J Physiol 2009 Mitchell 3573 85

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J Physiol587.14 (2009) pp 35733585 3573

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

Sharon E. Mitchell1, Ruben Nogueiras2,3, Amanda Morris1, Sulay Tovar2, Christine Grant1,

Morven Cruickshank1, D. Vernon Rayner1, Carlos Dieguez2,3 and Lynda M. Williams11Obesity and Metabolic Health Division, Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen AB21 9SB, UK2Department of Physiology, University of Santiago de Compostela, 15705 Santiago de Compostela, Spain3CIBER Fisiopatolog a de la Obesidad y Nutrici on, Instituto de Salud Carlos III, Santiago de Compostela, Spain

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 measuredusing quantitative 125I-labelledleptin in vitroautoradiography

and in situ hybridisation, respectively. Ob-R (all forms of leptin receptor) expression in the

choroidplexus(CP)wasunchangedbyhigh-fatdietorleptinchallenge,whereasfastingincreased

but refeeding failed to decrease expression.

125

I-labelled leptin binding to the CP was increasedby fasting and returned to basal levels on refeeding.125 I-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-Rbexpression 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.

(Received 31 March 2009; accepted after revision 30 May 2009; first published online 2 June 2009)

Corresponding authorL. Williams: Obesity and Metabolic Health Division, Rowett Institute of Nutrition and Health,

University of Aberdeen, Aberdeen AB21 9SB, UK. Email: [email protected]

AbbreviationsARC, arcuate nuclei of the hypothalamus; CP, choroid plexus; I.C.V., intracerebroventricular; JAK, Janus

kinase; Ob-R,all forms of theleptin receptor; Ob-Raand Ob-Rc, short forms of theleptin receptor; Ob-Rb, longsignalling

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.

Leptin secreted by adipocytes acts in the hypothalamusto regulate food intake and energy expenditure, therebylimiting adiposity (Campfield et al. 1995; Halaas et al.1995; Pelleymounter et al. 1995). It is now widelyrecognised that in obesity, where leptin levels are highbut fail to limit adiposity, leptin insensitivity occurs(Munzberg & Myers, 2005). This may be due to restrictedentry of leptin into the brain and/or failure in the responseof leptin receptive neurons (van Heek et al. 1997; ElHaschimi et al. 2000; Jeanrenaud & Rohner-Jeanrenaud,2001). The transport of leptin into the brain and neuronal

sensitivity to the leptin signal depend on the availabilityof leptin receptors at the bloodbrain barrier and on theleptin receptive neurons, respectively. Transport of leptininto the brain is thought to be via the short forms of theleptin receptor (Ob-Ra and Ob-Rc) present in the choroidplexus (CP) and brain microvessels (Bjorbaeket al.1998).Leptin signalling in neurons is dependent on the presenceof the long form of the leptin receptor (Ob-Rb) (Merceretal. 1996), which signals principally via the Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3) pathway (Hubschleet al.2001). Leptin challenge

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3574 S. E. Mitchell and others J Physiol587.14

rapidly induces expression of the suppressor ofcytokine signalling 3 (SOCS3), which inhibits leptinsignalling (Bjorbaek et al. 1999; Howard et al. 2004),and SOCS3 gene expression has been used as a markerfor leptin stimulation of the JAK/STAT pathway (Tupset al.2004). Alterations in both receptor gene expression,and the endocytosis and trafficking of ligand-activated

cell surface receptors can regulate receptor availability andresponse to ligand.

Relatively little is known about the co-regulation ofOb-Rand Ob-Rbgene expression and receptor number,or the impact of receptor regulation on leptin sensitivity.Both the lack of circulating leptin, as in the ob/obmouse,and the inability to respond to the leptin signal, as in theZucker rat, up-regulate Ob-Rb gene expression (Merceret al.1997; Bennettet al.1998), while leptin challenge hasbeen reported to down-regulateexpression.Fasting, whereleptin levels drop, up-regulates Ob-Rbgene expression(Baskin et al. 1998; Bennett et al. 1998) and increases125

I-labelled leptin binding in the rat arcuate nuclei (ARC)(Baskin et al. 1999). These studies indicate that leptinconcentration is important in regulating gene expressionand receptor number. However, changes in Ob-R andOb-Rb gene expression have also been shown to benutrition and leptin independent, with changes occurringin the ARC and ventromedial nuclei (VMH) during theoestrous cycle (Bennett et al. 1998, 1999), and in theVMH during pregnancy and lactation (Broganet al.2000;Ladyman & Grattan, 2005).

