Abstract
Leptin plays a major role in coordinating the integrated response of the brain to changes in nutritional state. Leptin receptor expressing neurons within the arcuate nucleus (ARC) of the hypothalamus sense circulating leptin and densely innervate other regions of the hypothalamus including the paraventricular nucleus (PVN). In the ARC leptin is known to alter the expression of genes with important roles in the control of energy balance, and our aim is to obtain a more comprehensive picture of leptin’s action in this nuclei. Mice were ad libitum fed, or fasted for 48 hours while receiving either sham or intraperitoneal (i.p.) leptin treatment. We used laser capture microdissection and microarrays to identify leptin regulated transcripts within the ARC. Expression of 639 genes are increased and 452 decreased within the fasted ARC. Leptin regulates 15% and 20% of these genes respectively. In addition to expected changes in Pomc, Agrp, Npy and Cart, pathway analysis indicated that leptin regulated other genes concerned with energy homeostasis and endocrine function. As previously reported for the PVN, leptin also altered the expression of genes involved in nervous system development and synaptic function. However, aside for a small number of such genes (e.g Gap43), leptin influenced the expression of different sets of neuronal developmental genes in the ARC and PVN. In conclusion, this study identifies a set of genes that are, at least in part, regulated by leptin in the ARC, highlighting these as candidates for possible roles in leptin action and resistance.
Introduction
Leptin is an adipocyte-derived hormone that plays a crucial role in the maintenance of energy balance. The circulating levels of leptin vary directly with the amount of fat stores in the body, thus reflecting its nutritional status. It is well established that the majority of leptin’s actions are mediated by the brain, in particular the hypothalamus as a crucial area involved in energy homeostasis. A major site of leptin action is the arcuate nucleus of the hypothalamus (ARC) which contains two distinct neuronal populations that both express the signalling form of the leptin receptor, ObRb, but have opposite effects on feeding. One population, which co-expresses the anorexigenic neuropeptides pro-opiomelanocortin (POMC) and cocaine & amphetamine related transcript (CART) is activated by leptin, while the other, which co-expresses the orexigenic neuropeptides NPY and AGRP is inhibited by leptin (1). The POMC derived peptides α and β-MSH signals to melanocortin 4 receptors (MC4Rs), which are highly expressed in the PVN to decrease food intake, while AGRP targets the MC4R as its endogenous antagonist (1). This pathway is well-characterized, and collectively known as the leptin-melanocortin pathway. Strikingly, mice with defective melanocortin signalling including Mc4r KO and agouti mice are not as obese as ob/ob mice (2), indicating that other leptin dependent neurocircuits independent of the melanocortin pathway are important for the control of energy balance. Additionally, the ARC also projects extensively to other hypothalamic nuclei and brain regions. Thus, in spite of recent progress, molecular mechanisms by which leptin affects energy homeostasis within this hypothalamic nucleus remain to be fully elucidated.
In this study, we used laser capture microdissection (LCM) coupled with transcriptional profiling to identify novel leptin regulated genes in the ARC and further characterize downstream pathways of leptin signalling. We have identified genes in the ARC whose expression is altered in fasting (a state of relative leptin deficiency) and restored with leptin treatment alone. In order to exclude non-specific changes in gene expression that are due to stress associated with fasting, we also analysed in these same animals the transcriptome of the cerebellum, a brain region which does not express the long isoform of leptin receptor, ObRb, and has no known role in the control of energy balance.
Methods
In vivo murine studies
All studies used male SV129 mice purchased from Charles River (Kent UK). Mice were kept at 22°C on 12 hour light/dark cycles (lights on 0700-1900). Prior to all procedures mice were acclimatized for at least one week. The animals were fed on standard laboratory chow (SDS diet) and had free access to water throughout. All mice were matched for bodyweight at the start of the experiment. All experimental procedures were in accordance with regulations and guidelines of the UK Home Office.
We used the protocol as described by Ahima and colleagues (3) for the administration of leptin during a fast. 10 week-old mice were divided into three weight-matched groups (n=4 for the microarray; n=9, n=14, n=9 for fed, fasted and leptin treated groups respectively for QPCR). The fed group had unlimited access to chow and the fasted group had food removed at the onset of light cycle and remained fasted for 48 hours. Both groups received twice daily intraperitoneal (i.p.) injections of saline. The fasted plus leptin group had all food removed at the onset of light cycle, remained fasted for 48 hours and received twice daily i.p. injections of recombinant murine leptin (Amgen) at a dose of 1 μg/g total body weight. At the end of the experiment, the body weights of ad libitum fed, fasted and fasted plus leptin mice were 26.4g ± 0.7g, 20.8g ± 0.4g, 21.5 g ± 0.6g, respectively. All animals were sacrificed by cervical dislocation 12 hours after the last injection. The brains were removed, snap-frozen on powdered dry ice and stored at -80°C until required.
LCM and RNA isolation
14μm coronal sections were sectioned by cryostat and mounted on RNase-free membrane-coated glass slides (P.A.L.M. Membrane Slides, P.A.L.M. MicrolaserTechnologies). Each slide was immediately placed on dry ice. Within 24 hours of sectioning, the frozen sections were thawed and fixed for 30 seconds in 95% ethanol, then rehydrated in 75% and 50% ethanol (30 seconds each).
