was induced by glucose deprivation and reduced by glucose excess in INS-1 cells, mimicking the response of the hypothalamus in vivo. PRL stimulated a 1.8- to 3.5-fold increase in AMPK mRNA levels in INS-1 cells (Fig. 6Go, top). The effects of PRL on AMPK mRNA were facilitated by glucose deprivation (Fig. 6Go, bottom). In contrast, DEX alone had a lesser effect on AMPK mRNA (20–75% increase) and did not potentiate the effect of PRL (Fig. 7Go and Table 3Go).


Figure 6
View larger version (23K):
[in this window]
[in a new window]

  		FIG. 6. Regulation of AMPK mRNA in INS-1 cells: effects of PRL and glucose. INS-1 cells were grown to 80% confluence in RPMI 1640 (11.1 mM glucose) containing 10% FBS (time 0). The medium was then changed to serum-free basal medium containing 2, 5.5, or 11 mM glucose in the presence or absence of varying concentrations of rat PRL. Control (diluent) values were arbitrarily set at 1.0. The top panel shows the effects of increasing concentrations of rat PRL in medium containing 5.5 mM glucose. The bottom panel shows the effects of varying concentrations of glucose on AMPK mRNA in cells incubated in the presence or absence of rat PRL. In all cases, the cellular RNA was processed at 48 h of incubation. AMPK mRNA levels were analyzed by real-time PCR. The labels 11, 5.5, and 2 mM signify 11, 5.5, and 2 mM glucose, respectively. PRL means that the cells were incubated with rat PRL, 500 ng/ml. Values show the mean ± SE of three samples; similar results were obtained in three separate experiments.

 

Figure 7
View larger version (15K):
[in this window]
[in a new window]

  		FIG. 7. Regulation of AMPK mRNA in INS-1 cells: interactions of PRL and DEX. INS-1 cells were grown to 80% confluence in RPMI 1640 (11.1 mM glucose) containing 10% FBS (time 0). The medium was then changed to serum-free basal medium (5.5 mM glucose) and the cells were incubated in the presence or absence of rat PRL (500 ng/ml), DEX (1 µM), a combination of the two, or diluent (control). RNA was processed at 24 or 48 h; control values at 24 h (day 1), normalized to riboprotein mRNA levels, were arbitrarily set at 1.0. Values show the mean ± SE of three samples; similar results were obtained in two separate experiments.

 

View this table:
[in this window]
[in a new window]

  		TABLE 3. Effect of AMPK inhibition on PRL and DEX action

 
Effects of AMPK inhibition on PRL and DEX action
To determine whether the effects of PRL or DEX on NPY or CART expression are mediated by induction of AMPK, we pretreated INS-1 cells with compound C (10 µM), a specific inhibitor of AMPK activity (49) and then exposed the cells to PRL, DEX, or diluent for 24 h. NPY and CART mRNA levels were then assessed by real-time PCR.

As shown in Table 3Go, compound C reduced slightly (–24.1%, P < 0.05) the induction of NPY mRNA by PRL and counteracted only slightly the inhibitory effect of PRL on CART mRNA (+14.8%, P < 0.05). Compound C had no effect on the induction of NPY by DEX but reversed the effects of DEX or of PRL plus DEX on CART mRNA.

Effects of other lactogenic and somatogenic hormones
To assess the specificity of the PRL effects on NPY, CART, and AMPK expression, we compared the effects of rat PRL with those of other lactogenic hormones (including mouse PRL, human PRL, ovine PRL, and human PL), which in the rat bind exclusively to PRL receptors, and of rat GH and mouse GH, which bind exclusively to GH receptors. As shown in Fig. 8Go, all of the lactogens and somatogens stimulated increases in NPY and reduced the levels of CART mRNA (all P < 0.001 vs. controls). The other lactogens and somatogens also stimulated increases in AMPK to levels comparable to that those observed after treatment with rat PRL (not shown). The half-maximal concentration of GH (0.5–1 nM) was comparable to that of PRL (0.5–2 nM), and at maximal or submaximal concentrations, rat PRL and rat GH in combination had no additive or synergistic effects on gene expression (Fig. 9Go). These findings suggest that the lactogens and somatogens modulate NPY, CART, and AMPK expression through similar cellular mechanisms.


