Skip to main content
NCBI home page
The Journal of Physiology logoLink to The Journal of Physiology
. 2009 Jun 2;587(Pt 14):3573–3585. doi: 10.1113/jphysiol.2009.173328

Leptin receptor gene expression and number in the brain are regulated by leptin level and nutritional status

Sharon E Mitchell 1, Ruben Nogueiras 2,3, Amanda Morris 1, Sulay Tovar 2, Christine Grant 1, Morven Cruickshank 1, D Vernon Rayner 1, Carlos Dieguez 2,3, Lynda M Williams 1
PMCID: PMC2742282  PMID: 19491239

Abstract

Hormone potency depends on receptor availability, regulated via gene expression and receptor trafficking. To ascertain how central leptin receptors are regulated, the effects of leptin challenge, high-fat diet, fasting and refeeding were measured on leptin receptor number and gene expression. These were measured using quantitative 125I-labelled leptin in vitro autoradiography and in situ hybridisation, respectively. Ob-R (all forms of leptin receptor) expression in the choroid plexus (CP) was unchanged by high-fat diet or leptin challenge, whereas fasting increased but refeeding failed to decrease expression. 125I-labelled leptin binding to the CP was increased by fasting and returned to basal levels on refeeding. 125I-Labelled leptin was reduced by leptin challenge and increased by high-fat feeding. Ob-Rb (signalling form) in the arcuate (ARC) and ventromedial (VMH) nuclei was increased after fasting and decreased by refeeding. Leptin challenge increased Ob-Rb expression in the ARC, but not after high-fat feeding. In general, changes in gene expression in the ARC and VMH appeared to be largely due to changes in area rather than density of labelling, indicating that the number of cells expressing Ob-Rb was the parameter that contributed most to these changes. Leptin stimulation of suppressor of cytokine signalling 3 (SOCS3), a marker of stimulation of the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway, was unchanged after high-fat diet. Thus, early loss of leptin sensitivity after high-fat feeding is unrelated to down-regulation of leptin receptor expression or number and does not involve the JAK/STAT pathway. The effect of leptin to decrease 125I-labelled leptin binding and the loss of ability of leptin to up-regulate Ob-Rb expression in the ARC after high-fat feeding offer potential mechanisms for the development of leptin insensitivity in response to both hyperleptinaemia and high-fat diet.

Leptin secreted by adipocytes acts in the hypothalamus to regulate food intake and energy expenditure, thereby limiting adiposity (Campfield et al. 1995; Halaas et al. 1995; Pelleymounter et al. 1995). It is now widely recognised that in obesity, where leptin levels are high but fail to limit adiposity, leptin insensitivity occurs (Munzberg & Myers, 2005). This may be due to restricted entry of leptin into the brain and/or failure in the response of leptin receptive neurons (van Heek et al. 1997; El Haschimi et al. 2000; Jeanrenaud & Rohner-Jeanrenaud, 2001). The transport of leptin into the brain and neuronal sensitivity to the leptin signal depend on the availability of leptin receptors at the blood–brain barrier and on the leptin receptive neurons, respectively. Transport of leptin into the brain is thought to be via the short forms of the leptin receptor (Ob-Ra and Ob-Rc) present in the choroid plexus (CP) and brain microvessels (Bjorbaek et al. 1998). Leptin signalling in neurons is dependent on the presence of the long form of the leptin receptor (Ob-Rb) (Mercer et al. 1996), which signals principally via the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway (Hubschle et al. 2001). Leptin challenge rapidly induces expression of the suppressor of cytokine signalling 3 (SOCS3), which inhibits leptin signalling (Bjorbaek et al. 1999; Howard et al. 2004), and SOCS3 gene expression has been used as a marker for leptin stimulation of the JAK/STAT pathway (Tups et al. 2004). Alterations in both receptor gene expression, and the endocytosis and trafficking of ligand-activated cell surface receptors can regulate receptor availability and response to ligand.

Relatively little is known about the co-regulation of Ob-R and Ob-Rb gene expression and receptor number, or the impact of receptor regulation on leptin sensitivity. Both the lack of circulating leptin, as in the ob/ob mouse, and the inability to respond to the leptin signal, as in the Zucker rat, up-regulate Ob-Rb gene expression (Mercer et al. 1997; Bennett et al. 1998), while leptin challenge has been reported to down-regulate expression. Fasting, where leptin levels drop, up-regulates Ob-Rb gene expression (Baskin et al. 1998; Bennett et al. 1998) and increases 125I-labelled leptin binding in the rat arcuate nuclei (ARC) (Baskin et al. 1999). These studies indicate that leptin concentration is important in regulating gene expression and receptor number. However, changes in Ob-R and Ob-Rb gene expression have also been shown to be nutrition and leptin independent, with changes occurring in the ARC and ventromedial nuclei (VMH) during the oestrous cycle (Bennett et al. 1998, 1999), and in the VMH during pregnancy and lactation (Brogan et al. 2000; Ladyman & Grattan, 2005).

Diet induced obesity in both humans and rodents results in increased circulating levels of leptin, and leptin insensitivity (Frederich et al. 1995). Both hyperleptinaemia (Montez et al. 2005; Scarpace et al. 2005) and high-fat feeding (Tulipano et al. 2004) have been shown to contribute to leptin insensitivity, while calorie restriction increases sensitivity to leptin (Wilsey & Scarpace, 2004). Ob-R gene expression has been reported to increase initially in leptin insensitivity induced by high-fat feeding, followed by a subsequent down-regulation (Lin et al. 2000). Also, reduced leptin receptor gene expression and receptor protein levels have been associated with diet induced obesity in rodents (Zhang & Scarpace, 2006). However, no change in Ob-Rb gene expression was found in C57Bl/6 mice on a high-fat diet (Haltiner et al. 2004; Munzberg et al. 2004), even when shown to be fully leptin resistant (Enriori et al. 2007). Also, at variance with the expected down-regulation of receptor expression resulting from ligand stimulation, Ob-Rb gene expression has been shown to increase in response to leptin challenge (Haltiner et al. 2004; Tang et al. 2007; Di Yorio et al. 2008).