After fixation, sections were stained with cresyl violet (Ambion) for 30 seconds, then dehydrated in a graded ethanol series (50%, 75%, 95% and 2x100%, 30 seconds each) followed by Histoclear for 5 minutes. All solutions were prepared with RNase-free water (Ambion), all percentages are volume/volume. LCM was performed using a P.A.L.M. MicrolaserSystem (P.A.L.M. Microlaser Technologies). The hypothalamic ARC was microdissected covering the region from -1.46 to - 1.97mm caudal to bregma (36 sections) as defined by Paxinos and Franklin (2001). Following microdissection, the captured cells were kept in RNAlater (Ambion) prior to RNA isolation. Total RNA was isolated from individual nuclei according to the manufacturer’s protocol using the RNAqueous®-Micro Kit (Ambion). Quality and quantity of total RNA was determined using the Agilent BioAnalyzer PicoChip (Agilent; according to manufacturer’s instructions).
RNA amplification and microarray hybridization
Before use in downstream applications, RNA isolated from individual nuclei was amplified using to two rounds of T7-based in vitro transcription (IVT) as we have previously described (4). Briefly, RNA was primed with a T7 promoter-oligo (dT) primer and reverse transcribed to generate first strand cDNA, which was used as a template to synthesize second strand cDNA by DNA polymerase (Two-cycles cDNA Synthesis Kit, Affymetrix UK Ltd.). T7 polymerase is used to transcribe antisense amplified RNA (aRNA; MEGAscript T7 kit, Ambion). The aRNA is then randomly primed to make single strand cDNA, which in turn serves as the template for second strand cDNA synthesis, primed, as in the first round, with a T7 promoter-oligo dT primer to make ds cDNA containing a T7 promoter site. A second T7 IVT step produced the second round of aRNA with biotin labelled ribonucleotide (GeneChip IVT labelling Kit, Affymetrix). The biotin-labeled cRNA was fragmented and hybridized to Affymetrix Murine 430 2.0 GeneChips. The hybridized arrays are stained with streptavidin phycoerythrin conjugate and scanned on an Affymetrix GeneChip 7G scanner.
Microarray analysis
Raw image data was converted to CEL files using Affymetrix GeneChip Operating Software. All downstream analysis of microarray data was performed using GeneSpring GX 7.3 (Agilent). The CEL files were used for both the RMA (5) and GCRMA (6) analyses. After importing the data, each chip was normalized to the 50th centile of the measurements taken from that chip and gene expression data are reported as fold-change from the ‘Fed’ state. Genes were considered to be leptin regulated if they were significantly up- or down-regulated by at least 1.3-fold in the fasted state and returned to within 1.2-fold of the ‘Fed’ state with leptin treatment. Statistical analysis was performed using a one-sample Student’s t-test, looking for statistically differentially expressed genes within each condition. The test was applied to the mean of each normalized value against the baseline value of 1, where genes do not show any differential expression with respect to the control. We considered a p ≤ 0.05 to be significant. Only genes which met the above criteria using RMA and GCRMA were taken forward for further study.
To create the scatter plots, values normalized to RMA, i.e. the log2 values, were used within the Partek Genomics Suite v6.4 programme (Partek Inc., St Louis, MO). The mean intensity values of all genes that were differentially regulated with fasting and leptin treatment were plotted against the values for the fasted state. The values for the leptin treated state were superimposed on the obtained scatter plot.
Quantitative PCR analysis
QPCR analysis was performed using MicroFluidic Cards (Applied Biosystems). Total RNA was amplified as described above, but without the biotin labelling. 100ng of amplified RNA from LCM samples were used in a random-primed first strand cDNA synthesis reaction, using SuperscriptII RT (Invitrogen). The resulting cDNA was diluted 5-fold and TaqMan mastermix (Applied Biosystems) was added. 100 μl of the sample-specific PCR mix were loaded onto each fill reservoir following the manufacturer’s protocol. Quantitative PCR reactions were performed using an ABI 7900HT (Applied Biosystems). Expression results were normalized to Gapdh, β-actin, B2m and 18S. QPCR statistical analysis was performed using Microsoft Excel. P-values were calculated using an unpaired Student’s T-Test. Data is expressed as mean ±SEM.
Results
Comparison of global gene expression changes in the ARC and the PVN
The ARC was removed from each brain using LCM and RNA was extracted and amplified as previously described (4) (Fig1). ARC RNA samples were then labelled and hybridized to murine whole genome oligonucleotide arrays (Affymetrix, Santa Clara). We analyzed the data using two different algorithms: Robust Multiarray Average (RMA) (5) and GC-RMA (6). We first considered the data using RMA. We identified 292 genes (158 negatively regulated; 134 positively regulated) using RMA, that were differentially expressed in a 48 hour fast and were regulated by leptin in the ARC, compared to 2556 genes in the PVN (4). When these data are presented as scatter plots it is clear that the number of genes that are leptin regulated and the magnitude of change in expression are far greater in the PVN than they are in the ARC (Fig1, FigS1).
Figure 1.
Left panel: LCM ARC; Coronal Nissl stained mouse brain section after lasercapture microdissection of the ARC, VMN and DMN at approximately Bregma -1.70 mm. Right panel: Scatter plot of ARC microarray data; Each dot represents a gene and is obtained by plotting the log of the average normalized signal intensities of the leptin regulated gene in the fasted state against the fed state. Red dots represent genes that are downregulated with fasting and the blue ones those that are upregulated in fasting. Signal intensities in the leptin treated state are superimposed in green.