Figure 8
View larger version (21K):
[in this window]
[in a new window]

  		FIG. 8. Effects of diverse lactogenic and somatogenic hormones on NPY and CART mRNA levels in INS-1 cells. INS-1 cells were grown to 80% confluence in RPMI 1640 (11.1 mM glucose) containing 10% FBS (time 0). The medium was then changed to serum-free basal medium (5.5 mM glucose), and the cells were incubated for 48 h with various hormones (all 500 ng/ml) or diluent. The figure shows the percent change in NPY or CART mRNA relative to diluent-treated cells. Values show the mean ± SE of three samples. The changes were highly significant for all hormones tested (P < 0.001). Similar results were obtained in two separate experiments. m, Mouse; r, rat; o, ovine.

 

Figure 9
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 9. Comparison of the effects of rat PRL and rat GH on NPY and CART mRNAs. INS-1 cells were grown to 80% confluence in RPMI 1640 (11.1 mM glucose) containing 10% FBS (time 0). The medium was then changed to seum-free basal medium (5.5 mM glucose), and the cells were incubated for 48 h with various concentrations of rat PRL, rat GH, a combination of the two, or diluent. Values show the mean ± SE of three samples. Similar results were obtained in two separate experiments. In a separate experiment using submaximal hormone concentrations (50 ng/ml) during a 48-h incubation, rat PRL stimulated a 4.3-fold increase in NPY mRNA, rat GH stimulated a 4.8-fold increase in NPY mRNA, and the combination of rat PRL and rat GH stimulated a 4.4-fold increase in NPY mRNA (all P < 0.01 vs. controls). In the same experiment, rat PRL (50 ng/ml) reduced CART mRNA by 46% (P < 0.01 vs. control), rat GH reduced CART mRNA by 79% (P < 0.01 vs. control), and the combination of the two reduced CART mRNA by 83.5% (P > 0.05 vs. GH alone).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Feeding behavior is regulated by the complex interactions of hormones and nutrients with neuropeptides that are expressed in hypothalamic centers including the ARH, DMH, PVN, and VMH. Through expression of the orexigens NPY and AgRP and the anorexigens CART and POMC, and through neuronal projections to the PVN and DMH, the ARH integrates feeding activity and body metabolism (18). The rise in plasma glucose, insulin, and amylin and the fall in plasma ghrelin during feeding reduce the expression of NPY and AgRP in the ARH and increase expression of POMC and CART. These changes in neuropeptide expression, in concert with increases in plasma GLP-1, PYY, and cholecystokinin, suppress food intake in the immediate postprandial period (18, 26, 51, 52, 53). Conversely, the fall in plasma glucose, insulin, amylin, and leptin during fasting, in concert with increases in plasma cortisol and ghrelin (26), increase NPY and AgRP expression in the ARH and reduce expression of POMC and CART. These changes, in concert with reductions in plasma GLP-1, PYY, and cholecystokinin, promote food intake in the postabsorptive (fasting) state.

Maternal food consumption is a critical determinant of fetal and neonatal development and growth (1, 2, 3, 4). Nevertheless, the regulation of food intake and neuropeptide expression in pregnancy and lactation is poorly understood. Food consumption rises after midgestation despite increases in maternal plasma insulin and leptin concentrations. These findings suggest a central state of insulin and leptin resistance, which may reflect reductions in hypothalamic expression of insulin and leptin receptors (54, 55, 56, 57, 58). Food intake increases more dramatically during lactation, when plasma insulin and leptin levels decline. Some investigators have demonstrated increases in NPY and/or AgRP mRNA levels in the ARH/VMH during pregnancy and lactation (12, 19, 21, 22, 23, 59). Lactation is reported to be associated with a reduction in CART expression in the ARH/VMH (20) but an increase in CART expression in the pituitary gland and supraoptic nucleus.

Previous studies demonstrating orexigenic effects of PRL and PL (11, 12, 13, 14, 15, 16, 17) suggest that the lactogens promote maternal food consumption during the latter half of pregnancy and lactation. Studies in the ring dove Streptopelia risoria implicate a central mode of action: intracerebroventricular administration of PRL stimulates increases in infundibular NPY and AgRP immunoreactivity and promotes food intake (12, 15, 16, 17).

Other investigators find that systemic administration of PRL induces NPY mRNA in the DMH but not the ARH (26). Direct effects of lactogens on hypothalamic function are also suggested by studies of rodent maternal behavior, which can be induced by injection of PRL or PL into the medial preoptic area (41, 42). Yet the mechanisms by which the lactogenic hormones stimulate food intake have not been elucidated, in part because investigators have lacked cellular models to study lactogen-dependent regulation of neuropeptide gene expression.