Diet induced obesity in both humans and rodentsresults in increased circulating levels of leptin, andleptin insensitivity (Frederich et al. 1995). Both hyper-leptinaemia (Montezet al.2005; Scarpaceet al.2005) andhigh-fat feeding (Tulipano et al. 2004) have been shown tocontribute to leptin insensitivity, while calorie restrictionincreases sensitivity to leptin (Wilsey & Scarpace, 2004).Ob-R gene expression has been reported to increaseinitially in leptin insensitivity induced by high-fat feeding,followed by a subsequent down-regulation (Lin et al.2000). Also, reduced leptin receptor gene expression andreceptor protein levels have been associated with dietinduced obesity in rodents (Zhang & Scarpace, 2006).However, no change inOb-Rbgene expression was foundin C57Bl/6 mice on a high-fat diet (Haltiner et al.2004;

Munzberget al.2004), even when shown to be fully leptinresistant (Enriori et al. 2007). Also, at variance with theexpected down-regulationof receptor expression resultingfrom ligand stimulation,Ob-Rbgene expression has beenshown to increase in response to leptin challenge (Haltineret al.2004; Tanget al.2007; Di Yorioet al.2008).

Thus, the present study aims to define the control ofOb-Rbgene expression in the mouse by nutritional statusand leptin challenge in the hypothalamic ARC and VMH,areas important in appetite control, together with Ob-Rgene expression andreceptor number in theCP, part of the

system transporting leptin into the brain. The impact ofrelatively short-term high-fat feeding on leptin receptorregulation in the C57Bl/6 mouse was also investigated.Neuronal insensitivity to leptin has been reported in thismodel after 4 weeks on a high-fat diet (Munzberget al.2004).

As both alterations in receptor gene expression,

and endocytosis and trafficking of ligand-activated cellsurface receptors can modulate the expression of thereceptor protein, we measured both gene expressionand the number of leptin receptors available to bindligand using in situ hybridisation and 125I-labelledleptin binding, respectively. Leptin was delivered to theanimal systemically via intraperitoneal (I.P.) injectionand centrally by intracerebroventricular (I.C.V.) injectionsbypassing the leptin transport system in the bloodbrainbarrier.

Methods

Experimental animals

In the UK, all animal studies were licensed underthe Animal (Scientific Procedures) Act 1986 and wereapproved by the Ethical Review Committee of the RowettResearch Institute. In Spain, all animal experimentalprocedures were regulated by Santiago de CompostelaMedical School Animal Care Research Committee.The I.C.V. experiments and the fasting and refeedingexperiments were carried out in Spain. All otherexperiments were carried out in the UK.

Male C57BL/6 mice were part of a first generation bredat the Rowett Research Institute from mice obtained fromHarlan (Oxon,UK). In Spain C57BL/6 mice were obtainedfrom Harlan Iberica (Barcelona, Spain). All mice were1012 weeks of age at the start of experiment. Animalswere maintained on a 12 h lightdark cycle at 22 1Cand water was available ad libitum. Mice were killed, in thelate morning or early afternoon, by cervical dislocationfollowed by decapitation. Brains were rapidly removed,frozen in isopentane chilled over dry ice and stored at80C until cryo-sectioned and processed for in situhybridisation or quantitative in vitro autoradiography.n= 68 in all experimental groups. Trunk blood wascollected into heparinised tubes on ice, centrifuged andplasma collected and stored at 80C until use.

The influence of high-fat diet and both short- and

long-term leptin challenge. C57BL/6 mice were eitherfed 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 weeksin total. It has been reported that mice on high-fatdiet for 4 weeks develop leptin resistance in the ARC


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J Physiol587.14 Leptin receptor regulation in mice 3575

(Munzberget al. 2004). Leptin challenge in the presentstudy was at a concentration widely used to measureleptin responsiveness in diet induced obesity(Enriori et al.2007). Mice were challenged with murine leptin deliveredby either intraperitoneal (I.P.) injection (2 mg (kg bodyweight)1) (R&D Systems, Abingdon, UK) or I.C.V., todistinguish whether theleptinresistance was dueto neuro-

nal insensitivity or to inhibited leptin transport into thebrain. One group was injected with leptin I.P. twice dailyfor 1 week and killed 1 h after the final leptin injection, asecond group received I.P. vehicle injections twice dailyfor 1 week and a single leptin injection 1 h prior tokilling, and a third group received vehicle injections I.P.twice daily for 1 week and were killed 1 h after the finalvehicle injection. Two separate groups of mice fed dietsas described above for 4 weeks were anaesthetised by anI.P. injection of ketamine/xylazine (ketamine 100 mg (kgBW)1 plus xylazine 15 mg (kg BW)1) and then receivedeither a single I.C.V. injection of leptin (2g per mouse) or