Next, we considered only genes whose expression patterns in both analyses were identical, as both gave very different results from the same experimental data. Using RMA, expression of 934 genes were significantly up-regulated, while using GCRMA, 1211 genes were up-regulated 1.3-fold in fasting, with 702 genes having the same expression pattern across both algorithms. Similarly, RMA and GC-RMA analyses determined that 675 and 1036 genes were down-regulated by fasting, respectively, with only 553 of these genes behaving similarly across both analyses (Fig 2; Tables S1a & S1b). Thus, in the ARC, significantly more genes are up-regulated than down-regulated in fasting. This contrasts with the PVN, where nearly ten times more genes are down-regulated as opposed to up-regulated during a 48-hour fast (4). Of the 543 genes that were down-regulated and 692 genes that were up-regulated in the ARC with fasting, 91 (16.7%) and 53 (7.6%) genes respectively were similarly regulated within the cerebellum and were excluded from further study (Tables S1c and S1d). Of the 452 genes that are down-regulated by fasting specifically in the ARC, 90 (19.9%) were positively rescued by leptin treatment; while of 639 genes up-regulated by fasting, 95 (14.8%) were found to be negatively regulated by leptin (Fig2).
Figure 2. Venn diagrams showing genes that are nutritionally regulated in the ARC according to RMA and GCRMA analysis.
A) 553 genes are down-regulated by fasting, whereas B) 702 genes are up-regulated by fasting according to both analyses. However, after removal of genes that are similarly regulated in the cerebellum, 452 and 639 genes appear to be down- and up-regulated by fasting, respectively. Of those, 90 genes are positively and 95 negatively regulated by leptin.
Genes positively regulated by leptin
Figure 4a lists the top 25 transcripts ranked according to the magnitude their fasting expressions are rescued by leptin treatment. The full list of genes is listed in table S1a. Amongst the genes positively regulated by leptin, are the anorexigenic Cart and Pomc, which are known to be down-regulated in fasting and positively leptin regulated in the ARC (1). This pattern of expression was confirmed using an independent set of biological replicates and TaqMan quantitative RT-PCR (QPCR) (Fig3). Additionally, consistent with the work of Bjorbaek and colleagues, Socs3 (suppressor of cytokine signaling), the seventh gene on the list, is positively regulated by leptin (7) (Table 1). The presence of these genes in the top ranked positively regulated genes validates our approach in this study.
Figure 4.
Confirmation of genes involved in A) energy homeostasis, B) reproduction and C) neuronal development and function to be leptin regulated by TaqMan quantitative RT-PCR. Gene expression was normalized to the housekeeping control Gapdh and values represent the means +/-SEM of n≥5 in each group. Changes in expression were regarded to be significant if the p-value obtained using a one-tailed, unpaired student’s t-test was < 0.05.
Figure 3. TaqMan was used to confirm that Cart, Pomc, AgRP and Npy change expression as expected.
Gene expression was normalized to the housekeeping control Gapdh and values represent the means +/- SEM of n≥5 in each group. Changes in expression were regarded to be significant if the p-value obtained using a one-tailed, unpaired student’s t-test was < 0.05.
Table 1. Top 25 genes that are positively regulated by leptin.
Genes were considered to be leptin regulated if they significantly changed expression at least 1.3 fold with fasting and returned at least 1.2 fold towards levels measured in the fed state according to GCRMA and RMA. They were ranked by the absolute fold change in expression in the fasted state as compared to the leptin treated state.
| Affymetrix ID | Fed/Fast | Fast/Lep | Genbank | Gene | Description |
|---|---|---|---|---|---|
| 1433413_at | -1.78 | 2.50 | AK006863 | Nrxn1 | neurexin I |
| 1456316_a_at | -1.86 | 2.24 | BI965035 | Acbd3 | acyl-Coenzyme A binding domain containing 3 |
| 1419593_at | -2.65 | 1.89 | NM_015764 | Greb1 | gene regulated by estrogen in breast cancer protein |
| 1417812_a_at | -2.17 | 1.89 | NM_008484 | Lamb3 | laminin, beta 3 |
| 1456212_x_at | -1.92 | 1.80 | BB831725 | Socs3 | Suppressor of cytokine signaling 3 |
| 1423171_at | -1.53 | 1.73 | BE947345 | Gpr88 | G-protein coupled receptor 88 |
| 1453145_at | -2.00 | 1.69 | AK007420 | 4933439C20Rik | RIKEN cDNA 4933439C20 gene |
| 1430387_at | -2.07 | 1.67 | AK007978 | 1810073O08Rik | RIKEN cDNA 1810073O08 gene |
| 1421471_at | -1.55 | 1.64 | NM_010934 | Npy1r | neuropeptide Y receptor Y1 |
| 1437409_s_at | -1.84 | 1.62 | BB812574 | Gpr126 | G protein-coupled receptor 126 |
| 1455899_x_at | -2.23 | 1.57 | BB241535 | Socs3 | Suppressor of cytokine signaling 3 |
| 1441588_at | -1.47 | 1.56 | BB487239 | Kcnq1 | Potassium voltage-gated channel, subfamily Q, member 1 |
| 1416266_at | -1.54 | 1.56 | AF026537 | Pdyn | prodynorphin |
| 1435353_a_at | -1.78 | 1.56 | BI454991 | 4933439C20Rik | CDNA, clone:Y1G0128F22, strand:plus |
| 1429076_a_at | -1.78 | 1.55 | BB550907 | Gdpd2 | Glycerophosphodiester phosphodiesterase domain containing 2 |
| 1418478_at | -2.20 | 1.55 | NM_057173 | Lmo1 | LIM domain only 1 |
| 1449876_at | -1.45 | 1.52 | NM_011160 | Prkg1 | protein kinase, cGMP-dependent, type I |
| 1439568_at | -2.66 | 1.48 | AV373997 | Greb1 | gene regulated by estrogen in breast cancer protein |
| 1450798_at | -1.46 | 1.44 | NM_031176 | Tnxb | tenascin XB |
| 1422825_at | -1.68 | 1.43 | NM_013732 | Cartpt | CART prepropeptide |
| 1419008_at | -1.38 | 1.42 | NM_016708 | Npy5r | neuropeptide Y receptor Y5 |
| 1425148_a_at | -1.33 | 1.42 | BC025911 | Snx6 | sorting nexin 6 |
| 1435110_at | -1.79 | 1.42 | BG065285 | Unc5b | unc-5 homolog B (C. elegans) |
| 1417760_at | -2.17 | 1.42 | NM_007430 | Nr0b1 | nuclear receptor subfamily 0, group B, member 1 |
| 1439725_at | -1.29 | 1.41 | BB384963 | Ptprt | Protein tyrosine phosphatase, receptor type, T |
Interestingly, the top two genes on this list play important roles in neuronal development and function (Table 1). Neurexin1 (Nrxn1), encodes a cell surface molecule with major synaptogenic activity (8), while Acyl coenzyme-A binding domain-containing 3 (Acbd3), the second gene on the list, has been implicated in the control of neuronal cell fate (9).