Thus, the present study aims to define the control of Ob-Rb gene expression in the mouse by nutritional status and leptin challenge in the hypothalamic ARC and VMH, areas important in appetite control, together with Ob-R gene expression and receptor number in the CP, part of the system transporting leptin into the brain. The impact of relatively short-term high-fat feeding on leptin receptor regulation in the C57Bl/6 mouse was also investigated. Neuronal insensitivity to leptin has been reported in this model after 4 weeks on a high-fat diet (Munzberg et al. 2004).

As both alterations in receptor gene expression, and endocytosis and trafficking of ligand-activated cell surface receptors can modulate the expression of the receptor protein, we measured both gene expression and the number of leptin receptors available to bind ligand using in situ hybridisation and 125I-labelled leptin binding, respectively. Leptin was delivered to the animal systemically via intraperitoneal (i.p.) injection and centrally by intracerebroventricular (i.c.v.) injections bypassing the leptin transport system in the blood–brain barrier.

Methods

Experimental animals

In the UK, all animal studies were licensed under the Animal (Scientific Procedures) Act 1986 and were approved by the Ethical Review Committee of the Rowett Research Institute. In Spain, all animal experimental procedures were regulated by Santiago de Compostela Medical School Animal Care Research Committee. The i.c.v. experiments and the fasting and refeeding experiments were carried out in Spain. All other experiments were carried out in the UK.

Male C57BL/6 mice were part of a first generation bred at the Rowett Research Institute from mice obtained from Harlan (Oxon, UK). In Spain C57BL/6 mice were obtained from Harlan Ibérica (Barcelona, Spain). All mice were 10–12 weeks of age at the start of experiment. Animals were maintained on a 12 h light–dark cycle at 22 ± 1°C and water was available ad libitum. Mice were killed, in the late morning or early afternoon, by cervical dislocation followed by decapitation. Brains were rapidly removed, frozen in isopentane chilled over dry ice and stored at −80°C until cryo-sectioned and processed for in situ hybridisation or quantitative in vitro autoradiography. n= 6–8 in all experimental groups. Trunk blood was collected into heparinised tubes on ice, centrifuged and plasma collected and stored at −80°C until use.

The influence of high-fat diet and both short- and long-term leptin challenge

C57BL/6 mice were either fed a high-fat diet (45% by energy) or a low-fat diet (10% by energy) (D12451 and D12450B, respectively; Research Diets, New Brunswick, NJ, USA) for 4 weeks in total. It has been reported that mice on high-fat diet for 4 weeks develop leptin resistance in the ARC (Munzberg et al. 2004). Leptin challenge in the present study was at a concentration widely used to measure leptin responsiveness in diet induced obesity (Enriori et al. 2007). Mice were challenged with murine leptin delivered by either intraperitoneal (i.p.) injection (2 mg (kg body weight)−1) (R&D Systems, Abingdon, UK) or i.c.v., to distinguish whether the leptin resistance was due to neuronal insensitivity or to inhibited leptin transport into the brain. One group was injected with leptin i.p. twice daily for 1 week and killed 1 h after the final leptin injection, a second group received i.p. vehicle injections twice daily for 1 week and a single leptin injection 1 h prior to killing, and a third group received vehicle injections i.p. twice daily for 1 week and were killed 1 h after the final vehicle injection. Two separate groups of mice fed diets as described above for 4 weeks were anaesthetised by an i.p. injection of ketamine/xylazine (ketamine 100 mg (kg BW)−1 plus xylazine 15 mg (kg BW)−1) and then received either a single i.c.v. injection of leptin (2 μg per mouse) or vehicle 1 h prior to killing. Brain infusion cannulae were stereotaxically placed with their tips in the lateral cerebral ventricle using the following coordinates: 0.7 mm posterior to bregma, 1.2 mm lateral to the midsagittal suture, and to a depth of 2.0 mm, with bregma and lambda at the same vertical dimension. Mice were allowed access to food and water throughout these challenges.

The influence of fasting and refeeding

C57BL/6 mice were fasted for 24 or 48 h or fed ad libitum on standard mouse diet prior to killing. Refed animals were allowed access to food ad libitum for a period of 24 h after fasting.

In situ hybridisation

Specific probes for Ob-R, Ob-Rb and SOCS3 were used as detailed previously (Adam et al. 2000; Mercer et al. 2000; Nilaweera et al. 2002; Nogueiras et al. 2004; Tups et al. 2004). Automated sequencing was performed to verify the sequences. Messenger RNA levels were quantified by in situ hybridisation, on 20 μm thick coronal hypothalamic cryo-sections, using techniques described in detail elsewhere (Mitchell et al. 2002). Briefly, slides were fixed in 4% (w/v) paraformaldehyde in 0.1 mol l−1 phosphate-buffered saline (PBS) for 20 min at room temperature, washed in PBS, incubated in 0.1 mmol l−1 triethanolamine for 2 min and acetylated in 0.1 mmol l−1 triethanolamine and 0.25% (v/v) acetic anhydride for 10 min. Sections were dehydrated in ethanol and dried under vacuum before hybridisation with riboprobes at 106 c.p.m. ml−1 for 18 h at 58°C. After hybridisation, sections were desalted through a series of washes in standard saline citrate (SSC) to a final stringency of 0.1× SSC at 60°C for 30 min, treated with RNase A and dehydrated in ethanol. Slides were apposed to Biomax MR (Sigma, Poole, Dorset, UK) together with 14C micro-scale standards (Amersham/GE Healthcare, Little Chalfont, UK) at room temperature for varying lengths of time depending on the probes used.

In vitro autoradiography

Sections were acid prewashed in low-pH, high-salt (pH 2, 0.5 m NaCl) Hepes buffer to remove any endogenous leptin bound to receptors, prior to a brief wash in Hepes, and were then incubated with 50 pm125I-labelled leptin (PerkinElmer LAS, UK) with the specific activity adjusted to 250 000 cpm pm−1 in Hepes for 2 h at room temp. Control slides were incubated with 125I-labelled leptin as detailed above plus 1 μm leptin. Slides were then thoroughly washed in Hepes buffer followed by distilled water at 4°C and air dried before being apposed to Biomax MR (Sigma) together with 125I micro-scale standards (Amersham/GE Healthcare) at room temperature. Characterisation of 125I-labelled leptin binding was carried out by increasing the times of incubation between 5 min and 6 h. A saturation isotherm was produced by incubating sections with increasing concentrations of 125I-labelled leptin between approximately 5 and 500 pm.