vehicle 1 h prior to killing. Brain infusion cannulae werestereotaxically placed with their tips in the lateral cerebralventricle using the following coordinates: 0.7 mm post-erior to bregma, 1.2 mm lateral to the midsagittal suture,and to a depth of 2.0 mm, with bregma and lambda at thesame vertical dimension. Mice were allowed access to foodand water throughout these challenges.

The influence of fasting and refeeding. C57BL/6 micewere fasted for 24 or 48 h or fed ad libitum on standardmouse diet prior to killing. Refed animals were allowedaccess to food ad libitum for a period of 24 h after fasting.

In situhybridisation

Specific probes for Ob-R, Ob-Rb and SOCS3 were used asdetailed previously (Adamet al.2000; Merceret al.2000;Nilaweeraet al. 2002; Nogueiras et al. 2004; Tups et al.2004). Automated sequencing was performed to verifythe sequences. Messenger RNA levels were quantifiedby in situhybridisation, on 20 m thick coronal hypo-thalamic cryo-sections, using techniques described indetail elsewhere (Mitchell et al. 2002). Briefly, slideswere fixed in 4% (w/v) paraformaldehyde in 0.1 mol l1

phosphate-buffered saline (PBS) for 20 min at roomtemperature, washed in PBS, incubated in 0.1 mmol l1

triethanolamine for 2 min and acetylated in 0.1 mmol l1

triethanolamine and 0.25% (v/v) acetic anhydride for10 min. Sections were dehydrated in ethanol and driedunder vacuum before hybridisation with riboprobes at106 c.p.m. ml1 for 18h at 58C. After hybridisation,sections were desalted through a series of washes instandard saline citrate (SSC) to a final stringency of0.1 SSC at 60C for 30 min, treated with RNase A anddehydrated in ethanol. Slides were apposed to Biomax MR

(Sigma, Poole, Dorset, UK) together with 14C micro-scalestandards (Amersham/GE Healthcare, Little Chalfont,UK) at room temperature for varying lengths of timedepending on the probes used.

In vitroautoradiography

Sections were acid prewashed in low-pH, high-salt (pH2, 0.5 M NaCl) Hepes buffer to remove any endogenousleptin bound to receptors, prior to a brief wash inHepes, and were then incubated with 50 pM 125I-labelledleptin (PerkinElmer LAS, UK) with the specific activityadjusted to 250 000 cpm pM1 in Hepes for 2 h at roomtemp. Control slides were incubated with 125I-labelledleptin as detailed above plus 1 M leptin. Slides werethen thoroughly washed in Hepes buffer followed bydistilled water at 4C and air dried before beingapposed to Biomax MR (Sigma) together with 125Imicro-scale standards (Amersham/GE Healthcare) atroom temperature. Characterisation of125I-labelled leptinbinding was carried out by increasing the times ofincubation between 5 min and 6 h. A saturation iso-therm was produced by incubating sections withincreasing concentrations of 125I-labelled leptin betweenapproximately 5 and 500 pM.

Quantification of in situhybridisation and in vitro

autoradiography

Autoradiographs were scanned on a Umax Power LookII (UMAX Data Systems, Fremont, CA, USA). Integratedoptical densities (IOD), area and optical densityof images,were quantified using the Image Pro-plus system (MediaCybernetics, Bethesda, MD, USA). IOD was converted tonCi g1 using 14C microscale standard curves for measuresof total gene expression. For quantification of125I-labelledleptin binding to the CP, mean optical densities weremeasured and converted to nCi g1 using 125I microscalestandard curves.

Plasma leptin

Plasma leptin was measured by ELISA (BioVendor GmbH,Heidelberg, Germany) according to the manufacturersinstructions.

Statistical analysis

Data are presented as means S.E.M. and were analysedusingGenStat (GenStat, 5th edn (2005),VSN InternationalLtd, Hemel Hempstead, UK). In the case of experimentstesting the influence of a single factor a one-way ANOVAwas performed. Where two factors were compared in a





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single experiment, as in the case of high-fat diet and leptinchallenge, a two-way ANOVA followed bypost hoc t-testsbasedon the LSD were performed. Inthis case the ANOVAresults are expressed in the figure legend and the result ofthemultiple comparison test are represented on thegraph.