Genes negatively regulated by leptin
Table 2 shows the top 25 genes upregulated in fasting and negatively regulated by leptin. As expected, expression of orexigenic Agrp and Npy, as determined by both microarray (Table 2 and Table S1b) and QPCR (Fig 3), increases with fasting and is rescued by leptin administration. Intriguingly, Sycp3, the second gene on the list, and Svs2, the sixth gene on the list, have been implicated in the control of male fertility (10, 11). However, nothing is known about their function in the brain, and no interaction with leptin has yet been described.
Table 2. Top 25 genes that are negatively regulated by leptin.
Genes were considered to be leptin regulated if they significantly changed expression at least 1.3 fold with fasting and returned at least 1.2 fold towards levels measured in the fed state according to GCRMA and RMA. They were ranked by the absolute fold change in expression in the fasted state as compared to the leptin treated state.
| Affymetrix ID | Fed/Fast | Fast/Lep | Genbank | Gene | Description |
|---|---|---|---|---|---|
| 1429379_at | 6.60 | -3.07 | AV124537 | Xlkd1 | extra cellular link domain-containing 1 |
| 1449534_at | 2.69 | -1.77 | NM_011517 | Sycp3 | synaptonemal complex protein 3 |
| 1444763_at | 2.37 | -1.72 | BB667296 | Ptprk | Transcribed locus |
| 1449347_a_at | 3.16 | -1.70 | NM_021365 | Xlr4b | X-linked lymphocyte-regulated 4B |
| 1439795_at | 3.92 | -1.62 | AV242919 | Gpr64 | G protein-coupled receptor 64 |
| 1422427_a_at | 7.53 | -1.61 | NM_017390 | Svs2 | seminal vesicle secretory protein 2 |
| 1449457_at | 1.91 | -1.58 | AB078618 | Acot12 | acyl-CoA thioesterase 12 |
| 1458148_at | 2.82 | -1.54 | BB466171 | Nlrc3 | NLR family, CARD domain containing 3 |
| 1453332_at | 2.56 | -1.53 | AK012141 | 2410002O22Rik | RIKEN cDNA 2410002O22 gene |
| 1438295_at | 2.40 | -1.49 | BM247146 | Glcci1 | Mus musculus cDNA clone NIA:K0747G12 |
| 1421690_s_at | 2.90 | -1.49 | NM_007427 | Agrp | agouti related protein |
| 1460053_at | 2.30 | -1.49 | BB021163 | Smyd4 | SET and MYND domain containing 4 |
| 1417483_at | 2.97 | -1.49 | AB026551 | Nfkbiz | nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, zeta |
| 1420416_at | 1.94 | -1.47 | NM_009152 | Sema3a | semaphorin 3A |
| 1434585_at | 2.04 | -1.45 | BB667130 | BB667130 | RIKEN cDNA 2210038L17 gene |
| 1419405_at | 2.19 | -1.44 | NM_026523 | Nmb | neuromedin B |
| 1454079_at | 1.94 | -1.44 | AK016591 | 4933400F03Rik | RIKEN cDNA 4933400F03 gene |
| 1434653_at | 1.55 | -1.43 | AV026976 | Ptk2b | PTK2 protein tyrosine kinase 2 beta |
| 1449164_at | 2.08 | -1.43 | BC021637 | Cd68 | CD68 antigen |
| 1421818_at | 1.99 | -1.40 | U41465 | Bcl6 | B-cell leukemia/lymphoma 6 |
| 1453713_s_at | 2.66 | -1.40 | AK016052 | 4930546H06Rik | RIKEN cDNA 4930546H06 gene |
| 1449827_at | 2.14 | -1.40 | NM_007424 | Acan | aggrecan |
| 1458397_at | 2.00 | -1.39 | BB260315 | Dnaja1 | DnaJ (Hsp40) homolog, subfamily A, member 1 |
| 1446840_at | 1.79 | -1.39 | BQ032894 | Thrap1 | Transcribed locus |
| 1444569_at | 1.60 | -1.38 | BB479211 | EG666752 | 13 days embryo heart cDNA |
Pathway analysis
Next, we analyzed the data using pathway analysis (Ingenuity Pathway Analysis), which detects groups of functionally-related annotated genes. Of the molecular and cellular functions; behaviour, digestive system development & function, and nutritional disease & metabolic disease were the top functions positively regulated by leptin (Table 3a; Tables S2a & S3a). Genes involved in ‘behaviour’ are bi-directionally regulated by leptin as they also appear in the list of functions negatively regulated by leptin (Table 3b). These functional groupings encompass many of the known genes that are key players in the regulation of energy homeostasis (e.g. Pomc, Cart, Agrp & Npy) and are known to be nutritionally and leptin regulated within the ARC. Secondly, genes involved in endocrine system development and function, in particular a number of those involved in the regulation of reproduction, also appear to be regulated by leptin in both directions. Congruent with this, the estrogen receptor signalling pathway was identified as significantly positively regulated by leptin (Table3a; Table S2b). Finally, the third group of genes we identify to be significantly regulated by leptin bi-directionally within the ARC are involved in nervous system development and function.