Quantification of in situ hybridisation and in vitro autoradiography

Autoradiographs were scanned on a Umax Power Look II (UMAX Data Systems, Fremont, CA, USA). Integrated optical densities (IOD), area and optical density of images, were quantified using the Image Pro-plus system (Media Cybernetics, Bethesda, MD, USA). IOD was converted to nCi g−1 using 14C microscale standard curves for measures of total gene expression. For quantification of 125I-labelled leptin binding to the CP, mean optical densities were measured and converted to nCi g−1 using 125I microscale standard curves.

Plasma leptin

Plasma leptin was measured by ELISA (BioVendor GmbH, Heidelberg, Germany) according to the manufacturer's instructions.

Statistical analysis

Data are presented as means ±s.e.m. and were analysed using GenStat (GenStat, 5th edn (2005), VSN International Ltd, Hemel Hempstead, UK). In the case of experiments testing the influence of a single factor a one-way ANOVA was performed. Where two factors were compared in a single experiment, as in the case of high-fat diet and leptin challenge, a two-way ANOVA followed by post hoc t-tests based on the LSD were performed. In this case the ANOVA results are expressed in the figure legend and the result of the multiple comparison test are represented on the graph. The type of statistical test carried out is stated in the figure legends. P < 0.05 was considered statistically significant.

Results

Localisation of gene expression and 125I-labelled leptin binding in mouse brain

Ob-R gene expression was present over the medial and lateral CP, the ARC and the VMH (Fig. 1A). Specific 125I-labelled leptin binding was clearly seen over both the medial and the lateral CP. No 125I-labelled leptin binding was measurable over hypothalamic nuclei (Fig. 1B). High levels of Ob-Rb gene expression are seen over the ARC and VMH. No discernable Ob-Rb gene expression was found over the CP (Fig. 1C) and no measurable induction of SOCS3 mRNA in this region was seen in response to leptin challenge (Fig. 1D).

Figure 1. Representative autoradiographs of mouse brain showing the areas measured in the present study.

Figure 1

Open in a new tab

A, Ob-R gene expression. B, 125I-labelled leptin binding. C, Ob-Rb gene expression. D, SOCS3 gene induction in a leptin challenged mouse. Arcuate (ARC) and ventromedial (VMH) nuclei. Lateral and medial choroid plexus (CP). Bar = 0.2 mm for all images.

Plasma leptin levels and body weight changes on a high-fat diet

The body weight (Fig. 2A) and body weight change (Fig. 2B) were higher in mice on a high-fat diet in comparison with mice on a low-fat diet (P < 0.001). Basal leptin levels were also higher in the mice on a high-fat diet vs. mice on a low-fat diet (P < 0.001) (Fig. 2C).

Figure 2. Body weight and leptin levels.

Figure 2

Open in a new tab

A, increase in body weight of C57Bl/6 mice fed either a low- or high-fat diet for 4 weeks. B, body weight difference in mice after 4 weeks on a low- or high-fat diet, ***P < 0.001. C, plasma leptin concentration in C57Bl/6 mice on a low- or high-fat diet, ***P < 0.001.

Regulation of Ob-R gene expression in the CP

Ob-R gene expression in the CP remained unchanged throughout the leptin challenge protocols used in the present study (data presented are for lateral CP: Ob-R gene expression in the medial CP showed the same pattern). Two-way ANOVA revealed no effect of high-fat feeding for 4 weeks and no effect of i.p. or i.c.v. leptin challenge on the CP in C57Bl/6 mice (Fig. 3A) (data not shown for i.c.v.). However, fasting for 24 h caused a significant increase in Ob-R expression (P < 0.001) that was not further increased after 48 h fasting. Refeeding did not return Ob-R expression levels to those of fed animals (P < 0.05 fed vs. refed) (Fig. 3B).

Figure 3. Levels of Ob-R expression in the lateral CP.

Figure 3

Open in a new tab

Statistical analysis was by two-way ANOVA followed by post hoc t-tests based on the LSD for A and E and one-way ANOVA for B and F. A, C57Bl/6 mice on low- or high-fat diets; after 1 h and 1 week i.p. leptin challenge. There was no effect of diet type or leptin. NS, no significant difference. B, C57Bl/6 fasted for either 24 or 48 h plus refed for 24 h. *P < 0.05, ***P < 0.001. Representative autoradiographs of Ob-R gene expression in the CP underlie the bar graph. C and D, characterisation of 125I-labelled leptin binding to the lateral CP. C, time course of specific 125I-labelled leptin binding. D, typical saturation isotherm giving Kd= 298 ± 63.15 pm and a Bmax of 30.01 ± 2.5 fm mg−1 protein. Levels of 125I-labelled leptin binding to the lateral CP. E, C57Bl/6 on low- or high-fat diets after 1 h or 1 week i.p. leptin challenge. There was a significant effect of diet (P < 0.05) and a significant effect of leptin (P < 0.001); there was no interaction between diet and leptin. **P < 0.01, ***P < 0.001. F, C57Bl/6 fasted for either 24 or 48 h plus refed for 24 h. *P < 0.05, **P < 0.01, ***P < 0.001. Representative autoradiographs of 125I-labelled leptin binding to the CP underlie the bar graphs.

Characterisation of 125I-labelled leptin binding to the CP

In vitro autoradiography of 125I-labelled leptin binding over the CP was time and concentration dependent (Fig. 3C and D). Although both hypothalamic and extra-hypothalamic 125I-labelled leptin binding is apparent in the rat brain in our hands and as reported by other groups (Baskin et al. 1999; Irani et al. 2007), the very low levels of 125I-labelled leptin binding in the hypothalamus of the mice in the present study were below the levels that could be accurately measured.

Regulation of 125I-labelled leptin binding to the CP

The level of specific 125I-labelled leptin binding to the CP was increased in C57Bl/6 mice fed a high-fat diet for 4 weeks (P < 0.05). An effect of leptin was also apparent (P < 0.001), with a rapid decrease of 125I-labelled leptin binding in the C57Bl/6 mouse on both high- and low-fat diets 1 h after a single i.p. or i.c.v. leptin challenge, which was maintained after a week of twice daily leptin injections (Fig. 3E) (Data not shown for i.c.v.). Fasting for 24 h resulted in an increase of 125I-labelled leptin binding to the CP (P < 0.001) that was significantly increased after 48 h (P < 0.05). Levels of 125I-labelled leptin binding were decreased by refeeding to significantly less than baseline levels after 24 h (P < 0.05 fed vs refed) (Fig. 3F).