Figure 1. Representative autoradiographs of mouse brain

showing the areas measured in the present study

A,Ob-Rgene expression.B, 125 I-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.

The type of statistical test carried out is stated in the figurelegends.P< 0.05 was considered statistically significant.

Results

Localisation of gene expression and 125 I-labelled

leptin binding in mouse brain

Ob-Rgene expression was present over the medial andlateral CP, the ARC and the VMH (Fig. 1A). Specific125I-labelled leptin binding was clearly seen over both themedial and the lateral CP. No 125I-labelled leptin bindingwas measurable over hypothalamic nuclei (Fig. 1B). Highlevels ofOb-Rb gene expression are seen over the ARCand VMH. No discernable Ob-Rb gene expression wasfound over the CP (Fig. 1C) and no measurable inductionof SOCS3 mRNA in this region was seen in response toleptin challenge (Fig. 1D).

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 incomparison with mice on a low-fat diet (P< 0.001). Basalleptin levels were also higher in the mice on a high-fat dietvs. mice on a low-fat diet (P< 0.001) (Fig. 2C).

Regulation of Ob-Rgene expression in the CP

Ob-R gene expression in the CP remained unchangedthroughout the leptin challenge protocols used in thepresent study (data presented arefor lateral CP: Ob-Rgeneexpression in the medial CP showed the same pattern).Two-way ANOVA revealed no effect of high-fat feedingfor 4 weeks and no effect of I.P. or I.C.V. leptin challengeon the CP in C57Bl/6 mice (Fig. 3A) (data not shownfor I.C.V.). However, fasting for 24 h caused a significantincrease in Ob-R expression (P< 0.001) that was notfurther increased after 48 h fasting. Refeeding did notreturn Ob-R expression levels to those of fed animals(P< 0.05 fedvs.refed) (Fig. 3B).

Characterisation of 125 I-labelled leptin binding

to the CP

In vitro autoradiography of 125I-labelled leptin bindingover the CP was time and concentration dependent(Fig. 3C and D). Although both hypothalamic andextra-hypothalamic 125I-labelled leptin binding isapparent in the rat brain in our hands and as reportedby other groups (Baskin et al. 1999; Irani et al. 2007),the very low levels of 125I-labelled leptin binding in the





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hypothalamus of the mice in the present study were belowthe levels that could be accurately measured.

Regulation of 125 I-labelled leptin binding to the CP

The level of specific 125I-labelled leptin binding to theCP was increased in C57Bl/6 mice fed a high-fat diet for4 weeks (P< 0.05). An effect of leptin was also apparent(P< 0.001), with a rapid decrease of 125I-labelled leptinbinding in the C57Bl/6 mouse on both high- and low-fatdiets 1 h after a singleI.P. or I.C.V. leptin challenge, whichwas maintained after a week of twice daily leptin injections(Fig. 3E) (Data not shown for I.C.V.). Fasting for 24 hresulted in an increase of 125I-labelled leptin binding tothe CP (P< 0.001) that was significantly increased after48h (P< 0.05). Levels of125I-labelled leptin binding weredecreased by refeeding to significantly less than baselinelevels after 24 h (P< 0.05 fedvsrefed) (Fig. 3F).

Regulation of Ob-Rbgene expression in the ARC

and VMH

There was no effect of diet on basal levels of totalexpression ofOb-Rbin the ARC or VMH. There was asignificant effect of leptin (P< 0.05) to up-regulate thelevel of expression ofOb-Rbin the ARC and a significantinteraction between diet and leptin (P< 0.05), with

Figure 2. Body weight and leptin levels

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.

high-fat diet preventing up-regulation of Ob-Rb geneexpression by leptin (P< 0.05) (Fig. 4A). There was asignificant effect of diet (P< 0.01), but not leptin, on theOb-Rbgene expression in the VMH (Fig. 4A). Density ofriboprobe labelling is consistently higher on a high-fatdiet in both the ARC and the VMH reflecting thelevel of gene expression per cell (Fig. 4B), while leptin

challenge appeared to increase the area of labelling, i.e. thenumber of cells expressing the receptor only in the ARC(Fig. 4C). Fasting for 24 h caused a significant increasein Ob-Rbexpression in the ARC (P< 0.001) and VMH(P< 0.001) that was maintained but not increased by48 h fasting in the VMH, but further increased in theARC (P< 0.01). Refeeding caused a significant decreasein Ob-Rb expression that had returned to basal levelsafter 24 h ad libitum access to food in the VMH, anddecreased below the levels of the fed animal in the ARC(P< 0.05) (Fig. 5A). Gene expression changes in the ARCand VMH (Fig. 5BandC) appear to be a combination of

both changes in density and area. However, the changes inarea are much larger than those seen for density indicatingthat it is the number of cells expressing the receptor thatcontributes most to changes in gene expression (Fig. 5BandC).