Table 3. Ingenuity pathway analysis of A) positively and B) negatively leptin regulated genes.
Genes involved in the regulation of energy homeostasis, reproduction and neuronal development and function are significantly and of note, bi-directionally leptin regulated in the ARC.
| A | B | |||
|---|---|---|---|---|
| Molecular and Cellular Fuction | p-value | Molecular and Cellular Function | p-value | |
| Behavior | 4.87E-09 | Cell Morphology | 3.99E-05 | |
| Digestive System Development and Function | 2.36E-07 | Cellular Compromise | 3.99E-05 | |
| Nutritional Disease | 1.68E-06 | Hepatic System Development and Function | 3.99E-05 | |
| Metabolic Disease | 2.99E-05 | Infection Mechanism | 1.19E-04 | |
| Nervous System Development and Function | 3.20E -05 | Lipid Metabolism | 1.19E-04 | |
| Small Molecule Biochemistry | 1.19E-04 | |||
| Tissue Morphology | 3.20E-05 | Amino Acid Metabolism | 1.30E-04 | |
| Organismal Development | 6.48E-05 | Molecular Transport | 1.30E-04 | |
| Genetic Disorder | 9.83E-05 | Endocrine System Development and Function | 1.88E-04 | |
| Small Molecule Biochemistry | 1.29E-04 | Cell Cycle | 2.38E-04 | |
| Carbohydrate Metabolism | 1.55E-04 | Cellular Assembly and Organization | 2.38E-04 | |
| Cell Morphology | 1.55E-04 | Connective Tissue Development and Function | 2.38E-04 | |
| Cellular Movement | 1.72E-04 | Skeleton and Muscular System Development and Function | 2.38E 04 | |
| Endocrine System Development and Function | 1.92E-04 | Behavior | 3.20E-04 | |
| Organ Development | 2.77E-04 | Nervous System Development and Function | 3.20E-04 | |
| Pathways | p-value | Pathways | p-value | |
| IL-9 Signaling | 0.020893 | Glycosphingolipid Biosynthesis - Neolactoseries | 0.008511 | |
| Calcium Signaling | 0.033884 | Fructose and Mannose Metabolism | 0.019498 | |
| Estrogen Receptor Signaling | 0.040738 | IL-2 Signaling | 0.044668 | |
QPCR validation of microarray data
Thus, the three categories of genes that are regulated by leptin within the ARC are involved in a.) control of energy homeostasis; b.) regulation of reproduction and c.) nervous system development and function. We selected a number of genes from these three functional groupings and validated their expression profile by QPCR in samples obtained from biological replicates. For the energy homeostasis category, in addition to Pomc, Cart, Agrp & Npy, which we discuss above, Npy5r and galanin are positively regulated, while Acot 12 and Nmb are negatively by leptin (Fig4a). The expression of Nmbr was highly increased in fasting with a trend towards down-regulation by leptin. NrOb1 and Svs2, which are implicated in the maintenance of fertility were confirmed to be positively and negatively regulated by leptin respectively (Fig4b). Finally, of the genes involved in neuronal development and function, Neurexin 1, Lmo1 and Acbd3 are all down-regulated in the fasted state and are positively regulated by leptin, although only Acbd3 reached statistical significance. Trt was up-regulated in fasting and was negatively regulated by leptin (Fig4c).
Differential regulation of genes involved in neuronal development & function in ARC and PVN
The observation that genes involved in nervous system development are co-ordinately regulated by leptin in the ARC is congruent with data we have previously reported, that this similar class of genes are leptin regulated within the PVN (4). We tested, in our ARC samples, the expression of five genes that are positively regulated by leptin within the PVN and all have a role to play in synaptic function or plasticity (Table 4; FigS2). Gap43, a pivotal gene for synapse formation, shows a similar expression pattern in the ARC and PVN (Table 4) (4). ApoE and Gabarap were down-regulated in fasting in both nuclei, but were not leptin regulated in the ARC, while no changed in Nnat and Basigin expression were observed in the ARC. Thus, for the most part, there is little overlap between the individual nervous system genes that are leptin regulated in the ARC and PVN (Table 4). Additionally, Neurexin1, as discussed above, is positively regulated by leptin within the ARC, while another member of the Neurexin superfamily, Cntnap2 (Neurexin4) is negatively regulated by leptin in the PVN (4).
Table 4. Differential regulation of synaptic plasticity genes in the PVN and the ARC.