Regulation of Ob-Rb gene expression in the ARC and VMH

There was no effect of diet on basal levels of total expression of Ob-Rb in the ARC or VMH. There was a significant effect of leptin (P < 0.05) to up-regulate the level of expression of Ob-Rb in the ARC and a significant interaction between diet and leptin (P < 0.05), with high-fat diet preventing up-regulation of Ob-Rb gene expression by leptin (P < 0.05) (Fig. 4A). There was a significant effect of diet (P < 0.01), but not leptin, on the Ob-Rb gene expression in the VMH (Fig. 4A). Density of riboprobe labelling is consistently higher on a high-fat diet in both the ARC and the VMH reflecting the level of gene expression per cell (Fig. 4B), while leptin challenge appeared to increase the area of labelling, i.e. the number of cells expressing the receptor only in the ARC (Fig. 4C). Fasting for 24 h caused a significant increase in Ob-Rb expression in the ARC (P < 0.001) and VMH (P < 0.001) that was maintained but not increased by 48 h fasting in the VMH, but further increased in the ARC (P < 0.01). Refeeding caused a significant decrease in Ob-Rb expression that had returned to basal levels after 24 h ad libitum access to food in the VMH, and decreased below the levels of the fed animal in the ARC (P < 0.05) (Fig. 5A). Gene expression changes in the ARC and VMH (Fig. 5B and C) appear to be a combination of both changes in density and area. However, the changes in area are much larger than those seen for density indicating that it is the number of cells expressing the receptor that contributes most to changes in gene expression (Fig. 5B and C).

Figure 4. Levels of Ob-Rb gene expression in the ARC and VMH on low- and high-fat diets after control, 1 h or 1 week i.p. leptin challenge, including representative autoradiographs.

Figure 4

Open in a new tab

Statistical analysis was by two-way ANOVA followed by post hoc t-tests based on the LSD. A, Ob-Rb gene expression in ARC and VMH of C57Bl/6 mice. In the ARC there was an effect of both diet (P < 0.001) and leptin (P < 0.05) and an interaction between diet and leptin (P < 0.05). In the VMH there was an effect of diet (P < 0.01), but no effect of leptin and no interaction. B, density of Ob-Rb gene expression in the ARC and VMH. In the ARC there was an effect of diet (P < 0.001) and of leptin (P < 0.05), but no interaction. In the VMH there was an effect of diet (P < 0.001), no effect of leptin and an interaction (P < 0.05). C, area of Ob-Rb gene expression in the ARC and VMH. In the ARC there was an effect of diet (P < 0.001) and leptin (P < 0.05) and an interaction (P < 0.01). In the VMH there was an effect of diet, no effect of leptin and no interaction. *P < 0.05, **P < 0.01, ***P < 0.001. NS, no significant difference.

Figure 5. Levels of Ob-Rb gene expression in the ARC and VMH of mice fasted for either 24 or 48 h and refed for 24 h including representative autoradiographs.

Figure 5

Open in a new tab

Statistical analysis was by one-way ANOVA. A, gene expression of Ob-Rb in the ARC and VMH. B, density of Ob-Rb gene expression in the ARC and VMH. C, area of Ob-Rb gene expression in the ARC and VMH. *P < 0.05, **P < 0.01, ***P < 0.001.

All changes in the level of total Ob-R and Ob-Rb gene expression and 125I-labelled leptin binding are summarised in Table 1.

Table 1.

Changes in Ob-R, Ob-Rb and 125I leptin labelling

Ob-Rb CP Leptin ↔
High vs. low-fat ↔
Fasted ↑
Refed ↔
125I-Leptin CP Leptin ↓
High vs. low-fat ↑
Fasted ↑
Refed ↓
Ob-Rb ARC Leptin ↑ Not high-fat
High vs. low-fat ↔
Fasted ↑
Refed ↓
Ob-Rb VMH Leptin ↔
High vs. low fat ↔
Fasted ↑
Refed ↓
Open in a new tab

Regulation of SOCS3 gene expression

Basal levels of SOCS3 gene expression were similar in low- and high-fat fed mice, and after 1 h and 1 week of leptin challenge in the ARC and VMH (Fig. 6). Two-way ANOVA revealed a significant effect of leptin (P < 0.05) with no significant effect of diet and no interaction.

Figure 6. Levels of SOCS3 gene expression ARC and VMH of C57Bl/6 mice after low- and high-fat diets after control, after 1 h or 1 week i.p. leptin challenge.

Figure 6

Open in a new tab

Statistical analysis was by two-way ANOVA. There was a significant effect of leptin P < 0.001 but no significant effect of diet and no interaction. For SOCS3 in VMH gene expression is shown in nCi g−1 as control values were not detectable (ND). **P < 0.01, ***P < 0.001

Discussion

The present study shows, for the first time, that leptin receptor gene expression and leptin receptor number in vivo, measured by 125I-labelled leptin binding, are differentially regulated by both leptin and high-fat diet. Two separate mechanisms have been demonstrated that may contribute to the development of leptin insensitivity. The first is the loss of ability of leptin to increase Ob-Rb gene expression in the ARC after 4 weeks of high-fat feeding in the C57Bl/6 mouse. The second is the rapid decrease in leptin receptor number in response to leptin challenge. A better understanding of the mechanisms responsible for leptin insensitivity may help to identify potential targets in the treatment of obesity.

Under the experimental protocols used in this study the level of Ob-R gene expression on the CP remained unchanged after leptin challenge and in C57Bl/6 mice fed a high-fat diet. However, fasting induced an increase in Ob-R gene expression in the CP, but refeeding for 24 h failed to reverse the increase in expression. Although leptin levels drop precipitously in response to fasting and rise again on refeeding (Ishii et al. 2000), the insensitivity of Ob-R gene expression in the CP to long and short-term leptin challenge indicates that factors that change with fasting and refeeding, other than leptin, may regulate Ob-R expression on the CP. Conversely, it may be that the fasting-induced drop in leptin up-regulates gene expression while increasing levels of leptin on refeeding fail to down-regulate gene expression or may do so more slowly.