As noted previously, INS-1 cells, like pancreatic ß-cells from perinatal rats and mice, express mRNAs encoding NPY and CART as well as a number of other hypothalamic peptides including tyrosine hydroxylase, neuron-specific enolase, TRH, orexin A, urocortin III, and the receptors for insulin, leptin, GLP-1, IL-1, corticotropin-releasing-factor, and the glucocorticoids (28, 29, 30, 31, 32, 33, 34, 35, 36, 37). Moreover, the expression of NPY in INS-1 cells in vitro, as in the rodent hypothalamus in vivo, is induced by glucose deprivation or DEX (this study). Thus, INS-1 cells have functional similarities with hypothalamic neurons and may serve as a useful, although imperfect, paradigm for studying the control of neuropeptide gene expression.

The studies described in the current manuscript demonstrate that lactogenic hormones regulate directly the expression of NPY and CART in INS-1 cells. The rodent and human PRLs and human PL stimulate increases in NPY mRNA levels and reduce dramatically the expression of CART. These actions are achieved at concentrations within the physiological range and are specific, because PRL regulates neither pancreatic polypeptide nor PYY mRNAs.

The molecular mechanisms by which lactogens regulate NPY and CART mRNA levels are currently unknown. The effects of the lactogens on NPY expression are potentiated by glucose deprivation and DEX, although in different ways; glucose deprivation and PRL in combination appear to have additive effects on NPY mRNA levels, whereas DEX and PRL clearly act synergistically to increase NPY expression. DEX may also potentiate the inhibitory effect of PRL on CART mRNA, although our studies failed to demonstrate (P = 0.06) that the effect of DEX plus PRL on CART mRNA was significantly different from that of PRL alone. The effect of PRL on CART expression does not appear to be modulated by glucose. Considered together, these observations suggest that the mechanism(s) of lactogen action differ, at least in part, from those of glucose and glucocorticoids.

Support for this hypothesis is provided by studies of the effects of the hormones on AMPK expression. PRL and DEX had differential effects on AMPK mRNA levels, and compound C, a pharmacological inhibitor of AMPK activity, reversed entirely the effect of DEX on CART mRNA but blunted only slightly the effects of PRL on CART or NPY. Interestingly, compound C also reversed the effect of PRL plus DEX on CART mRNA but not NPY mRNA; thus, the pathways by which lactogens and glucocorticoids regulate CART may overlap or converge at some level.

The effects of the lactogens on NPY and CART expression were mirrored by those of rat and mouse GH. This finding is consistent with studies by Chan et al. (39), who demonstrated that NPY mRNA is reduced in ARH neurons of hypophysectomized male rats and restored by systemic GH treatment. Because maximal concentrations of PRL and GH have neither additive nor synergistic effects on NPY, CART, or AMPK expression in INS-1 cells, the lactogens and somatogens likely exert their effects through similar mechanisms. Nevertheless, we have not been able to compare the effects of the lactogens on AgRP production or MSH signaling with those of GH because INS-1 cells express little or no AgRP, POMC, or melanocortin receptors 3 or 4 (melanocortin receptor data not shown). Effects on AgRP or MSH signaling may be important, because systemic administration of PRL is reported to increase tuberal AgRP immunoreactivity in the ring dove (17).

Together with previous investigations that demonstrated increases in hypothalamic NPY expression and reductions in CART expression in states of lactogen excess, including pregnancy and lactation (12, 19, 20, 21, 22, 23, 24, 28), our findings concord with the hypothesis that lactogenic hormones stimulate food intake through direct effects on neuropeptide gene expression. The potentiation of lactogen effects by glucose deprivation and DEX and the lactogen induction of AMPK, a sensor of nutrient deficiency, suggest that PRL and PL may stimulate food intake in concert with hypoglycemia and glucocorticoids. Given the increase in maternal PL concentrations during fasting (5, 60) and the rise in plasma PRL during insulin-induced hypoglycemia (61, 62), we speculate that the lactogens promote food consumption in response to fasting or nutrient deprivation (Fig. 10Go), when glucose levels fall and cortisol levels rise.


Figure 10
View larger version (19K):
[in this window]
[in a new window]

  		FIG. 10. Hypothetical depiction of the possible roles of lactogenic hormones, glucose deprivation, and glucocorticoids in the regulation of feeding behavior.