All changes in the level of total Ob-R and Ob-Rbgene expression and 125I-labelled leptin binding aresummarised in Table 1.





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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 leptinchallenge in theARC andVMH (Fig. 6). Two-way ANOVArevealed a significant effect of leptin (P< 0.05) with nosignificant effect of diet and no interaction.

Discussion

The present study shows, for the first time, that leptinreceptor gene expression and leptin receptor numberin vivo, measured by 125I-labelled leptin binding, aredifferentially regulated by both leptin and high-fat diet.Two separate mechanisms have been demonstrated thatmay contribute to the development of leptin insensitivity.The first is the loss of ability of leptin to increase Ob-Rbgene expression in the ARC after 4 weeks of high-fatfeeding in the C57Bl/6 mouse. The second is the rapid

decrease in leptin receptor number in response to leptinchallenge. A better understanding of the mechanismsresponsible for leptin insensitivity may help to identifypotential targets in the treatment of obesity.

Under the experimental protocols used in this studythe level ofOb-R gene expression on the CP remainedunchanged after leptin challenge and in C57Bl/6 mice feda high-fat diet. However, fasting induced an increase inOb-R gene expression in the CP, but refeeding for 24 hfailed to reverse theincreasein expression. Although leptinlevels drop precipitously in response to fasting and riseagain on refeeding (Ishii et al. 2000), the insensitivity ofOb-Rgene expression in the CP to long and short-term

leptin challenge indicates that factors that change withfasting and refeeding, other than leptin, may regulateOb-Rexpression on the CP. Conversely, it may be thatthe fasting-induced drop in leptin up-regulates geneexpression while increasing levels of leptin on refeedingfail to down-regulate gene expression or may do so moreslowly.

In contrast to theregulation ofOb-Rgene expression ontheCP, leptin receptor number changes rapidlyin responseto changes in circulating leptin. The up-regulation of125I-labelled leptin binding to the CP in response to

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

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 BandF.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 mg1 protein. Levels of 125 I-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 125 I-labelled leptin binding to the CP underlie the bar graphs.

fasting and down-regulation to reach lower than base-line levels in response to refeeding together with theresponse to leptin challenge, detailed above, indicate thatthe drop in circulating leptin levels with fasting andincrease in leptin levels with refeeding (Ahrenet al.1997;Havel, 2001) drive these changes in receptor number. Thepresent study shows that receptor number on the CP

increases in response to fasting and also in response toa high-fat diet where serum triglyceride levels are elevated(Schreyer et al. 1998), indicating that receptor numberis not limiting transport of leptin into the brain. Oneexplanation for the increased level of 125I-labelled leptinbinding to the CPin conditions whereleptintransportintothe brain is reportedly decreased (Kastin & Akerstrom,2000; Bankset al.2004) is that inhibition of the transportprocess results in a subsequent accumulation of receptorsat the cell surface. However, this hypothesis remains to betested.

The increase in 125I-labelled leptin binding to the CP

after 4 weeks on a high-fat diet precedes the reportedincrease inOb-Rgene expression in the CP after 8 weeksof high-fat diet (Lin et al. 2000) and indicates thatchanges in receptor number occur prior to changesin gene expression. Apart from this counter-intuitiveincrease in leptin receptor number on the high-fat dietwhere leptin levels increase, our findings largely confirmprevious studies where receptor numbers drop in responseto increased leptin levels (Uotani et al.1999; Smallwoodet al. 2007) and increase in response to decreased leptinlevels in fasting (Baskin et al. 1999). However, 125I-labelledleptin binding to the hypothalamus has been shownto be lower in rats with a genetic predisposition todevelop obesity on a high-energy diet compared withrats resistant to diet induced obesity (Irani et al. 2007),and in that study 125I-labelled leptin binding appearsto be independent of the level of circulating leptin,indicating that leptin-independent changes in receptornumber also occur. The present study demonstrates thatboth leptin-dependent and leptin-independent changes inleptin receptor number take place.

Ob-Rbgene expression is specific for the long signallingform of theleptinreceptor necessary for second messengersignalling to be induced. Ob-Rb gene expression was





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Figure 4. Levels ofOb-Rbgene 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

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 ofOb-Rbgene 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 ofOb-Rbgene 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.