In contrast to the regulation of Ob-R gene expression on the CP, leptin receptor number changes rapidly in response to changes in circulating leptin. The up-regulation of 125I-labelled leptin binding to the CP in response to fasting and down-regulation to reach lower than baseline levels in response to refeeding together with the response to leptin challenge, detailed above, indicate that the drop in circulating leptin levels with fasting and increase in leptin levels with refeeding (Ahren et al. 1997; Havel, 2001) drive these changes in receptor number. The present study shows that receptor number on the CP increases in response to fasting and also in response to a high-fat diet where serum triglyceride levels are elevated (Schreyer et al. 1998), indicating that receptor number is not limiting transport of leptin into the brain. One explanation for the increased level of 125I-labelled leptin binding to the CP in conditions where leptin transport into the brain is reportedly decreased (Kastin & Akerstrom, 2000; Banks et al. 2004) is that inhibition of the transport process results in a subsequent accumulation of receptors at the cell surface. However, this hypothesis remains to be tested.

The increase in 125I-labelled leptin binding to the CP after 4 weeks on a high-fat diet precedes the reported increase in Ob-R gene expression in the CP after 8 weeks of high-fat diet (Lin et al. 2000) and indicates that changes in receptor number occur prior to changes in gene expression. Apart from this counter-intuitive increase in leptin receptor number on the high-fat diet where leptin levels increase, our findings largely confirm previous studies where receptor numbers drop in response to increased leptin levels (Uotani et al. 1999; Smallwood et al. 2007) and increase in response to decreased leptin levels in fasting (Baskin et al. 1999). However, 125I-labelled leptin binding to the hypothalamus has been shown to be lower in rats with a genetic predisposition to develop obesity on a high-energy diet compared with rats resistant to diet induced obesity (Irani et al. 2007), and in that study 125I-labelled leptin binding appears to be independent of the level of circulating leptin, indicating that leptin-independent changes in receptor number also occur. The present study demonstrates that both leptin-dependent and leptin-independent changes in leptin receptor number take place.

Ob-Rb gene expression is specific for the long signalling form of the leptin receptor necessary for second messenger signalling to be induced. Ob-Rb gene expression was undetectable over the CP, although its presence has been reported on the ovine CP (Merino et al. 2006) and nuclear translocation of STAT3 has been reported in the rat CP in response to leptin challenge (Mutze et al. 2006). Also, we did not detect induction of SOCS3 gene expression in the CP in response to leptin challenge, further indicating that Ob-Rb is not present in the mouse CP. However, the possibility remains that low levels of Ob-Rb, below the sensitivity of the techniques used in the present study, may be present.

Ob-Rb gene expression in the ARC and VMH was simultaneously up-regulated by fasting and reversed by refeeding. However, leptin challenge caused a rapid (1 h) increase in Ob-Rb gene expression, which may be specific to the ARC. Changes in Ob-Rb gene expression in the VMH, in response to leptin challenge, were more difficult to interpret but may be the result of smaller changes which, in the present study, fail to reach statistical significance. There also appears to be separate and different effects of high-fat diet and leptin challenge when the density and area of Ob-Rb expression are considered, rather than total gene expression. High-fat diet consistently increases the density of Ob-Rb expression in the ARC and the VMH, while leptin challenge increases the area of gene expression, but only significantly in the ARC and not on a high-fat diet. Nonetheless, the increase in total Ob-Rb expression in the ARC was clear and was maintained after 1 week of leptin challenge in agreement with previous reports (Haltiner et al. 2004; Di Yorio et al. 2008). In the present study this response is lost after high-fat feeding, indicating that the high-fat diet induces neuronal insensitivity to leptin. However, the induction of SOCS3 gene expression in these mice in response to leptin remained intact indicating that the JAK/STAT pathway was not compromised.

In contrast to the up-regulation of Ob-Rb in the ARC by leptin challenge, fasting, which results in a drop in circulating leptin, also up-regulated Ob-Rb gene expression and refeeding, in which circulating levels of leptin are increased, and down-regulated Ob-Rb gene expression, not only in the ARC but also in the VMH. This indicates that factors other than leptin appear to be responsible for the regulation of Ob-Rb gene expression in response to nutritional status. One possibility is the change in the level of corticosterone, which is known to rise and fall with fasting and refeeding (Makimura et al. 2003) and also to regulate both leptin signalling (Ishida-Takahashi et al. 2004) and the level of gene expression of appetite regulatory signals in the hypothalamus (Makimura et al. 2003). Dexamethasone has been shown to either up-regulate or have no effect on Ob-Rb and Ob-Ra expression, depending on the tissue or cell type tested (Hosoi et al. 2003; Liu et al. 2004; Wyrwoll et al. 2005).

In summary, the present findings demonstrate that, in the mouse, regulation of leptin receptor number and receptor gene expression by leptin and nutritional status is region and receptor sub-type specific. Fasting and refeeding, along with the concomitant drop and subsequent rise in circulating leptin, appears to be the only treatment that had a consistent effect on both receptor number and gene expression indicating that a drop in leptin is important in the regulation of both receptor number and Ob-R and Ob-Rb gene expression, and that a drop in leptin levels is necessary for subsequently increasing leptin levels to down-regulate receptor gene expression. Increases in leptin level give rise to more complex effects which may be due to an interaction between leptin and diet or different effects of increasing concentrations of leptin (Di Yorio et al. 2008). However, taking these results together, we conclude that (a) high levels of leptin decrease 125I-labelled leptin binding, offering a potential mechanism for leptin insensitivity in response to hyperleptinaemia; (b) Ob-R down-regulation is not an essential mechanism in the development of leptin insensitivity; and (c) high-fat diet somehow blunts the ability of leptin to regulate Ob-Rb gene expression in the ARC.

Acknowledgments

We would like to thank Dr Graham Horgan, Biostatistics Scotland (BIOSS) for expert advice and Dr Gerald Lobley (RINH) for critically reading the manuscript. This work was supported by the Ministerio de Ciencia y Tecnologia in Spain and the Scottish Government Rural and Environment Research and Analysis Directorate (RERAD) in the UK. Sharon Mitchell and Christine Grant were funded by EC Framework V Grant QLK6-2002-02288. Rubén Nogueiras was a recipient of a Research Training Grant at Obesechool funded by EC Framework V Program HPMT-CT-2001-00410. Lynda Williams was a recipient of a British Heart Foundation Grant No. PG/98179.

Glossary

Abbreviations

ARC

arcuate nuclei of the hypothalamus

CP

choroid plexus

i.c.v.

intracerebroventricular

JAK

Janus kinase

Ob-R

all forms of the leptin receptor

Ob-Ra and Ob-Rc

short forms of the leptin receptor

Ob-Rb

long signalling form of the leptin receptor

SSC

standard saline citrate

STAT

signal transducer and activator of transcription

SOCS

suppresser of cytokine signalling

VMH

ventromedial nuclei of the hypothalamus.

Author contribution

S.E.M., R.N., C.D. and L.M.W. contributed to the conception and design and interpretation of the data. A.M., S.T., C.G., M.C., D.V.R. contributed to the analysis and interpretation of the data. All authors contributed to the drafting and revision of the article for important intellectual content and final approval of the version to be published. Animal experiments were carried out at the Rowett Institute of Nutrition and Health and in the Department of Physiology, University of Santiago de Compostela. Tissues were analysed at the Rowett Institute of Nutrition and Health.

References

  1. Adam CL, Moar KM, Logie TJ, Ross AW, Barrett P, Morgan PJ, Mercer JG. Photoperiod regulates growth, puberty and hypothalamic neuropeptide and receptor gene expression in female Siberian hamsters. Endocrinology. 2000;141:4349–4356. doi: 10.1210/endo.141.12.7807. [DOI] [PubMed] [Google Scholar]
  2. Ahren B, Mansson S, Gingerich RL, Havel PJ. Regulation of plasma leptin in mice: influence of age, high- fat diet, and fasting. Am J Physiol Regul Integr Comp Physiol. 1997;273:R113–R120. doi: 10.1152/ajpregu.1997.273.1.R113. [DOI] [PubMed] [Google Scholar]
  3. Banks WA, Coon AB, Robinson SM, Moinuddin A, Shultz JM, Nakaoke R, Morley JE. Triglycerides induce leptin resistance at the blood-brain barrier. Diabetes. 2004;53:1253–1260. doi: 10.2337/diabetes.53.5.1253. [DOI] [PubMed] [Google Scholar]
  4. Baskin DG, Breininger JF, Bonigut S, Miller MA. Leptin binding in the arcuate nucleus is increased during fasting. Brain Res. 1999;828:154–158. doi: 10.1016/s0006-8993(99)01252-4. [DOI] [PubMed] [Google Scholar]
  5. Baskin DG, Seeley RJ, Kuijper JL, Lok S, Weigle DS, Erickson JC, Palmiter RD, Schwartz MW. Increased expression of mRNA for the long form of the leptin receptor in the hypothalamus is associated with leptin hypersensitivity and fasting. Diabetes. 1998;47:538–543. doi: 10.2337/diabetes.47.4.538. [DOI] [PubMed] [Google Scholar]
  6. Bennett PA, Lindell K, Karlsson C, Robinson IC, Carlsson LM, Carlsson B. Differential expression and regulation of leptin receptor isoforms in the rat brain: effects of fasting and oestrogen. Neuroendocrinology. 1998;67:29–36. doi: 10.1159/000054295. [DOI] [PubMed] [Google Scholar]
  7. Bennett PA, Lindell K, Wilson C, Carlsson LMS, Carlsson B, Robinson ICAF. Cyclical variations in the abundance of leptin receptors, but not in circulating leptin, correlate with NPY expression during the oestrous cycle. Neuroendocrinology. 1999;69:417–423. doi: 10.1159/000054444. [DOI] [PubMed] [Google Scholar]
  8. Bjorbaek C, El Haschimi K, Frantz JD, Flier JS. The role of SOCS-3 in leptin signalling and leptin resistance. J Biol Chem. 1999;274:30059–30065. doi: 10.1074/jbc.274.42.30059. [DOI] [PubMed] [Google Scholar]
  9. Bjorbaek C, Elmquist JK, Michl P, Ahima RS, van Bueren A, McCall AL, Flier JS. Expression of leptin receptor isoforms in rat brain microvessels. Endocrinology. 1998;139:3485–3491. doi: 10.1210/endo.139.8.6154. [DOI] [PubMed] [Google Scholar]
  10. Brogan RS, Grove KL, Smith MS. Differential regulation of leptin receptor but not orexin in the hypothalamus of the lactating rat. J Neuroendocrinol. 2000;12:1077–1086. doi: 10.1046/j.1365-2826.2000.00559.x. [DOI] [PubMed] [Google Scholar]
  11. Campfield LA, Smith FJ, Guisez Y, Devos R, Burn P. Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science. 1995;269:546–549. doi: 10.1126/science.7624778. [DOI] [PubMed] [Google Scholar]
  12. Di Yorio MP, Bilbao MG, Pustovrh MC, Prestifilippo JP, Faletti AG. Leptin modulates the expression of its receptors in the hypothalamic-pituitary-ovarian axis in a differential way. J Endocrinol. 2008;198:355–366. doi: 10.1677/JOE-07-0622. [DOI] [PubMed] [Google Scholar]
  13. El Haschimi K, Pierroz DD, Hileman SM, Bjorbaek C, Flier JS. Two defects contribute to hypothalamic leptin resistance in mice with diet-induced obesity. J Clin Invest. 2000;105:1827–1832. doi: 10.1172/JCI9842. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Enriori PJ, Evans AE, Sinnayah P, Jobst EE, Tonelli-Lemos L, Billes SK, Glavas MM, Grayson BE, Perello M, Nillni EA, Grove KL, Cowley MA. Diet-induced obesity causes severe but reversible leptin resistance in arcuate melanocortin neurons. Cell Metab. 2007;5:181–194. doi: 10.1016/j.cmet.2007.02.004. [DOI] [PubMed] [Google Scholar]
  15. Frederich RC, Hamann A, Anderson S, Lollmann B, Lowell BB, Flier JS. Leptin levels reflect body lipid-content in mice – evidence for diet-induced resistance to leptin action. Nat Med. 1995;1:1311–1314. doi: 10.1038/nm1295-1311. [DOI] [PubMed] [Google Scholar]
  16. Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, Lallone RL, Burley SK, Friedman JM. Weight-reducing effects of the plasma-protein encoded by the obese gene. Science. 1995;269:543–546. doi: 10.1126/science.7624777. [DOI] [PubMed] [Google Scholar]
  17. Haltiner AL, Mitchell TD, Harris RBS. Leptin action is modified by an interaction between dietary fat content and ambient temperature. Am J Physiol Regul Integr Comp Physiol. 2004;287:R1250–R1255. doi: 10.1152/ajpregu.00313.2004. [DOI] [PubMed] [Google Scholar]
  18. Havel PJ. Peripheral signals conveying metabolic information to the brain: Short-term and long-term regulation of food intake and energy homeostasis. Exp Biol and Med. 2001;226:963–977. doi: 10.1177/153537020122601102. [DOI] [PubMed] [Google Scholar]
  19. Hosoi T, Okuma Y, Wada S, Nomura Y. Inhibition of leptin-induced IL-1β expression by glucocorticoids in the brain. Brain Res. 2003;969:95–101. doi: 10.1016/s0006-8993(03)02282-0. [DOI] [PubMed] [Google Scholar]
  20. Howard JK, Cave BJ, Oksanen LJ, Tzameli I, Bjorbaek C, Flier JS. Enhanced leptin sensitivity and attenuation of diet-induced obesity in mice with haploinsufficiency of Socs3. Nat Med. 2004;10:734–738. doi: 10.1038/nm1072. [DOI] [PubMed] [Google Scholar]
  21. Hubschle T, Thom E, Watson A, Roth J, Klaus S, Meyerhof W. Leptin-induced nuclear translocation of STAT3 immunoreactivity in hypothalamic nuclei involved in body weight regulation. J Neurosci. 2001;21:2413–2424. doi: 10.1523/JNEUROSCI.21-07-02413.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Irani BG, Dunn-Meynell AA, Levin BE. Altered hypothalamic leptin, insulin, and melanocortin binding associated with moderate-fat diet and predisposition to obesity. Endocrinology. 2007;148:310–316. doi: 10.1210/en.2006-1126. [DOI] [PubMed] [Google Scholar]
  23. Ishida-Takahashi R, Uotani S, Abe T, Degawa-Yamauchi M, Fukushima T, Fujita N, Sakamaki H, Yamasaki H, Yamaguchi Y, Eguchi K. Rapid inhibition of leptin signalling by glucocorticoids in vitro and in vivo. J Biol Chem. 2004;279:19658–19664. doi: 10.1074/jbc.M310864200. [DOI] [PubMed] [Google Scholar]
  24. Ishii S, Shibasaki T, Murakami T, Shima K, Wakabayashi I. Response of leptin mRNA to 24-h food deprivation and refeeding is influenced by age in rats. Reg Pep. 2000;92:45–50. doi: 10.1016/s0167-0115(00)00148-8. [DOI] [PubMed] [Google Scholar]
  25. Jeanrenaud B, Rohner-Jeanrenaud F. Effects of neuropeptides and leptin on nutrient partitioning: Dysregulations in obesity. Annu Rev Med. 2001;52:339–351. doi: 10.1146/annurev.med.52.1.339. [DOI] [PubMed] [Google Scholar]
  26. Kastin AJ, Akerstrom V. Fasting, but not adrenalectomy, reduces transport of leptin into the brain. Peptides. 2000;21:679–682. doi: 10.1016/s0196-9781(00)00195-9. [DOI] [PubMed] [Google Scholar]
  27. Ladyman SR, Grattan DR. Suppression of leptin receptor messenger ribonucleic acid and leptin responsiveness in the ventromedial nucleus of the hypothalamus during pregnancy in the rat. Endocrinology. 2005;146:3868–3874. doi: 10.1210/en.2005-0194. [DOI] [PubMed] [Google Scholar]
  28. Lin S, Storlien LH, Huang XF. Leptin receptor, NPY, POMC mRNA expression in the diet-induced obese mouse brain. Brain Res. 2000;875:89–95. doi: 10.1016/s0006-8993(00)02580-4. [DOI] [PubMed] [Google Scholar]
  29. Liu ZJ, Endoh A, Li R, Ohzeki T. Effects of leptin and dexamethasone on long and short leptin receptor mRNA. Ped Int. 2004;46:561–564. doi: 10.1111/j.1442-200x.2004.01945.x. [DOI] [PubMed] [Google Scholar]
  30. Makimura H, Mizuno T, Isoda F, Beasley J, Silverstein J, Mobbs C. Role of glucocorticoids in mediating effects of fasting and diabetes on hypothalamic gene expression. BMC Physiol. 2003;3:5. doi: 10.1186/1472-6793-3-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mercer JG, Hoggard N, Williams LM, Lawrence CB, Hannah LT, Trayhurn P. Localization of leptin receptor mRNA and the long form splice variant (Ob-Rb) in mouse hypothalamus and adjacent brain regions by in situ hybridization. FEBS Lett. 1996;387:113–116. doi: 10.1016/0014-5793(96)00473-5. [DOI] [PubMed] [Google Scholar]
  32. Mercer JG, Moar KM, Rayner DV, Trayhurn P, Hoggard N. Regulation of leptin receptor and NPY gene expression in hypothalamus of leptin-treated obese (ob/ob) and cold-exposed lean mice. FEBS Lett. 1997;402:185–188. doi: 10.1016/s0014-5793(96)01525-6. [DOI] [PubMed] [Google Scholar]
  33. Mercer JG, Moar KM, Ross AW, Hoggard N, Morgan PJ. Photoperiod regulates arcuate nucleus POMC, AGRP, and leptin receptor mRNA in Siberian hamster hypothalamus. Am J Physiol Regul Integr Comp Physiol. 2000;278:R271–R281. doi: 10.1152/ajpregu.2000.278.1.R271. [DOI] [PubMed] [Google Scholar]
  34. Merino B, Diez-Fernandez C, Ruiz-Gayo M, Somoza B. Choroid plexus epithelial cells co-express the long and short form of the leptin receptor. Neurosci Lett. 2006;393:269–272. doi: 10.1016/j.neulet.2005.10.003. [DOI] [PubMed] [Google Scholar]
  35. Mitchell SE, Robinson JJ, King ME, McKelvey WAC, Williams LM. Interleukin 8 in the cervix of non-pregnant ewes. Reproduction. 2002;124:409–416. [PubMed] [Google Scholar]
  36. Montez JM, Soukas A, Asilmaz E, Fayzikhodjaeva G, Fantuzzi G, Friedman JM. Acute leptin deficiency, leptin resistance, and the physiologic response to leptin withdrawal. Proc Natl Acad Sci U S A. 2005;102:2537–2542. doi: 10.1073/pnas.0409530102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Munzberg H, Myers MG. Molecular and anatomical determinants of central leptin resistance. Nat Neurosci. 2005;8:566–570. doi: 10.1038/nn1454. [DOI] [PubMed] [Google Scholar]
  38. Munzberg H, Flier JS, Bjorbaek C. Region-specific leptin resistance within the hypothalamus of diet-induced obese mice. Endocrinology. 2004;145:4880–4889. doi: 10.1210/en.2004-0726. [DOI] [PubMed] [Google Scholar]
  39. Mutze J, Roth J, Gerstberger M, Matsumura K, Hubschle T. Immunohistochemical evidence of functional leptin receptor expression in neuronal and endothelial cells of the rat brain. Neurosci Lett. 2006;394:105–110. doi: 10.1016/j.neulet.2005.10.031. [DOI] [PubMed] [Google Scholar]
  40. Nilaweera KN, Ellis C, Barrett P, Mercer JG, Morgan PJ. Hypothalamic bHLH transcription factors are novel candidates in the regulation of energy balance. Eur J Neurosci. 2002;15:644–650. doi: 10.1046/j.1460-9568.2002.01894.x. [DOI] [PubMed] [Google Scholar]
  41. Nogueiras R, Tovar S, Mitchell SE, Rayner DV, Archer ZA, Dieguez C, Williams LM. Regulation of growth hormone secretagogue receptor gene expression in the arcuate nuclei of the rat by leptin and ghrelin. Diabetes. 2004;53:2552–2558. doi: 10.2337/diabetes.53.10.2552. [DOI] [PubMed] [Google Scholar]
  42. Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, Collins F. Effects of the obese gene- product on body-weight regulation in ob/ob mice. Science. 1995;269:540–543. doi: 10.1126/science.7624776. [DOI] [PubMed] [Google Scholar]
  43. Scarpace PJ, Matheny M, Tumer N, Cheng KY, Zhang Y. Leptin resistance exacerbates diet-induced obesity and is associated with diminished maximal leptin signalling capacity in rats. Diabetologia. 2005;48:1075–1083. doi: 10.1007/s00125-005-1763-x. [DOI] [PubMed] [Google Scholar]
  44. Schreyer SA, Wilson DL, LeBoeuf RC. C57BL/6 mice fed high fat diets as models for diabetes-accelerated atherosclerosis. Atherosclerosis. 1998;136:17–24. doi: 10.1016/s0021-9150(97)00165-2. [DOI] [PubMed] [Google Scholar]
  45. Smallwood S, Pyrski M, Morris A, Barrett P, Mertani HC, Raccurt M, Morel G, Meyerhof W, Williams LM. Endocytosis of the long signalling form of the leptin receptor (Ob-Rb) is necessary for full leptin signalling. ENDO 2007: program and abstracts of the 89th annual meeting of the Endocrine Society, abstract (P1–47)
  46. Tang CH, Lu DY, Yang RS, Tsai HY, Kao MC, Fu WM, Chen YF. Leptin-induced IL-6 production is mediated by leptin receptor, insulin receptor substrate-1, phosphatidylinositol 3-kinase, Akt, NF-κB, and p300 pathway in microglia. J Immunol. 2007;179:1292–1302. doi: 10.4049/jimmunol.179.2.1292. [DOI] [PubMed] [Google Scholar]
  47. Tulipano G, Vergoni AV, Soldi D, Muller EE, Cocchi D. Characterization of the resistance to the anorectic and endocrine effects of leptin in obesity-prone and obesity-resistant rats fed a high-fat diet. J Endocrinol. 2004;183:289–298. doi: 10.1677/joe.1.05819. [DOI] [PubMed] [Google Scholar]
  48. Tups A, Ellis C, Moar KM, Logie TJ, Adam CL, Mercer JG, Klingenspor M. Photoperiodic regulation of leptin sensitivity in the Siberian hamster, Phodopus sungorus, is reflected in arcuate nucleus SOCS-3 (suppressor of cytokine signalling) gene expression. Endocrinology. 2004;145:1185–1193. doi: 10.1210/en.2003-1382. [DOI] [PubMed] [Google Scholar]
  49. Uotani S, Bjorbaek C, Tornoe J, Flier JS. Functional properties of leptin receptor isoforms: internalization and degradation of leptin and ligand-induced receptor downregulation. Diabetes. 1999;48:279–286. doi: 10.2337/diabetes.48.2.279. [DOI] [PubMed] [Google Scholar]
  50. van Heek M, Compton DS, France CF, Tedesco RP, Fawzi AB, Graziano MP, Sybertz EJ, Strader CD, Davis HRJ. Diet-induced obese mice develop peripheral, but not central, resistance to leptin. J Clin Invest. 1997;99:385–390. doi: 10.1172/JCI119171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wilsey J, Scarpace PJ. Caloric restriction reverses the deficits in leptin receptor protein and leptin signalling capacity associated with diet-induced obesity: role of leptin in the regulation of hypothalamic long-form leptin receptor expression. J Endocrinol. 2004;181:297–306. doi: 10.1677/joe.0.1810297. [DOI] [PubMed] [Google Scholar]
  52. Wyrwoll CS, Mark PJ, Waddell BJ. Directional secretion and transport of leptin and expression of leptin receptor isoforms in human placental BeWo cells. Mol Cell Endocrinol. 2005;241:73–79. doi: 10.1016/j.mce.2005.05.003. [DOI] [PubMed] [Google Scholar]
  53. Zhang Y, Scarpace PJ. The role of leptin in leptin resistance and obesity. Physiol Behav. 2006;88:249–256. doi: 10.1016/j.physbeh.2006.05.038. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Physiology are provided here courtesy of The Physiological Society

RESOURCES