 
It is currently unclear whether PRL-dependent changes in NPY, CART, or AMPK expression affect pancreatic ß-cell development or function per se. NPY is coexpressed with insulin in the perinatal mouse pancreas between embryonic day 16 (e16) and p0 (day of birth); at e20–21, the majority of insulin-producing cells express NPY (32, 34, 63, 64). Islet NPY immunoreactivity declines thereafter, and in the postnatal period the peptide localizes primarily to pancreatic nerves (65); however, ß-cell expression of NPY can be induced in adult pancreatic islets by treatment with DEX in vivo or in vitro (34, 63, 64). The temporal expression of NPY in the rodent pancreas coincides with a surge of fetal glucocorticoids and a wave of islet cell proliferation during the perinatal period; simultaneously, the PRL receptor, which binds PL as well as PRL, first appears in pancreatic ß-cells (66). A recent study reports that NPY (67), like PRL and PL (46), stimulates ß-cell mitogenesis; thus, the lactogens and NPY may act in concert to increase ß-cell mass during the perinatal period. Interestingly, NPY and PRL have divergent effects on ß-cell insulin secretion: NPY inhibits glucose-dependent insulin release (68), whereas the lactogens promote insulin production (47, 69). A similar dichotomy has been observed with GLP-1, which stimulates INS-1 cell NPY expression but facilitates glucose-dependent insulin secretion (68). It has been proposed that the induction of NPY may restrain the effect of GLP-1 on islet cell insulin secretion; a similar interaction between NPY and the lactogens might favor ß-cell proliferation at the expense of insulin secretion during the perinatal period.

The roles of CART and AMPK in lactogen or somatogen action in the ß-cell are unclear. In the rat (33, 36, 37), CART colocalizes with insulin and glucagon from e13 to p10 and with somatostatin from e15 through adulthood; expression of CART in insulin-producing cells peaks on the day of birth and declines thereafter to undetectable levels. After p12, CART is found only in somatostatin cells and in pancreatic nerves. CART stimulates pancreatic exocrine secretion in adult rats (70), but its effects on ß-cell development and function and its relationship to lactogen action are unknown.

The same may be said of the role of AMPK in lactogen or somatogen action in the ß-cell. Although PRL stimulates AMPK expression in rat INS-1 cells, our studies using compound C suggest that factors other than or in addition to AMPK activation mediate the effects of PRL on ß-cell NPY and CART mRNAs. Moreover, in contrast to the effects of PRL and PL in isolated islets and insulinoma cell lines, AMPK activation suppresses islet glucose oxidation, reduces insulin gene transcription and glucose-dependent insulin secretion, and promotes ß-cell apoptosis (71, 72). The interactions of lactogens, somatogens, and AMPK in the regulation of ß-cell function will therefore require additional study.


   Acknowledgments
 
We thank Drs. Michael Muehlbauer and Christopher Newgard, and the Sarah W. Stedman Nutrition and Metabolism Center at Duke University for measurements of NPY by RIA.


   Footnotes
 
This work was supported by grants to M.F. from the National Institute of Child Health and Development (HD24192) and Pfizer Corp. and by a grant to R.A. from the Duke Children’s Miracle Network.

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 5, 2006

Abbreviations: AgRP, Agouti-related peptide; AMPK, AMP-activated protein kinase; ARH, arcuate nucleus of the hypothalamus; CART, cocaine- and amphetamine-regulated transcript; CT, threshold cycle; DEX, dexamethasone; DMH, dorsomedial hypothalamus; e, embryonic day; FBS, fetal bovine serum; GLP-1, glucagon-like peptide 1; NPY, neuropeptide Y; p0, day of birth; PL, placental lactogen; POMC, proopiomelanocortin; PRL, prolactin; PVN, paraventricular nucleus; PYY, peptide YY; VMH, ventromedial hypothalamus.

Received July 5, 2006.