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Figure 5. Levels of Ob-Rbgene expression in the ARC and VMH of mice fasted for either 24 or 48 h and

refed for 24 h including representative autoradiographs

Statistical analysis was by one-way ANOVA. A , gene expression of Ob-Rb in the ARC and VMH. B , density of

Ob-Rbgene expression in the ARC and VMH.C, area ofOb-Rbgene expression in the ARC and VMH. P< 0.05,P< 0.01, P< 0.001.





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Table 1. Changes in Ob-R, Ob-Rb and125 I leptin labelling

Ob-Rb CP Leptin

High vs. low-fat

Fasted

Refed

125I-Leptin CP Leptin

Highvs.

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

undetectable over the CP, although its presence has been

reported on the ovine CP (Merino et al. 2006) and nucleartranslocation of STAT3 has been reported in the rat CPin response to leptin challenge (Mutzeet al.2006). Also,we did not detect induction of SOCS3 gene expression inthe CP in response to leptin challenge, further indicatingthatOb-Rbis not present in the mouse CP. However, thepossibility remains that low levels ofOb-Rb, below thesensitivity of the techniques used in the present study,may be present.

Ob-Rb gene expression in the ARC and VMH wassimultaneously up-regulated by fasting and reversed by

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

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

refeeding. However, leptin challenge caused a rapid (1 h)increase inOb-Rbgene expression, which may be specificto the ARC. Changes in Ob-Rb gene expression in theVMH, in response to leptin challenge, were more difficultto interpret butmay be theresultof smallerchanges 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 andarea ofOb-Rbexpression are considered, rather than totalgene expression. High-fat diet consistently increases thedensity ofOb-Rbexpression in the ARC and the VMH,whileleptinchallengeincreasestheareaofgeneexpression,but only significantly in the ARC and not on a high-fatdiet. Nonetheless, the increase in total Ob-Rbexpressionin the ARC was clear and was maintained after 1 weekof leptin challenge in agreement with previous reports(Haltineret al.2004; Di Yorio et al.2008). In the presentstudy this response is lost after high-fat feeding, indicatingthat the high-fat diet induces neuronal insensitivity

to leptin. However, the induction of SOCS3 geneexpression in these mice in response to leptin remainedintact indicating that the JAK/STAT pathway was notcompromised.

In contrast to the up-regulation of Ob-Rb in theARC by leptin challenge, fasting, which results in adrop in circulating leptin, also up-regulated Ob-Rbgeneexpression and refeeding, in which circulating levelsof leptin are increased, and down-regulated Ob-Rbgene expression, not only in the ARC but also in theVMH. This indicates that factors other than leptin





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appear to be responsible for the regulation of Ob-Rbgene expression in response to nutritional status. Onepossibility is the change in the level of corticosterone,which is known to rise and fall with fasting and refeeding(Makimuraet al. 2003) and also to regulate both leptinsignalling (Ishida-Takahashi et al. 2004) and the levelof gene expression of appetite regulatory signals in the

hypothalamus (Makimura et al. 2003). Dexamethasonehas been shown to either up-regulate or have no effect onOb-RbandOb-Raexpression, depending on the tissue orcell type tested (Hosoiet al.2003; Liuet al.2004; Wyrwollet al.2005).

In summary, the present findings demonstrate that,in the mouse, regulation of leptin receptor numberand receptor gene expression by leptin and nutritionalstatus is region and receptor sub-type specific. Fastingand refeeding, along with the concomitant drop andsubsequent rise in circulating leptin, appears to be theonlytreatment that had a consistent effect on both receptor

number and gene expression indicating that a drop inleptin is important in the regulation of both receptornumber andOb-Rand Ob-Rbgene expression, and thata drop in leptin levels is necessary for subsequentlyincreasing leptin levels to down-regulate receptor geneexpression. Increases in leptin level give rise to morecomplex effects which may be due to an interactionbetween leptin and diet or different effects of increasingconcentrations of leptin (Di Yorio et al. 2008). However,taking these results together, we conclude that (a) highlevels of leptin decrease 125I-labelled leptin binding,offering a potential mechanism for leptin insensitivity inresponse to hyperleptinaemia; (b)Ob-Rdown-regulationis not an essential mechanism in thedevelopment of leptininsensitivity; and (c) high-fat diet somehow blunts theability of leptin to regulateOb-Rbgene expression in theARC.

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