Gap43 is significantly downregulated with fasting and rescued toward levels in the fed state with leptin treatment in both the ARC and the PVN. However, ApoE, Gabarap, Nnat and Basigin undergo differential regulation in the PVN and the ARC.
| PVN | ARC | |||
|---|---|---|---|---|
| Gene of interest | Fasting | Leptin | Fasting | Leptin |
| Gap43 |
 * |
|
* |
|
| ApoE |
* |
|
* |
|
| Gabarap |
* |
* |
|
|
| Nnat |
* |
|
|
|
| Basigin |
* |
* |
|
|
Discussion
In this study, we compared the expression profiles of laser captured ARC from ad libitum fed mice, fasted mice and mice given leptin during a similar fast, in order to gain insights into molecules and pathways mediating downstream signalling actions of leptin. We compare the expression profile of the ARC to that of the PVN in order to reveal global differences in how each nuclei responds to fasting and leptin treatment. However, these gene changes may be due to direct and/or indirect actions of leptin on these neurons, and these studies do not distinguish between the two.
The ARC and PVN respond differently to fasting and leptin treatment
It is unsurprising that the ARC and PVN would display different gene expression profiles in response to fasting and leptin treatment. These are, after all, two distinct regions of the hypothalamus, expressing different genes with different roles to play in the control of energy homeostasis. What is surprising are the differences that occur on a global level. Many more genes are regulated by leptin in the PVN than ARC. Although a similar number of genes were negatively regulated by leptin in both nuclei, ten-fold more genes are positively regulated by leptin in the PVN. Further, scatter plots demonstrate that not only are more genes regulated by leptin in the PVN, but the magnitude of change is far greater than for those in the ARC.
The changes in gene expression might be less dramatic in the ARC because of the presence of two populations of leptin responsive neurons; anorectic Pomc/Cart, which are activated and orexigenic Agrp/Npy, which are inhibited by leptin. Consistent with this, Agrp and Npy were found to be negatively regulated by leptin in the ARC, whereas Pomc and Cart were regulated in a reciprocal manner. Even though LCM allows us to specifically avoid contamination with neighbouring nuclei, it was not used in this experiment to isolate single cells expressing defined neuropeptides. Hence, although changes in gene expression are likely to be as marked in specific ARC neuronal populations as they are the PVN, they are masked by the fact that the anorectic Pomc/Cart and orexigenic Agrp/Npy neurons regulate a similar set of genes but in opposing directions, thus diminishing the net effect on change in transcript levels. The PVN however, densely expresses MC4R and canonically plays more of a role in satiety. Thus, the major effect of leptin in the PVN is to positively regulate genes whose expression levels were down-regulated by fasting.
As we have previously reported, the oxidative phosphorylation pathway is the most significantly leptin regulated pathway in the PVN, which led us to speculate that modulation of OXPHOS is an important feature of the brain’s response to food deprivation and leptinergic tone. Interestingly, this pathway does not appear to be nutritionally or leptin regulated in the ARC. If however, the positive regulation of OXPHOS genes by leptin activation of PVN neurons forms a critical part of the satiety response, it is entirely possible that in neurons inhibited by leptin, this same pathway would be regulated in a reciprocal manner. Thus, in the ARC, with the presence of neuronal populations both activated and inhibited by leptin, the changes in OXPHOS gene expression might, in effect, cancel themselves out.
Bi-directional regulation of gene pathways within the ARC
The genes which are nutritionally and leptin regulated within the ARC can be broadly classified into those involved in a.) energy homeostasis; b.) regulation of reproduction and c.) neuronal development and function. Unlike the PVN, where pathways or functions are enriched in either positively or negatively regulated groups of genes, these three categories of genes are bi-directionally regulated by leptin within the ARC.
It is well-established that the ARC plays a crucial role in the central control of energy balance; it is therefore unsurprising that molecules that are crucial for the maintenance of energy homeostasis are differentially regulated by leptin in both directions within this nucleus. We have already discussed Pomc, Cart, Npy and Agrp. Other genes in this category include Socs3, which exerts a negative effect on leptin receptor signalling and, consistent with previous findings (12), is positively regulated by leptin in the ARC. Of note, Npy1r and Npy5r, both cognate receptors of Npy, are regulated in a similar manner in the ARC. In contrast, neuromedin B (Nmb) and its receptor are negatively regulated by leptin as confirmed by QPCR. Nmb has recently been shown to be a potent activator of Npy neurons in the mediobasal hypothalamus (13). Thus, it is plausible that Nmb expression, enhanced during fasting, promotes the neuronal activity of Npy neurons, resulting in an increase of food intake.
As has been demonstrated in both mice and humans, leptin, in addition to its role in the control of energy homeostasis, also has a well established in the control of reproduction. Leptin deficiency results in infertility in both species and can be restored by administration of exogenous leptin (14, 15). One of the top genes which we identify in this study is NrOb1, which encodes a nuclear receptor transcription factor of critical importance for the development of reproductive organs, and is down-regulated with fasting and rescued with leptin treatment. Mice and humans lacking this gene display with a complex disease spectrum including hypogonadotrophic hypogonadism (16, 17). Of note, NrOb1 knockout mice with a male genotype are characterized by defects in testis development as early as in the fetal stage, resulting in complete gonadal sex reversal (18). Concordant with the animal model, a male to female sex reversal phenotype has been reported for a subset of humans with dysregulated NROB1 levels (19).