Accepted for publication September 25, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Butte NF, Wong WW, Treuth MS, Ellis KJ, O’Brian Smith E 2004 Energy requirements during pregnancy based on total energy expenditure and energy deposition. Am J Clin Nutr 79:1078–1087[Abstract/Free Full Text]
  2. Ramos MP, Crespo-Solans MD, del Campo S, Cacho J, Herrera E 2003 Fat accumulation in the rat during early pregnancy is modulated by enhanced insulin responsiveness. Am J Physiol Endocrinol Metab 285:E318–E328
  3. Lopez-Luna P, Munoz T, Herrera E 1986 Body fat in pregnant rats at mid- and late-gestation. Life Sci 39:1389–1393[CrossRef][Medline]
  4. Freemark M 2006 Regulation of maternal metabolism by pituitary and placental hormones: roles in fetal development and metabolic programming. Horm Res 65(Suppl 3):40–48
  5. Freemark M 1999 The roles of growth hormone, prolactin and placental lactogen in human fetal development. Totowa, NJ: Humana Press; 57–83
  6. Bakowska JC, Morrell JI 2003 The distribution of mRNA for the short form of the prolactin receptor in the forebrain of the female rat. Brain Res Mol Brain Res 116:50–58[Medline]
  7. Augustine RA, Kokay IC, Andrews ZB, Ladyman SR, Grattan DR 2003 Quantitation of prolactin receptor mRNA in the maternal rat brain during pregnancy and lactation. J Mol Endocrinol 31:221–232[Abstract]
  8. Grattan DR 2001 The actions of prolactin in the brain during pregnancy and lactation. Prog Brain Res 133:153–171[Medline]
  9. Kokay IC, Grattan DR 2005 Expression of mRNA for prolactin receptor (long form) in dopamine and pro-opiomelanocortin neurones in the arcuate nucleus of non-pregnant and lactating rats. J Neuroendocrinol 17:827–835[Medline]
  10. Mann PE, Bridges RS 2002 Prolactin receptor gene expression in the forebrain of pregnant and lactating rats. Brain Res Mol Brain Res 105:136–145[Medline]
  11. Byatt JC, Staten NR, Salsgiver WJ, Kostelc JG, Collier RJ 1993 Stimulation of food intake and weight gain in mature female rats by bovine prolactin and bovine growth hormone. Am J Physiol 264:E986–E992
  12. Buntin JD, Hnasko RM, Zuzick PH 1999 Role of the ventromedial hypothalamus in prolactin-induced hyperphagia in ring doves. Physiol Behav 66:255–261[CrossRef][Medline]
  13. Gerardo-Gettens T, Moore BJ, Stern JS, Horwitz BA 1989 Prolactin stimulates food intake in a dose-dependent manner. Am J Physiol 256:R276–R280
  14. Goodridge AG, Ball EG 1967 The effect of prolactin on lipogenesis in the pigeon. In vivo studies. Biochemistry 6:1676–1682[CrossRef][Medline]
  15. Sauve D, Woodside B 1996 The effect of central administration of prolactin on food intake in virgin female rats is dose-dependent, occurs in the absence of ovarian hormones and the latency to onset varies with feeding regimen. Brain Res 729:75–81[Medline]
  16. Strader AD, Buntin JD 2001 Neuropeptide-Y: a possible mediator of prolactin-induced feeding and regulator of energy balance in the ring dove (Streptopelia risoria). J Neuroendocrinol 13:386–392[CrossRef][Medline]
  17. Strader AD, Buntin JD 2003 Changes in agouti-related peptide during the ring dove breeding cycle in relation to prolactin and parental hyperphagia. J Neuroendocrinol 15:1046–1053[CrossRef][Medline]
  18. Cone RD, Cowley MA, Butler AA, Fan W, Marks DL, Low MJ 2001 The arcuate nucleus as a conduit for diverse signals relevant to energy homeostasis. Int J Obes Relat Metab Disord 25(Suppl 5):S63–S67
  19. Garcia MC, Lopez M, Gualillo O, Seoane LM, Dieguez C, Senaris RM 2003 Hypothalamic levels of NPY, MCH, and prepro-orexin mRNA during pregnancy and lactation in the rat: role of prolactin. FASEB J 17:1392–1400[Abstract/Free Full Text]
  20. Xiao XQ, Grove KL, Lau SY, McWeeney S, Smith MS 2005 Deoxyribonucleic acid microarray analysis of gene expression pattern in the arcuate nucleus/ventromedial nucleus of hypothalamus during lactation. Endocrinology 146:4391–4398[Abstract/Free Full Text]
  21. Rocha M, Bing C, Williams G, Puerta M 2003 Pregnancy-induced hyperphagia is associated with increased gene expression of hypothalamic agouti-related peptide in rats. Regul Pept 114:159–165[CrossRef][Medline]
  22. Wilding JP, Ajala MO, Lambert PD, Bloom SR 1997 Additive effects of lactation and food restriction to increase hypothalamic neuropeptide Y mRNA in rats. J Endocrinol 152:365–369[Abstract]
  23. Chen P, Li C, Haskell-Luevano C, Cone RD, Smith MS 1999 Altered expression of agouti-related protein and its colocalization with neuropeptide Y in the arcuate nucleus of the hypothalamus during lactation. Endocrinology 140:2645–2650[Abstract/Free Full Text]
  24. Smith MS 1993 Lactation alters neuropeptide-Y and proopiomelanocortin gene expression in the arcuate nucleus of the rat. Endocrinology 133:1258–1265[Abstract]
  25. Cave BJ, Norman M, Flynn A, Townsend J, Wakerley JB, Tortonese DJ 2005 Prolactin-induced activation of STAT5 within the hypothalamic arcuate nucleus. Neuroreport 16:1423–1426[CrossRef][Medline]
  26. Chen HY, Trumbauer ME, Chen AS, Weingarth DT, Adams J R, Frazier EG, Shen Z, Marsh DJ, Feighner SD, Guan XM, Ye Z, Nargund RP, Smith RG, Van der Ploeg LH, Howard A D, MacNeil DJ, Qian, S 2004 Orexigenic action of peripheral ghrelin is mediated by neuropeptide Y and agouti-related protein. Endocrinology 145:2607–2612[Abstract/Free Full Text]
  27. Hurley DL, Birch DV, Almond MC, Estrada IJ, Phelps CJ 2003 Reduced hypothalamic neuropeptide Y expression in growth hormone- and prolactin-deficient Ames and Snell dwarf mice. Endocrinology 144:4783–4789[Abstract/Free Full Text]
  28. Fragner P, Quette J, Aratan-Spire S 1998 Thyroid status and the regulation of thyrotropin-releasing hormone synthesis in rat pancreatic islets: comparison with insulin regulation. Biochem Biophys Res Commun 247:564–568[CrossRef][Medline]
  29. Delaunay F, Khan A, Cintra A, Davani B, Ling ZC, Andersson A, Ostenson C G, Gustafsson J, Efendic S, Okret S 1997 Pancreatic ß-cells are important targets for the diabetogenic effects of glucocorticoids. J Clin Invest 100:2094–2098[Medline]
  30. Ouedraogo R, Naslund E, Kirchgessner AL 2003 Glucose regulates the release of orexin-a from the endocrine pancreas. Diabetes 52:111–117[Abstract/Free Full Text]
  31. Scarim AL, Arnush M, Hill JR, Marshall CA, Baldwin A, McDaniel ML, Corbett JA 1997 Evidence for the presence of type I IL-1 receptors on ß-cells of islets of Langerhans. Biochim Biophys Acta 1361:313–320[Medline]
  32. Teitelman G, Alpert S, Polak JM, Martinez A, Hanahan D 1993 Precursor cells of mouse endocrine pancreas coexpress insulin, glucagon and the neuronal proteins tyrosine hydroxylase and neuropeptide Y, but not pancreatic polypeptide. Development 118:1031–1039[Abstract]
  33. Jensen PB, Kristensen P, Clausen JT, Judge ME, Hastrup S, Thim L, Wulff BS, Foged C, Jensen J, Holst JJ, Madsen OD 1999 The hypothalamic satiety peptide CART is expressed in anorectic and non-anorectic pancreatic islet tumors and in the normal islet of Langerhans. FEBS Lett 447:139–143[CrossRef][Medline]
  34. Myrsen-Axcrona U, Ekblad E, Sundler F 1997 Developmental expression of NPY, PYY and PP in the rat pancreas and their coexistence with islet hormones. Regul Pept 68:165–175[CrossRef][Medline]
  35. Tadayyon M, Welters HJ, Haynes AC, Cluderay JE, Hervieu G 2000 Expression of melanin-concentrating hormone receptors in insulin-producing cells: MCH stimulates insulin release in RINm5F and CRI-G1 cell-lines. Biochem Biophys Res Commun 275:709–712[CrossRef][Medline]
  36. Wierup N, Bjorkqvist M, Kuhar MJ, Mulder H, Sundler F 2006 CART regulates islet hormone secretion and is expressed in the ß-cells of type 2 diabetic rats. Diabetes 55:305–311[Abstract/Free Full Text]
  37. Wierup N, Kuhar M, Nilsson BO, Mulder H, Ekblad E, Sundler F 2004 Cocaine- and amphetamine-regulated transcript (CART) is expressed in several islet cell types during rat development. J Histochem Cytochem 52:169–177[Abstract/Free Full Text]
  38. Marty N, Dallaporta M, Foretz M, Emery, M, Tarussio D, Bady I, Binnert C, Beermann F, Thorens B 2005 Regulation of glucagon secretion by glucose transporter type 2 (glut2) and astrocyte-dependent glucose sensors. J Clin Invest 115:3545–3553[CrossRef][Medline]
  39. Chan YY, Clifton DK, Steiner RA 1996 Role of NPY neurones in GH-dependent feedback signalling to the brain. Horm Res 45(Suppl 1):12–14