The opioid hormone prodynorphin, which affects multiple functions including reproduction was also positively regulated by leptin in the ARC. Work by Navarro and colleagues suggests that prodynorphin exerts its role in central regulation of reproduction by mediating estradiol negative feedback on gonadotropin-releasing hormone (GnRH) neurons leading to reduced LH secretion (20).
Distinct genes involved in synaptic plasticity are leptin regulated in the ARC and the PVN
Despite differences in the transcriptome of the ARC and PVN, pathway analysis revealed genes involved in neuronal development and function to be co-ordinately leptin regulated in both nuclei (4). It is becoming clear that in addition to engaging classical ‘neuropeptide/receptor’ systems within the brain, leptin also rapidly modifies synaptic connections between neurons. Leptin has been demonstrated to be necessary for both normal development of neuronal projections within the hypothalamus (21) as well as in the regulation of synaptic plasticity (22). Thus, it is interesting that the top positively regulated gene by leptin within the ARC, neurexin1, plays a role in the formation of synaptic contacts through binding of neuroligins (8). Recently, the deletion of this gene was shown to cause autism in humans (23). A role in synaptic function has also been suggested for the semaphorin Sema3a, which is upregulated with fasting and comes back down to fed levels with leptin treatment according to the microarray data. In addition to its role in axon guidance, Sema3a is critical for dentritic spine maturation, thus modulating neuronal connectivity in the cerebral cortex (24).
When we tested the expression of PVN leptin regulated neuronal genes within the ARC, Gap43, a pivotal gene for synapse function, shows the same pattern of regulation in both nuclei. Gap43 is an intrinsic determinant of neuronal development (25), and its co-regulation by leptin implies that leptin mediated synaptic plasticity is physiologically relevant in both PVN and ARC. However, most synaptic plasticity genes that were identified to be leptin regulated in the PVN are not differentially regulated in the ARC, suggesting that the molecular mechanisms underlying synaptic rewiring in response to leptin is different for ARC and the PVN.
Conclusion
The adipokine leptin regulates the transcriptome of the ARC and PVN. Genes involved in energy homeostasis, reproduction and neuronal function & development are regulated by leptin in a bi-directional manner within the ARC. Notably, comparison of global gene expression changes in the ARC and PVN in response to fasting and leptin treatment reveals striking differences, which are of critical importance to mediating leptin signalling in the hypothalamus. Changes in gene expression arising from fasting and leptin treatment are modest in the ARC as compared to PVN, both in terms of numbers of genes and their magnitude of change. Despite these differences, genes mediating synaptic remodelling are co-ordinately leptin regulated in both nuclei. However, the ARC and PVN appear to recruit different sets of synaptic plasticity genes to rewire the neuronal connections in response to leptin. Further work will be needed to illuminate the underlying molecular machinery necessary for leptin’s actions on synaptic plasticity and remodelling within the ARC and PVN.
Supplementary Material
Acknowledgements
This study was supported by the UK Medical Research Council Centre for Obesity and Related metabolic Disorders (MRC-CORD), the Wellcome Trust, the EU (FP6: EUGENE2, LSHM-CT-2004-512013 and FP7- HEALTH- 2009- 241592 EurOCHIP). ZJ is a Cambridge Gates Scholar.
References
- 1.Coll AP, Farooqi IS, O’Rahilly S. The hormonal control of food intake. Cell. 2007;129(2):251–62. doi: 10.1016/j.cell.2007.04.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Huszar D, Lynch CA, Fairchild-Huntress V, Dunmore JH, Fang Q, Berkemeier LR, Gu W, Kesterson RA, Boston BA, Cone RD, Smith FJ, et al. Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell. 1997;88(1):131–41. doi: 10.1016/s0092-8674(00)81865-6. [DOI] [PubMed] [Google Scholar]
- 3.Ahima RS, Prabakaran D, Mantzoros C, Qu D, Lowell B, Maratos-Flier E, Flier JS. Role of leptin in the neuroendocrine response to fasting. Nature. 1996;382(6588):250–2. doi: 10.1038/382250a0. [DOI] [PubMed] [Google Scholar]
- 4.Tung YC, Ma M, Piper S, Coll A, O’Rahilly S, Yeo GS. Novel leptin-regulated genes revealed by transcriptional profiling of the hypothalamic paraventricular nucleus. J Neurosci. 2008;28(47):12419–26. doi: 10.1523/JNEUROSCI.3412-08.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res. 2003;31(4):e15. doi: 10.1093/nar/gng015. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Wu ZJ, Irizarry RA, Gentleman R, Martinez-Murillo F, Spencer F. A Model-Based Background Adjustment for Oligonucleotide Expression Arrays. Journal of American Statistical Association. 2004;99:909–17. [Google Scholar]
- 7.Bjorbaek C, El-Haschimi K, Frantz JD, Flier JS. The role of SOCS-3 in leptin signaling and leptin resistance. J Biol Chem. 1999;274(42):30059–65. doi: 10.1074/jbc.274.42.30059. [DOI] [PubMed] [Google Scholar]
- 8.Graf ER, Zhang X, Jin SX, Linhoff MW, Craig AM. Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins. Cell. 2004;119(7):1013–26. doi: 10.1016/j.cell.2004.11.035. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Zhou Y, Atkins JB, Rompani SB, Bancescu DL, Petersen PH, Tang H, Zou K, Stewart SB, Zhong W. The mammalian Golgi regulates numb signaling in asymmetric cell division by releasing ACBD3 during mitosis. Cell. 2007;129(1):163–78. doi: 10.1016/j.cell.2007.02.037. [DOI] [PubMed] [Google Scholar]
- 10.Kawano N, Yoshida M. Semen-coagulating protein, SVS2, in mouse seminal plasma controls sperm fertility. Biol Reprod. 2007;76(3):353–61. doi: 10.1095/biolreprod.106.056887. [DOI] [PubMed] [Google Scholar]
- 11.Miyamoto T, Hasuike S, Yogev L, Maduro MR, Ishikawa M, Westphal H, Lamb DJ. Azoospermia in patients heterozygous for a mutation in SYCP3. Lancet. 2003;362(9397):1714–9. doi: 10.1016/S0140-6736(03)14845-3. [DOI] [PubMed] [Google Scholar]
- 12.Baskin DG, Breininger JF, Schwartz MW. SOCS-3 expression in leptin-sensitive neurons of the hypothalamus of fed and fasted rats. Regul Pept. 2000;92(1-3):9–15. doi: 10.1016/s0167-0115(00)00143-9. [DOI] [PubMed] [Google Scholar]
- 13.van den Pol AN, Yao Y, Fu LY, Foo K, Huang H, Coppari R, Lowell BB, Broberger C. Neuromedin B and gastrin-releasing peptide excite arcuate nucleus neuropeptide Y neurons in a novel transgenic mouse expressing strong Renilla green fluorescent protein in NPY neurons. J Neurosci. 2009;29(14):4622–39. doi: 10.1523/JNEUROSCI.3249-08.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Chehab FF, Lim ME, Lu R. Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nat Genet. 1996;12(3):318–20. doi: 10.1038/ng0396-318. [DOI] [PubMed] [Google Scholar]
- 15.Farooqi IS, Matarese G, Lord GM, Keogh JM, Lawrence E, Agwu C, Sanna V, Jebb SA, Perna F, Fontana S, Lechler RI, et al. Beneficial effects of leptin on obesity, T cell hyporesponsiveness, and neuroendocrine/metabolic dysfunction of human congenital leptin deficiency. J Clin Invest. 2002;110(8):1093–103. doi: 10.1172/JCI15693. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Muscatelli F, Strom TM, Walker AP, Zanaria E, Recan D, Meindl A, Bardoni B, Guioli S, Zehetner G, Rabl W, et al. Mutations in the DAX-1 gene give rise to both X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism. Nature. 1994;372(6507):672–6. doi: 10.1038/372672a0. [DOI] [PubMed] [Google Scholar]
- 17.Trivedi P, Yu H, MacNeil DJ, Van der Ploeg LH, Guan XM. Distribution of orexin receptor mRNA in the rat brain. FEBS Lett. 1998;438(1-2):71–5. doi: 10.1016/s0014-5793(98)01266-6. [DOI] [PubMed] [Google Scholar]
- 18.Bouma GJ, Albrecht KH, Washburn LL, Recknagel AK, Churchill GA, Eicher EM. Gonadal sex reversal in mutant Dax1 XY mice: a failure to upregulate Sox9 in pre-Sertoli cells. Development. 2005;132(13):3045–54. doi: 10.1242/dev.01890. [DOI] [PubMed] [Google Scholar]
- 19.Smyk M, Berg JS, Pursley A, Curtis FK, Fernandez BA, Bien-Willner GA, Lupski JR, Cheung SW, Stankiewicz P. Male-to-female sex reversal associated with an approximately 250 kb deletion upstream of NR0B1 (DAX1. Hum Genet. 2007;122(1):63–70. doi: 10.1007/s00439-007-0373-8. [DOI] [PubMed] [Google Scholar]
- 20.Navarro VM, Gottsch ML, Chavkin C, Okamura H, Clifton DK, Steiner RA. Regulation of gonadotropin-releasing hormone secretion by kisspeptin/dynorphin/neurokinin B neurons in the arcuate nucleus of the mouse. J Neurosci. 2009;29(38):11859–66. doi: 10.1523/JNEUROSCI.1569-09.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Bouret SG, Draper SJ, Simerly RB. Trophic action of leptin on hypothalamic neurons that regulate feeding. Science. 2004;304(5667):108–10. doi: 10.1126/science.1095004. [DOI] [PubMed] [Google Scholar]
- 22.Pinto S, Roseberry AG, Liu H, Diano S, Shanabrough M, Cai X, Friedman JM, Horvath TL. Rapid rewiring of arcuate nucleus feeding circuits by leptin. Science. 2004;304(5667):110–5. doi: 10.1126/science.1089459. [DOI] [PubMed] [Google Scholar]
- 23.Kim HG, Kishikawa S, Higgins AW, Seong IS, Donovan DJ, Shen Y, Lally E, Weiss LA, Najm J, Kutsche K, Descartes M, et al. Disruption of neurexin 1 associated with autism spectrum disorder. Am J Hum Genet. 2008;82(1):199–207. doi: 10.1016/j.ajhg.2007.09.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Morita A, Yamashita N, Sasaki Y, Uchida Y, Nakajima O, Nakamura F, Yagi T, Taniguchi M, Usui H, Katoh-Semba R, Takei K, et al. Regulation of dendritic branching and spine maturation by semaphorin3A-Fyn signaling. J Neurosci. 2006;26(11):2971–80. doi: 10.1523/JNEUROSCI.5453-05.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Benowitz LI, Routtenberg A. GAP-43: an intrinsic determinant of neuronal development and plasticity. Trends Neurosci. 1997;20(2):84–91. doi: 10.1016/s0166-2236(96)10072-2. [DOI] [PubMed] [Google Scholar]
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