  40. Pihoker C, Robertson MC, Freemark M 1993 Rat placental lactogen-I binds to the choroid plexus and hypothalamus of the pregnant rat. J Endocrinol 139:235–242[Abstract]
  41. Bridges RS, Freemark MS 1995 Human placental lactogen infusions into the medial preoptic area stimulate maternal behavior in steroid-primed, nulliparous female rats. Horm Behav 29:216–226[CrossRef][Medline]
  42. Bridges RS, Robertson MC, Shiu RP, Sturgis JD, Henriquez BM, Mann PE 1997 Central lactogenic regulation of maternal behavior in rats: steroid dependence, hormone specificity, and behavioral potencies of rat prolactin and rat placental lactogen I. Endocrinology 138:756–763[Abstract/Free Full Text]
  43. Kamegai J, Minami S, Sugihara H, Hasegawa O, Higuchi H, Wakabayashi I 1996 Growth hormone receptor gene is expressed in neuropeptide Y neurons in hypothalamic arcuate nucleus of rats. Endocrinology 137:2109–2112[Abstract]
  44. Bohlooly-Y M, Olsson B, Bruder CE, Linden D, Sjogren K, Bjursell M, Egecioglu E, Svensson L, Brodin P, Waterton JC, Isaksson OG, Sundler F, Ahren B, Ohlsson C, Oscarsson J, Tornell J 2005 Growth hormone overexpression in the central nervous system results in hyperphagia-induced obesity associated with insulin resistance and dyslipidemia. Diabetes [Erratum (2005) 54:1249] 54:51–62
  45. Dheda K, Huggett JF, Bustin SA, Johnson MA, Rook G, Zumla A 2004 Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques 37:112–119[Medline]
  46. Friedrichsen BN, Galsgaard ED, Nielsen JH, Moldrup A 2001 Growth hormone- and prolactin-induced proliferation of insulinoma cells, INS-1, depends on activation of STAT5 (signal transducer and activator of transcription 5). Mol Endocrinol 15:136–148[Abstract/Free Full Text]
  47. Fleenor DE, Freemark M 2001 Prolactin induction of insulin gene transcription: roles of glucose and signal transducer and activator of transcription 5. Endocrinology 142:2805–2810[Abstract/Free Full Text]
  48. Chang GQ, Karatayev O, Davydova Z, Wortley K, Leibowitz SF 2005 Glucose injection reduces neuropeptide Y and agouti-related protein expression in the arcuate nucleus: a possible physiological role in eating behavior. Brain Res Mol Brain Res 135:69–80[Medline]
  49. Minokoshi Y, Alquier T, Furukawa N, Kim YB, Lee A, Xue B, Mu J, Foufelle F, Ferre P, Birnbaum MJ, Stuck BJ, Kahn BB 2004 AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus. Nature 428:569–574[CrossRef][Medline]
  50. Sindelar DK, Ste Marie L, Miura GI, Palmiter RD, McMinn JE, Morton GJ, Schwartz MW 2004 Neuropeptide Y is required for hyperphagic feeding in response to neuroglucopenia. Endocrinology 145:3363–3368[Abstract/Free Full Text]
  51. Adam TC, Westerterp-Plantenga MS 2005 Glucagon-like peptide-1 release and satiety after a nutrient challenge in normal-weight and obese subjects. Br J Nutr 93:845–851[CrossRef][Medline]
  52. Neary NM, Small CJ, Druce MR, Park AJ, Ellis SM, Semjonous NM, Dakin CL, Filipsson K, Wang F, Kent AS, Frost GS, Ghatei MA, Bloom SR 2005 Peptide YY3–36 and glucagon-like peptide-17–36 inhibit food intake additively. Endocrinology 146:5120–5127[Abstract/Free Full Text]
  53. Bi S, Scott KA, Kopin AS, Moran TH 2004 Differential roles for cholecystokinin a receptors in energy balance in rats and mice. Endocrinology 145:3873–3880[Abstract/Free Full Text]
  54. Johnstone LE, Higuchi T 2001 Food intake and leptin during pregnancy and lactation. Prog Brain Res 133:215–227[Medline]
  55. Ladyman SR, Grattan DR 2005 Suppression of leptin receptor messenger ribonucleic acid and leptin responsiveness in the ventromedial nucleus of the hypothalamus during pregnancy in the rat. Endocrinology 146:3868–3874[Abstract/Free Full Text]
  56. Obici S, Feng Z, Karkanias G, Baskin DG, Rossetti L 2002 Decreasing hypothalamic insulin receptors causes hyperphagia and insulin resistance in rats. Nat Neurosci 5:566–572[CrossRef][Medline]
  57. Seeber RM, Smith JT, Waddell BJ 2002 Plasma leptin-binding activity and hypothalamic leptin receptor expression during pregnancy and lactation in the rat. Biol Reprod 66:1762–1767[Abstract/Free Full Text]
  58. Garcia MD, Casanueva FF, Dieguez C, Senaris RM 2000 Gestational profile of leptin messenger ribonucleic acid (mRNA) content in the placenta and adipose tissue in the rat, and regulation of the mRNA levels of the leptin receptor subtypes in the hypothalamus during pregnancy and lactation. Biol Reprod 62:698–703[Abstract/Free Full Text]
  59. Oberto A, Mele P, Zammaretti F, Panzica G, Eva C 2003 Evidence of altered neuropeptide Y content and neuropeptide Y1 receptor gene expression in the hypothalamus of pregnant transgenic mice. Endocrinology 144:4826–4830[Abstract/Free Full Text]
  60. Fielder PJ, Ogren L, Edwards D, Talamantes F 1987 Effects of fasting on serum lactogenic hormone concentrations during mid- and late pregnancy in mice. Am J Physiol 253:E40–E44
  61. Gale EA, Bennett T, Macdonald IA, Holst JJ, Matthews JA 1983 The physiological effects of insulin-induced hypoglycaemia in man: responses at differing levels of blood glucose. Clin Sci (Lond) 65:263–271[Medline]
  62. Williams TC, Berelowitz M, Berk MA, Frohman LA 1987 Differential effects of insulin- and proinsulin-induced hypoglycemia on pituitary hormone and catecholamine secretion. Diabetes Care 10:278–285[Abstract]
  63. Myrsen U, Ahren B, Sundler F 1996 Dexamethasone-induced neuropeptide Y expression in rat islet endocrine cells. Rapid reversibility and partial prevention by insulin. Diabetes 45:1306–1316[Abstract]
  64. Waeber G, Thompson N, Waeber B, Brunner HR, Nicod P, Grouzmann E 1993 Neuropeptide Y expression and regulation in a differentiated rat insulin-secreting cell line. Endocrinology 133:1061–1067[Abstract]
  65. Myrsen-Axcrona U, Ahren B, Sundler F 1999 Modulatory role of adrenergic nerves on dexamethasone-induced islet cell NPY expression in the rat: evidence from chemical sympathectomy. Pancreas 18:180–188[Medline]
  66. Freemark M, Driscoll P, Maaskant R, Petryk A, Kelly PA 1997 Ontogenesis of prolactin receptors in the human fetus in early gestation. Implications for tissue differentiation and development. J Clin Invest 99:1107–1117[Medline]
  67. Cho YR, Kim CW 2004 Neuropeptide Y promotes ß-cell replication via extracellular signal-regulated kinase activation. Biochem Biophys Res Commun 314:773–780[CrossRef][Medline]
  68. Morgan DG, Kulkarni RN, Hurley JD, Wang ZL, Wang RM, Ghatei MA, Karlsen AE, Bloom SR, Smith DM 1998 Inhibition of glucose stimulated insulin secretion by neuropeptide Y is mediated via the Y1 receptor and inhibition of adenylyl cyclase in RIN 5AH rat insulinoma cells. Diabetologia 41:1482–1491[CrossRef][Medline]
  69. Brelje TC, Scharp D, Lacy PE, Ogren L, Talamantes F, Robertson M, Friesen HG, Sorenson RL 1993 Effect of homologous placental lactogens, prolactins, and growth hormones on islet B-cell division and insulin secretion in rat, mouse, and human islets: implication for placental lactogen regulation of islet function during pregnancy. Endocrinology 132:879–887[Abstract]
  70. Cowles RA, Segura BJ, Mulholland MW 2001 Stimulation of rat pancreatic exocrine secretion by cocaine- and amphetamine-regulated transcript peptide. Regul Pept 99:61–68[CrossRef][Medline]
  71. da Silva Xavier G, Leclerc I, Varadi A, Tsuboi T, Moule SK, Rutter GA 2003 Role for AMP-activated protein kinase in glucose-stimulated insulin secretion and preproinsulin gene expression. Biochem J 371:761–774[CrossRef][Medline]
  72. Richards SK, Parton LE, Leclerc I, Rutter GA, Smith RM 2005 Over-expression of AMP-activated protein kinase impairs pancreatic ß-cell function in vivo. J Endocrinol 187:225–235[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
R. A. Augustine and D. R. Grattan
Induction of Central Leptin Resistance in Hyperphagic Pseudopregnant Rats by Chronic Prolactin Infusion
Endocrinology, March 1, 2008; 149(3): 1049 - 1055.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
Y. Imai, H. R. Patel, E. J. Hawkins, N. M. Doliba, F. M. Matschinsky, and R. S. Ahima
Insulin Secretion Is Increased in Pancreatic Islets of Neuropeptide Y-Deficient Mice
Endocrinology, December 1, 2007; 148(12): 5716 - 5723.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/1/258    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow