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Ann. N.Y. Acad. Sci. ISSN 0077-8923

ANNALS OF THE NEW YORK ACADEMY OF SCIENCESIssue: The Year in Diabetes and Obesity

CNS leptin and insulin action in the control of energyhomeostasis

Bengt F. Belgardt and Jens C. BruningDepartment of Mouse Genetics and Metabolism, Institute for Genetics, Center for Molecular Medicine, Cologne ExcellenceCluster on Cellular Stress Responses in Aging-Associated Diseases, Second Department for Internal Medicine University ofCologne, and Max Planck Institute for the Biology of Ageing, Cologne, Germany

Address for correspondence: Jens C. Bruning, M.D., Institute for Genetics and Center for Molecular Medicine (CMMC),Department of Mouse Genetics and Metabolism, Zulpicher Str. 47a, 50674 Cologne, Germany. [email protected]

The obesity and diabetes pandemics have made it an urgent necessity to define the central nervous system (CNS)pathways controlling body weight, energy expenditure, and fuel metabolism. The pancreatic hormone insulin andthe adipose tissue–derived leptin are known to act on diverse neuronal circuits in the CNS to maintain body weightand metabolism in a variety of species, including humans. Because these homeostatic circuits are disrupted duringthe development of obesity, the pathomechanisms leading to CNS leptin and insulin resistance are a focal point ofresearch. In this review, we summarize the recent findings concerning the mechanisms and novel neuronal mediatorsof both insulin and leptin action in the CNS.

Keywords: obesity; leptin; insulin; central nervous system (CNS); pancreas; diabetes

Leptin and insulin as messengers ofperipheral energy levels to the CNS

The circulating levels of leptin and insulin are pos-itively correlated with adiposity and body weight,1

and are now broadly accepted to deliver informationon peripheral energy stores to the central nervoussystem (CNS) by acting on diverse neuron popu-lations. In line with this notion, intracerebroven-tricular (i.c.v.) injection of insulin or intranasal ap-plication of insulin, which selectively mirrors CNSinsulin concentration, decreases food intake andbody weight in mice,2 rats,3 baboons,4 and men,5

although a recent report failed to detect an effect onfood intake and body weight in rats.6 Comparablystronger than insulin’s effect, leptin’s ability to re-duce food intake and decrease body weight is wellestablished.7–9 It has been ascertained that insulinand leptin action in the CNS, and here especially onneurons, is essential for decreasing the food intake(anorexigenic) and eventually weight reducing ef-fects of these two hormones,10,11 although notably,the role of other cell types present in the CNS such asastrocytes or microglia, which do express insulin re-ceptors (IR) and leptin receptors (LEPR),12,13 is still

poorly understood in this regard. In addition to in-sulin’s critical roles in glucose and lipid metabolismin the periphery, leptin has also direct effects on pe-ripheral tissues, and the interested reader is directedto a recent review on this topic.14

Hypothalamic mediators of insulinand leptin action

After the discovery that leptin and insulin mediatetheir effects on body weight and fuel metabolismby acting on neurons, the specific neuronal pop-ulation impacted on by both hormones had tobe established. It was already known that in ro-dents, lesions in the hypothalamus could impactbody weight, either inducing weight gain or weightloss, depending on the specific region of the hy-pothalamus ablated. In line with this, both lep-tin and IR are strongly and broadly expressed inthe hypothalamus, insulin action in the hypothala-mus has been demonstrated to induce anorexia andweight loss,15 whereas inhibition of insulin signal-ing has the opposite effect.16 Similarly, hypothala-mic signaling is necessary for leptin’s effects on bodyweight.17,18

doi: 10.1111/j.1749-6632.2010.05799.xAnn. N.Y. Acad. Sci. 1212 (2010) 97–113 c© 2010 New York Academy of Sciences. 97

CNS leptin and insulin action Belgardt & Bruning

Figure 1. Circuitry of leptin and insulin responsive neurons. Neuron populations of the arcuate nucleus (ARC), ventromedialhypothalamus (VMH), paraventricular hypothalamus (PVH), lateral hypothalamus (LH), ventral tegmental area (VTA), raphenucleus (RAPHE), and nucleus tractus solitarius (NTS)—which are responsive to leptin, insulin, or both—are depicted, as well asrelevant synaptic contact between these neuron populations. POMC and AGRP neurons in the ARC are first-order neurons of bothleptin and insulin. Whereas �-aminobutric acid (GABA) released from AGRP neurons inhibits POMC neuron activation directly,POMC and AGRP neurons release �-MSH and AGRP respectively onto PVH neurons, which express thyroid-releasing hormone(TRH) or corticotrophin-releasing hormone (CRH), which are involved in thermogenesis and feeding, respectively. In the VMH,steroidogenic factor 1 (SF1) expressing neurons are bona fide leptin-sensitive neurons, and predominantly regulate food intake. Inthe lateral hypothalamus, leptin acts on GABAergic neurons, which are in synaptic contact with dopaminergic neurons in the VTA.Both leptin and insulin also regulate activity of orexin neurons in the LH through hyperpolarization (leptin) or transcriptionalrepression of orexin (insulin). Dopaminergic neurons in the VTA, which release dopamine in the striatum to control activity andreward, are also directly silenced by leptin signaling, whereas dopamine reuptake is under control of insulin through transcriptionalcontrol of the dopamine transporter. Although serotonergic neurons in the raphe nucleus release serotonin (5-HT) onto POMCneurons in the ARC (and striatal neurons), leptin acts directly onto 5-HT neurons as well. Finally, nucleus tractus solitarius (NTS)neurons relay and compute signals from the gastrointestinal (GI) tract and exchange information with the parabrachial nucleus(PBN). Additionally, GABA release in the PBN from AGRP neurons is essential for feeding. Note that some synaptic connectionsand target regions are not depicted for optimal clarity. Region descriptions are in bold and underlined, neuron descriptions are inbold, and neurotransmitters/neuropeptides are written in normal formatting. Leptin (L) or insulin (I) responsiveness is depictedin bold and italic.

The first breakthrough in defining primary tar-get neurons of leptin and insulin occured in2001, when it was demonstrated that proopiome-lanocortin (POMC)-expressing neurons are depo-larized by leptin treatment, which leads to an in-crease in neuronal activity.19 In the hypothalamic

melanocortin system, POMC-expressing neuronsrelease the POMC cleavage product �-melanocytestimulating hormone (�-MSH), which acts ondownstream target neurons (some of which locatedin the paraventricular hypothalamus, Figure 1) toreduce food intake, increase energy expenditure,

98 Ann. N.Y. Acad. Sci. 1212 (2010) 97–113 c© 2010 New York Academy of Sciences.

Belgardt & Bruning CNS leptin and insulin action

and regulate glucose metabolism.20–22 In fact, nullmutations in leptin, LEPR, POMC, its cleavage en-zymes (prohormone convertases), or downstreamreceptors (such as Melanocortin receptor 4, MC4R)relate to hyperphagia and obesity both in rodentsand humans.23 In contrast to POMC neurons, acti-vation of arcuate nucleus (ARC) neurons coexpress-ing agouti-related protein (AGRP), and neuropep-tide Y (NPY) induces feeding, reduce energy expen-diture, and impacts on activity.24–26 AGRP acts asan inverse agonist on the MC4R, whereas NPY reg-ulates neuronal activity by acting on several NPYreceptors subtypes, ultimately blocking �-MSH-mediated anorexia. Of note, AGRP/NPY neuronsalso directly inhibit POMC neurons through synap-tic release of inhibitory �-aminobutric acid (GABA)on POMC soma.27 The critical role of these twotypes of neurons has been demonstrated by acuteablation models, which lead to hyperphagia (POMCneuron ablation) or hypophagia (AGRP neuron ab-lation), and following these initial experiments, itwas revealed that AGRP neuron-released GABA inthe hindbrain parabrachial nucleus is necessary forfeeding.25,26,28

With the advent of the Cre/loxP system, neuronalpopulation-specific ablation of the IR or LEPR ortheir signaling components had been made possible,and mice with POMC or AGRP/NPY-specific abla-tion of these two receptors have been analyzed. Ab-lation of the leptin receptor in POMC, AGRP/NPYor both neurons led to elevated body weight andmild obesity, again underlining the role of themelanocortin pathway.29,30 On the other hand, itwas deducted from these experiments that thereare other neuronal populations (and possibly extra-hypothalamic sites) that are critically targeted byleptin to inhibit feeding.

The ablation of IR from POMC and AGRP neu-rons surprisingly did not lead to hyperphagia or obe-sity, although IR signaling in AGRP neurons has animportant role in systemic glucose metabolism (seebelow).31 This was unexpected, because insulin’sanorexigenic action is at least partially dependenton an intact melanocortin system,32 and insulinclearly regulates electrical activity and transcrip-tional events in POMC neurons, such as localizationof forkhead transcription factors.33,34 Several recentreports have further shed light on this notion. Bothinsulin and leptin were known to influence mem-brane polarization and firing only in a subset of

neurons, in the range of 50% of all POMC neuronstested, and in an opposite manner; whereas leptindepolarized POMC neurons,19 insulin clearly hy-perpolarized POMC neurons.33,35 This conundrumwas elegantly resolved by the finding that these twohormones impact on different subpopulations ofPOMC neurons, that is there are neurons sensitive toleptin, whereas other neurons respond to insulin.36

Moreover, combined leptin and insulin action inPOMC neurons has synergistic effects on glucosehomeostasis (and fertility), because mice lackingboth IR and LEPR on POMC neurons show drasticimpairment of steady state glucose metabolism.37

Finally, it appears that in addition to the existenceof subpopulations of POMC neurons with regard toreceptor expression and hormone sensitivity, thereare neurons which only shortly during developmentexpress POMC, some of which go on to express NPYduring adulthood.38 Nonetheless, in these neuronsloxP-flanked genes of interest have already been re-combined due to the short-term activation of thePOMC-Cre transgene, which may compromise theanalysis of the resulting phenotype. Taken together,the efforts to unanimously identify the hypothala-mic neurons necessary for insulin-induced anorexiahave proven fruitless so far,31 with the primary lep-tin targets being better understood.

If hypothalamic insulin action is necessary andof consequence for food intake and body weightdefense, of what nature are those neurons? First,there are several subhypothalamic nuclei, in whichanalysis of IR ablation has not been reported yet,for example the ventromedial and the paraventric-ular nuclei, which are insulin responsive and whosefunction is absolutely required for normal energyhomeostasis.39–41 Intriguingly, leptin signaling inventromedial hypothalamic (VMH) or lateral hy-pothalamic (LH) neurons is of equal importancewith regards to body weight control as is leptinsignaling in POMC neurons.42,43 The neuropep-tide orexin is expressed in the LH, and it is cru-cial for wakefulness, because mutations in orexinor its receptor are implicated in narcolepsy in miceand men.44 In the LH, leptin hyperpolarizes orexin-containing neurons, which is in line with the notionthat satiety allows for periods of rest and sleep.45

Similarly, a report found that insulin, throughnuclear export of the transcription factor FOXA,decreases orexin expression,46 whereas orexinapplication silences POMC neurons.47 Besides these

Ann. N.Y. Acad. Sci. 1212 (2010) 97–113 c© 2010 New York Academy of Sciences. 99


targets, there are likely more hypothalamic neuronpopulations important for weight regulation, butthere are no marker genes available yet to studythem in detail.

In contrast, the role of hypothalamic insulin sig-naling in control of peripheral glucose metabolismis well established,48,49 where electrical inhibitionof neurons expressing AGRP has been identified asa major component of hepatic glucose productionregulation likely through vagal innervations.31 Lep-tin’s well-known ability to improve systemic glu-cose metabolism has also been shown to dependon hypothalamic circuits.50,51 Indeed, there is nowconclusive evidence that leptin signaling in POMCneurons is predominantly necessary for regulationof systemic glucose homeostasis. This notion wasdeducted from the finding that reexpression of theLEPR only in POMC neurons, whereas all othercells lack the LEPR, was sufficient to mostly normal-ize glucose homeostasis (but not weight), whereasdb/db mice (lacking LEPR globally) suffer from earlyonset uncontrolled diabetes.52

Notably, it has not yet been proven that CNS in-sulin and leptin action play the same role in humans,because patients undergoing liver transplantationand therefore hepatic deenervation show only mod-est changes in glucose production and metabolism,although these findings are hard to qualify due tothe pathologies leading to the liver transplantationitself, immunosuppression therapy, and other en-docrine abnormalities detected after the transplan-tation.53 Moreover, one group demonstrated thatin dogs, CNS insulin action appears to have only asmall effect on glucose metabolism, underlying theneed for further studies in nonrodent species.54

In addition to glucose metabolism, both CNS in-sulin and leptin action has been demonstrated toregulate lipid uptake and/or metabolism in the whiteadipose tissue, highlighting the broad potency ofthese two hormones to coordinately orchestrate fuelpartitioning.55–57

Leptin and insulin intracellular signalingcascades

Because leptin and insulin have profound effects ontranscriptional and electrical events in neurons, thesignaling events evoked by these hormones are ofhigh interest (Figure 2). Leptin activates intracel-lular signaling cascades through the recruitment ofthe Janus kinase (JAK) to the LEPR, where it phos-

phorylates several key residues on the LEPR. Signaltransducer and activator of transcription (STAT) 3proteins binds to the phosphorylated LEPR, and arethemselves phosphorylated by JAKs.58 This allowsfor dimerisation, and subsequent translocalizationinto the nucleus, where the STAT3 proteins bind toand regulate transcription of target genes.24 The roleof this pathway especially in POMC transcriptionis well defined because leptin treatment increasesPOMC transcription.59 On the other hand, leptin isable to stimulate the phosphatidylinositol-3-kinase(PI3K), which is also insulin’s main intracellularsignaling cascade.60 Here, insulin will stimulatebinding of the regulatory subunit of the PI3K tophosphorylated insulin receptor substrates (IRS),which allows for activation of the catalytic subunitof the PI3K. PI3K catalyzes the phosphorylationof the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2) and thus generatesphosphatidylinositol-3,4,5-trisphosphate (PIP3).PIP3 can bind to and activate ion channels, but isalso recognized by phosphatidylinositol-dependentkinase 1, which phosphorylates several proteinssuch as the kinase AKT to elicit downstreamsignaling events.61 Although it is widely acceptedthat leptin activates PI3K signaling at least inspecific neurons, the signaling cascade linkingleptin stimulation to PI3K activation has not yetbeen fully resolved. Leptin action may also reducethe degradation of PIP3 to PIP2 by phosphorylationand thus deactivation of the PIP3 dephosphatasephosphatase and tensin homologue (PTEN).62 Onthe other hand, the adapter protein SH2B1 hasbeen shown to recruit JAK and IRS proteins in a su-percomplex, thus allowing crosstalk between thesetwo pathways.63 Importantly, SH2B1 has also beenlinked to human obesity.64 Leptin-induced PI3Ksignalling in the hypothalamus has been linked toperipheral glucose homeostasis and food intake,60,65

although the neuron populations mediating botheffects are not completely elucidated.

Another molecular target of both leptin and in-sulin is the AMP-dependent kinase (AMPK). Lowcellular energy levels will increase the AMP/ATP ra-tio, which is sensed by AMPK and converted intoa cellular response to induce ATP generation andreduce ATP consumption. In the hypothalamus,AMPK activation increases food intake, and bothleptin and insulin have been shown to decrease thephosphorylation and thus activation of AMPK in



Figure 2. Intracellular signaling cascades activated by insulin and leptin. Insulin and leptin are able to stimulate activity ofthe phosphatidylinositol-3-kinase (PI3K), which subsequently results in phosphorylation and nuclear exclusion of the forkheadtranscription factors FOXO1 and FOXA2. FOXO1 is a negative regulator of carboxypeptidase E (CPE) expression in POMCneurons, whereas it stimulates transcription of AGRP in AGRP neurons. Leptin-activated STAT3 binds to POMC and AGRPpromoters, where it stimulates (POMC) or inhibits (AGRP) expression via recruitment of histone modifying enzymes. FOXA2 wasdemonstrated to bind to the orexin and MCH promoters, where it stimulates expression of these neuropeptides. Insulin stimulationis known to increase expression of the dopamine transporter (DAT) gene in dopaminergic neurons. Both leptin and insulin areable to hyperpolarize neurons by PI3K-mediated opening of ATP-dependent potassium channels (KATP channels) and subsequentpotassium outflow. On the other hand, leptin is able to depolarize neurons and thus increase the firing rate by opening a nonspecificcation channel by a PI3K and JAK-dependent pathway, and pharmacological manipulation has led to the conclusion that thischannel may be the transient receptor potential (TRP) channel. Note that for clarity, several other pathways have been omitted (seetext).

the hypothalamus,66 although it is unresolved howexactly activation of AMPK is blocked by both hor-mones. AMPK action in POMC and AGRP neuronsplays a role in the neuron’s response to ambient lev-els of glucose, whereas leptin’s and insulin’s effect onneuron firing is not affected.67 Besides these path-ways, leptin (and insulin) are able to inducemitogen-activated protein kinases (MAPK) suchas extracellular signal-regulated kinase (ERK). Al-

though ERK signaling mediates some of the effectsof leptin on food intake in the hypothalamus,68 itis unknown if insulin uses this pathway to maintainbody weight.

Transcription, membrane potential,and synaptology

As true regulators of neuronal activity, leptin and in-sulin change membrane potential of target neurons



to control firing rate and thus neuropeptide andneurotransmitter release. Both leptin’s and insulin’seffect on POMC neuron firing has been extensivelystudied, with leptin depolarizing and insulin hyper-polarizing a subset of POMC neurons. With regardsto leptin, the cation-channel opened by leptin stim-ulation has been elusive for some years, with recentreports implicating both leptin-stimulated JAK andespecially PI3K signaling in opening of transient re-ceptor potential channels in POMC neurons, and itwill be highly informative to see if this holds truealso for other leptin-stimulated neurons, such asVMH neurons.69,70 On the other hand, both in-sulin and leptin have been shown to be able to ac-tivate ATP-dependent potassium (KATP) channels,which leads to potassium outflow, hyperpolariza-tion and a reduction of the firing rate.71,72 This hasbeen well demonstrated for AGRP neurons.31,73,74

Mechanistically, it has been proposed that PI3K ac-tivation leads to local accumulation of PIP3, whichbinds to KATP channels, increasing the probabilityfor an “open” channel, and reducing the affinity forATP.75 On the other hand, PIP3 generation alonemay not be sufficient, because actin filament sta-bilization prevents insulin-stimulated KATP channelopening, whereas the mechanisms induced by in-sulin (and leptin) to control actin filament dynam-ics are poorly understood.74 As the name suggests,KATP channels are sensitive to cellular levels of ATP,that is, they are closed by intracellular rises in ATP.Glucose-sensitive neurons (such as POMC neurons)are able to sense a rise in ambient glucose concentra-tions, because the increase in cytoplasmic ATP in re-sponse to glycolysis closes the KATP channels, whicheventually results in depolarization and an increasein firing rate.76 PI3K activation and insulin stimu-lation have also been reported to depolarize AGRPneurons, although the channels involved have notbeen identified, underscoring the diversity of find-ings regarding electrophysiological responses to in-sulin and leptin, as recently discussed.67,76,77 Takentogether, depending on the neuronal population,acute insulin and leptin application may depolar-ize or hyperpolarize target neurons, effects whichmay be accounted for by differential expression oftheir receptors, that of ion channels or intracellularsignaling intermediates determining the net result.

Both leptin and insulin directly control transcrip-tion of target genes, including neuropeptides. Asmentioned before, leptin-activated STAT3 signaling

controls POMC transcription in POMC neurons.59

In the same vein, leptin and insulin stimulationleads to phosphorylation and nuclear exclusion ofthe forkhead transcription factor FOXO1, allowingfor STAT3 binding to the promoter and transcrip-tion of POMC.33,34,78,79 FOXO1 is also a negativeregulator of carboxypeptidase E (CPE) expression,which is important for a distal step in processingPOMC into its cleavage products, i.e., �-MSH.80

Regarding expression of orexigenic neuropep-tides, FOXO1 and STAT3 again compete for bindingto the promoters of AGRP and NPY, with FOXO1being an activator of transcription of these orexi-genic neuropetides, and STAT3 being inhibitory.78,79

Indeed, leptin’s ability to reduce AGRP/NPY ex-pression depends on PI3K signaling,81 and insulinstimulation excludes FOXO1 from the nucleus ofAGRP neurons.34 Because mice with constitutiveSTAT3 activation only in AGRP neurons are hyper-active and lean, STAT3 signaling in these neuronssurprisingly regulates locomotor activity, althoughthe downstream neurons mediating this phenotypeare unknown.24 Interestingly, mice with constitutiveSTAT3 signaling in POMC neurons are hyperphagicand mildly obese, due to suppressor of cytokine sig-naling (SOCS) 3 overexpression.82 SOCS3 inhibitsactivation of the leptin signaling cascade at the levelof the receptor, and SOCS3 expression is under con-trol of STAT3 signaling, thus constituting a negativefeedback loop. Because leptin levels are chronicallyelevated in obesity, SOCS3 levels are increased inthe hypothalamus of obese mice,83 and ablation ofSOCS3 in the brain or POMC neurons ameliorateshigh fat diet- (HFD)induced obesity.84,85 Nonethe-less, hyperleptinemia alone does not induce SOCS3-mediated leptin resistance and consequentlyobesity, because leptin-transgenic mice remainleptin sensitive.86 Interestingly though, hyper-leptinemia predisposed leptin-transgenic mice toobesity when challenged with a HFD. SOCS3 is alsoan inhibitor of insulin signaling through degrada-tion of IRS proteins, thus (at least in POMC neu-rons) hyperleptinemia or STAT3 overactivation willconcurrently lead to cellular insulin resistance.82,87

Leptin also controls expression of multiple neu-ropeptides in neurons downstream of POMC andAGRP/NPY neurons, for example in the paraven-tricular neurons (PVN). Here, leptin has beenshown to increase expression of thyroid releas-ing hormone (TRH), which is a positive regulator



of energy expenditure.88 Moreover, leptin stimu-lation affects transcription of many more genes,nonetheless mice with leptin receptor deficiencyonly in the PVN have not been generated yet, thusthe direct and indirect targets (through neuropep-tide/neurotransmitters released by upstream neu-rons) are currently indistinguishable.89

Besides direct transcriptional control and mem-brane potential regulation, leptin (and purport-edly insulin) signaling is able to change thesynaptic input onto neurons. For example, micelacking leptin show decreased numbers of gluta-matergic (=excitatory) synapses on POMC neuronsand increased glutamatergic input on NPY neurons,both of which is rapidly, that is in hours, normal-ized upon leptin treatment.90 Other hormones, forexample estrogen, a known anorexigenic hormone,or ghrelin, which stimulates food intake, also regu-late synaptic input,90,91 thus synaptic rewiring maybe an important level of regulation used by manydifferent hormones involved in energy homeostasis.Nonetheless, it is still unresolved by which mech-anism and which attractant the synapses are re-cruited or repelled, and, if this is due to signaling onthe presynaptic or postsynaptic neurons. Althougha role for insulin in synaptic plasticity in neuronsdirectly linked to energy homeostasis has not beenreported, postsynaptic insulin action in hippocam-pal neurons is known to recruit GABA receptors andthus sensitize cells for this inhibitory neurotransmit-ter,92 which at least opens up the question if insulinplays a similar role in hypothalamic neurons.

Extrahypothalamic neurons targetedby insulin and leptin

Clear evidence that hypothalamic neurons do notcontribute all of the weight-regulating effects of bothleptin and insulin has opened the search for othernuclei expressing LEPR and IR, and analysis of theirimportance with regards to energy homeostasis. Allof these nuclei and neuropeptide circuits had beenpreviously shown to control body weight regula-tion, and it is now obvious that insulin and leptinact on almost all levels of feeding, including foodrecognition, food liking, and meal initiation.

DopamineThe generation of mice lacking dopamine in thebrain led to the discovery that these mice show re-duced activity, less food intake, and would die if

not treated with l-dopa, which is metabolized todopamine.93 Notably, if these mice are crossed tomice lacking leptin, the resulting animals are alsohypophagic and die.94 The role of the dopaminer-gic circuit concerning addiction to drugs such asalcohol, amphetamines or cocaine but also to therewarding aspect of food is well established,95 withdopaminergic neurons located in the substantia ni-gra (SN) and ventral tegmental area (VTA) pro-jecting to many brain nuclei involved in activity,decision-making and activity, such as the frontalcortex, hippocampus or the striatum. Intriguingly,there is now growing evidence that obesity is alsolinked to dysfunction of the dopaminergic system,as striatal dopamine D2 receptor binding is reducedin obese patients as measured by positron emis-sion tomography.96 Most importantly, leptin andinsulin impact on midbrain dopaminergic neuronsto regulate food-finding behavior and eventuallybody weight.95 Thus, LEPR are expressed on VTAdopaminergic neurons, which are hyperpolarizedafter stimulation with leptin ex vivo.97,98 Moreover,leptin microinjection into the VTA reduces foodintake, whereas ablation of the receptor only inthe VTA increased the sensitivity of these mice tothe rewarding aspect of highly palatable food, suchas sucrose.98 The crucial role for VTA dopamineneurons in the regulation of energy homeostasisis underlined by the finding that direct leptin ac-tion on LH neurons also signals to VTA dopamineneurons by synaptic contact, decreasing food in-take and thus body weight.43 Besides LEPR, theIR is also expressed on VTA (and SN) dopaminer-gic neurons.99 Intracerebroventricular insulin treat-ment has been demonstrated to increase expressionthe dopamine transporter (DAT) in dopaminergicneurons.100 Dopamine from the synaptic cleft istaken up by DAT back into the presynaptic neu-ron, and thus stops to stimulate postsynaptic neu-rons. Hence, insulin may act through this cascadeto decrease the rewarding effect of food, which is inline with the findings from multiple experimentalparadigms.95

SerotoninIn the CNS, serotonin (5-Hydroxytryptamin) is aneuropeptide expressed only in the raphe nucleuslocated in the midbrain. Besides being involved inmood regulation, serotonin clearly plays an impor-tant role in the control of weight. Thus, molecules



Figure 3. Mechanisms implicated in CNS leptin and insulin resistance. Both leptin and insulin must pass the blood–brain barrier(BBB) by a saturable and active transport mechanism. Binding of C-reactive protein (CRP), whose expression is elevated in obesityto leptin inhibits interaction of leptin with its receptors, and has been speculated to impair BBB transport of leptin. Hyperlipidemia,hyperglycemia, and obesity by itself have also been shown to impair insulin transport across the BBB. Elevated circulating levels ofsaturated fatty acids, such as palmitate, may activate toll-like receptor TLR / Myd88-dependent signaling, which results in inhibitorof NFKB kinase activation, which is able to both directly impair leptin signaling, but may also upregulate expression of suppressorof cytokine signaling (SOCS) 3, which then acts on the leptin receptor. IKK and SOCS3 are also involved in the induction of CNSinsulin resistance. Inflammatory signals, for example tumor necrosis factor-(TNF)� , can activate inflammatory signalling cascades,such as IKK but also c-Jun N-terminal kinase (JNK)1. JNK1 has been implicated in blocking insulin signaling directly at insulinreceptor substrates through inhibitory serine phosphorylation. Both IKK and JNK1 can be activated by high-fat diet (HFD) induced



increasing the bioavailability of serotonin (by in-hibiting reuptake into the presynaptic neuron) suchas fenfluramin were very effective in reducing bodyweight in patients, although side effects partially dueto serotonin receptor activation in the heart led toits withdrawal from the market. Interestingly, sero-tonin acts on the melanocortin system to reducehunger. Thus, serotonin nerve terminals are foundon POMC neurons, serotonin treatment increasesexcitation and thus the firing rate of POMC neu-rons, and mice with reexpression of serotonin recep-tor type 2 only on POMC neurons on a backgroundof serotonin type 2c receptor knockout greatly nor-malizes body weight compared to the obese HTR2creceptor knockout mice.101,102 Although previousreports had already detected significant LEPR ex-pression in the (dorsal) raphe nucleus,103 recentdata indicates that leptin acts directly on serotoner-gic neurons to control food intake and body weight,because mice lacking LEPR only on serotonergiccells showed similar obesity compared to mice lack-ing all LEPR (i.e., db/db mice).104 The authors couldfurthermore show that leptin treatment decreasedthe number of action potentials of serotonin neu-rons.104 Because serotonin release onto POMC neu-rons is thought to mediate its anorexigenic effects atleast in part, it is not immediately obvious how thisinhibitory effect of leptin would lead to an outcomeof reduced feeding and body weight.

Nucleus tractus solitarius (NTS)The NTS of the brainstem is a relay between affer-ent input from the gut, with synaptic connectionsto the hypothalamus, the parabrachialis nucleus, butalso higher brain regions such as the forebrain.105

Gut distention due to food intake in combinationwith release of gut hormones such as glucagon-likepeptide (GLP) signals to the brainstem via the va-gus nerve to induce satiety, and this input is thenforwarded and computed in the aforementionednuclei. Leptin signaling in the brainstem is clearlyrelevant for meal size regulation, because LEPR ex-pression is found in the brainstem, injection of lep-tin into the brainstem parenchyma at very small

doses induces phosphorylation of STAT3, and isable to reduce meal size and thus food intake.106

Importantly, a subpopulation of NTS neurons ex-ists that both integrates leptin action (through theLEPR) and gastrointestinal input (through vagalinnervation).105 Moreover, leptin potentiates gas-trointestinal (GI) signals; thus when gastric disten-tion and leptin injection into the 4th ventricle (inclose contact with the NTS), at levels that do notreduce food intake, are combined, a significant re-duction in food intake is achieved.105 The NTS alsoharbors a population of POMC neurons, which areactivated by the anorexigenic gut hormone chole-cystokinine, and signal to MC4R in the brainstemto subsequently reduce food intake.107 Although itis not clear of insulin has similar properties with re-spect to NTS-mediated food intake suppression, thesynergy between leptin (and possibly insulin) andGI signals is of high interest, because GI hormonessuch as GLP (and its derivates) or oxyntomodulinpositively affect glucose homeostasis and/or bodyweight, and importantly, obesity may not induceresistance against gut hormones to the same extentas against leptin and insulin.108

The mechanisms of CNS insulinand leptin resistance

Frustratingly, leptin and insulin’s ability to controlenergy homeostasis is abrogated both in obese ani-mal models and individuals suffering from obesity.Because lifestyle interventions alone are not suffi-cient to normalize body weight in most individuals,therapeutic interventions appear to be necessary fortreatment of obese patients. Thus, to understandthe mechanisms by which the weight-reducing ef-fects of leptin and insulin are blunted is of utmostimportance (Figure 3).

The first obstruction that both leptin and in-sulin must surpass is the blood–brain barrier (BBB),at least in brain regions where the BBB is tight(in the ARC, the BBB is only weakly developed).Both leptin and insulin are transported acrossthe BBB via saturable mechanisms, which are im-paired by obesity and associated pathologies such as

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−endoplasmatic reticulum (ER) stress, induced by accumulation of misfolded proteins in the ER, which leads to the unfolded proteinresponse (UPR). Palmitate also regulates localization and activation of protein kinase C theta (PKCΘ), which may induce insulinresistance through serine phosphorylation of IRS proteins. Low-grade inflammation will induce expression of protein phosphatase1B (PTP1B), which dephosphorylates and thereby deactivates IR, IRS, LEPR, and JAK proteins. Finally, HFD induces apoptosis ofhypothalamic neurons in rats, and it is unknown if this holds true also for other model organisms.



hypertriglyceridemia.109–112 A more direct impacton leptin signaling has been linked to the low-levelchronic inflammation found in obesity, that is C-reactive protein (CRP), secreted by the liver andincreased in obese patients, binds to leptin and in-hibits interaction with LEPR, thus leading to leptinresistance.113 Notably, leptin itself induces hepaticCRP expression, and as such hyperleptinemia it-self has been shown to be a negative regulator ofleptin sensitivity. Thus, chronic hyperleptinemia incombination with additional factors such as HFDwill lead to overexpression of SOCS3, which inducesboth a blockade of leptin signaling at the level of theleptin receptor,114 and insulin resistance, becauseSOCS3 targets IRS proteins for ubiquitin-mediateddegradation.87 Further to this, SOCS3 ablation fromall neurons strongly protects from diet-inducedobesity.84

Besides SOCS3, other negative regulators of leptinand insulin signaling have been shown to be involvedin the induction of resistance to these two hormones,such as protein phosphatase 1b (PTP1B).115 Hy-pothalamic PTP1B is increased upon obesity (andinterestingly aging), it may dephosphorylate JAKs,STATs, the IR as well as IRS proteins.116,117 BothSOCS3 and PTP1B are involved in inflammatoryprocesses. The notion that obesity induces low-levelinflammation in the periphery, especially the adi-pose tissue, has been known since the late 1980s,when it was demonstrated that adipose tissue of bothmurine obesity models and that of obese patientsexpresses higher levels of tumor-necrosis factor-�(TNF-�),118 which is released into circulation, andinduces insulin resistance in target tissues such asskeletal muscle and adipose tissue itself.119 Indeed,in that setting, macrophages invade the adipose tis-sue, likely recruited to take up overloaded and thusdying adipocytes,120 and release proinflammatorycytokines which in a positive feedback loop will at-tract more macrophages.121

Interestingly also in the hypothalamus, increasedexpression of both TNF-� and Interleukin 6 havebeen reported upon obesity development,122 ac-companied by activation of inflammation-sensitivekinases such as inhibitor of NFKB kinase (IKK) andc-Jun N-terminal kinase (JNK).122–124 Moreover,inhibition of IKK signaling improves leptin andinsulin sensitivity, whereas JNK1 inhibition amelio-rates hypothalamic insulin resistance123–125. Mech-anistically and in line with the accepted role for

SOCS3 in leptin resistance, IKK activation may posi-tively control SOCS3 expression, especially in AGRPneurons.124 JNK1 on the other hand, may phospho-rylate IRS proteins on serine residues, to inhibitactivating tyrosine phosphorylation.126 JNK1 mayalso inhibit TRH expression and thus energy expen-diture, although it is unknown if this is due to JNK1action directly in the PVN neurons.123,127

In this context, it is notable that TNF-� recep-tor knockout mice show increased energy expendi-ture potentially due to elevated TRH levels, whichmay suggest a TNF-�-> JNK1->TRH-regulatorycircuit.128 On first view, this data may indicate thatincreased TNF-� and Interleukin 6 signaling maybe underlying the activation of the proinflamma-tory signaling cascades found in the hypothalami ofobese animals, yet closer inspection may yield otherconclusions. First, mice lacking interleukin 6 de-velop adult-onset obesity,129 indicating that IL6 sig-naling is necessary for normal energy homeostasis.Second, it is unclear if in the mouse model systemcentrally applied TNF-� is anorexigenic or orexi-genic,130,131 which may be dependent on the con-centration used. Third, TNF-� signaling has beendemonstrated to protect brain tissue against multi-ple cellular stressors such as (glutamate) excitotox-icity, axonal injury or oxidative stress.132,133 In thiscontext it is important to note, that hyperglycemia,a hallmark of diabetes and obesity, induces neu-ronal oxidative stress,134 mitochondrial abnormali-ties,135 and most importantly, diet-induced obesityleads to loss of hypothalamic neurons due to apop-tosis in rats.136 It has also been noted that chroniccilliary neurotrophic factor (CNTF) administrationreduces body weight dependent on neurogenesis inthe hypothalamus,137 whereas the acute anorecticeffect of CNTF is mediated via gp130 receptor sig-naling in POMC neurons,138 indicating that gainor loss of neurons known to be relevant in energyhomeostasis may be involved in the developmentand prevention of obesity. Finally, it has not yetbeen revealed if cytokines produced in peripheraltissues such as adipose tissue are the source of thelow-level inflammation in the hypothalamus, or ifmicroglia, astrocytes and/or neurons produce thesecytokines, which then act in an autocrine/paracrinemanner. It is likely, that both peripheral and locallyproduced cytokines are involved in the inflamma-tory processes in hypothalamic tissue of obese an-imals. Further decisive experiments regarding the



diet-induced dysfunction and degeneration of neu-rons involved in energy homeostasis will be highlyinformative.

Besides cytokines, (saturated) fatty acids such aspalmitate have been directly implicated in both lep-tin and insulin resistance by eliciting activation ofinflammatory kinases such as IKKs or JNKs. It haslong been acknowledged that hyperlipidemia in-duces peripheral insulin resistance, partially due toactivation of toll-like receptor (TLR) 4 signaling.139

In the hypothalamus, i.c.v. injection of palmitateat a dose which does not affect food intake par-tially blocks the ability of leptin to activate STAT3signaling and reduce food intake.140 This was notthe case in mice lacking neuronal MYD88, a scaf-fold protein necessary for TLR2/4-mediated activa-tion of IKK and JNK signaling.140 Similar studies inrats could show that palmitate application inducedIKK activation, and subsequently hypothalamic in-sulin resistance.141 Notably, this study confirmedthat even when total caloric intake was similar be-tween groups, the group consuming HFD showedgreater CNS insulin resistance.141 Finally, fatty acidsmay induce local expression of cytokines, whichthen may lead to leptin resistance and obesity.142

Fatty acids also activate protein kinase (PKC) theta,which might then translocate to the plasma mem-brane and induce inhibitory serine phosphorylationon IRS proteins.143 This report underlined the no-tion that unsaturated fatty acids, such as oleate, donot induce CNS insulin resistance,143 and in factmay have anorexigenic properties, by inhibition ofKATP channels of POMC neurons.144 It should benoted, that as of now, there has been no conclusiveevidence that saturated fatty acids such as palmi-tate directly bind to TLRs, and moreover, there hasbeen the intriguing finding that under conditions ofobesity, small amounts of intestinal lipopolysaccha-ride (LPS, the prototypical TLR activating molecule)may enter the circulation due to a leakage of the in-testinal barrier function, which then might inducelow-grade inflammation.145

Besides both low-grade inflammation and hyper-lipidemia, another crucial event in the pathology ofperipheral insulin resistance is endoplasmatic retic-ulum (ER) stress. Protein folding in the ER is neces-sary for normal cellular homeostasis, as misfoldingof nascent proteins can have deleterious results, ulti-mately compounding cell viability. In obese animals,the need for translation is increased (at least in liver

and the pancreas), whereas at the same time fold-ing capability is limited. Accumulation of unfoldedproteins is then sensed by specific receptors, whichwill initiate an adaptive program, termed unfoldedprotein response (UPR). During the UPR, the ex-pression of chaperones, proteins assisting in proteinfolding will be increased, whereas global transla-tion is reduced to resolve the ER stress.146 Note thatupon enduring unresolved ER stress, the cell willinitiate an apoptosis program.146 In peripheral tis-sues, especially the liver, ER stress has been detectedin diabetic and obese mouse models, and appli-cation of chemical chaperones can greatly reduceglucose intolerance and insulin resistance in theseanimals.147,148 ER stress is not restricted to periph-eral tissues; instead it appears to play a major rolein the induction of hypothalamic leptin and insulinresistance. Neuronal ablation of XBP-1, a transcrip-tion factor involved in the UPR, leads to massiveleptin resistance, and obesity.149 Hence, i.c.v. ap-plication of chemical chaperones ameliorates leptinresistance.149 Moreover, i.c.v. application of chem-ical reagents such as tunicamycin or thapsigarginknown to induce ER stress partially inhibits the ef-ficacy of i.c.v. leptin and insulin as well.150 In linewith the notion that hyperlipidemia, inflammationand ER stress are interlinked, hyperlipidemia andinflammation may induce hypothalamic ER stress,and ER stress induces cytokine expression, whichmay be involved in a specific decrease of anorex-igenic POMC expression, possibly due to POMCneuron apoptosis.124,136,142,151 Although several re-ports now point to the induction of ER stress uponhyperlipidemic or inflammatory insults also in thehypothalamus, the mechanisms by which ER stressis induced, are less well defined (and vice versa,the mechanisms by which chaperone treatment im-proves hypothalamic leptin sensitivity). Saturatedfatty acids may directly impair ER homeostasis, forexample by changing the lipid composition of the ERmembrane, which is followed by calcium depletionand breakdown of ER function.152–154 Notably, over-abundance of saturated fatty acids such as palmitatemay also induce generation of ceramides, a lipidspecies involved in insulin resistance and apopto-sis. Ceramide generation has been linked to insulinresistance in peripheral tissues such as liver andskeletal muscle.155 Palmitate is necessary for the denovo generation of ceramides, palmitate supplementacutely induces ceramide generation, and ceramide



Table 1. Urgent questions

• Are there leptin- but not insulin-responsive subpopulations in all nuclei involved in energy homeostasis? How can

we identify and differentiate between these subpopulations in vivo by transgenesis to address their function?

• Does insulin influence synaptic rewiring of neurons? Is this cell-intrinsic or postsynaptically controlled? Can we

determine the molecular pathways controlled by leptin and insulin to affect axon guidance and synaptic contact?

• If leptin-mediated regulation of serotonin is crucial for energy homeostasis, is insulin involved in this as well?

• Does VTA/NTS/raphe leptin and insulin resistance exist? Do the same mechanisms occur as in the hypothalamus?

• Can we identify further extrahypothalamic targets mediating leptin’s and insulin’s effects, such as olfactory

neurons?

• How can we connect and integrate genes found by human genome–wide association studies (GWAS) into the

known signaling pathways of leptin and insulin? Is there a connection to be found at all?

• Can we identify marker for the neurons implicated in computing gastrointestinal signals and insulin/leptin

signaling? Are GI signals in the CNS modulated by insulin?

accumulation induces insulin resistance by block-ing AKT activation and dephosphorylation of acti-vated AKT in peripheral tissues or nonneuronal celllines.156–158

Conclusion

In this review, we attempt to give an informingoverview on the current concepts of insulin andleptin targets, the intracellular cascades activated,and the pathomechanisms leading to CNS resistanceagainst these two anorexigenic hormones. We havesummarized the most urgent questions in need ofclarification in Table 1. It is clear from the existingdata that although hypothalamic circuits are nec-essary for control of energy homeostasis, severalothers such as the dopaminergic and serotonergicneurons critically contribute to normal body weightregulation. It is also accepted that besides leptin andinsulin, there are dozens of other hormones suchas ghrelin, but also metabolites such as glucose andlipids, which directly impact weight regulation andglucose metabolism. Moreover, there is the worryingfact that obesity and diabetes, especially in the preg-nant mother, have negative and importantly long-term effects for the unborn child (see review in thisvolume by S. Ozanne, Ref. 159), which suggest thatin our rapidly aging and westernized societies, thediabesity pandemics will grow. Thus, although en-vironmental changes and lifestyle adaptation willhopefully be able to throttle the increase in obe-sity and diabetes with beneficial effects on otheraging-related diseases, we have to try to developpotential treatment options, which may eventually

include intranasal delivery of neuropeptides,160 orself-inactivating gene therapy.161

We would like to stress that although one maythink that we have identified all major brain sub-regions involved in energy homeostasis, we havelikely only identified a small proportion. Furtherprogress in the analysis of extra-hypothalamic nu-clei and integration of these findings including therole of clearly relevant genes discovered by humangenome–wide association studies (GWAS) of hu-man obesity (such as FTO)162,163 into our existingconcepts will eventually lead to novel therapeuti-cal targets and better treatments for the diabesitypandemic.

Acknowledgments

We apologize to all colleagues whose important con-tribution could not be cited due to space limitations.We thank G. Schmall and T. Rayle for excellent secre-tarial assistance and all members of the Bruning labfor helpful discussion of the manuscript. This workwas supported by grants from the CMMC (TV1)and the DFG (Br. 1492/7–1) to J.C.B. CECAD isfunded by the DFG within the Excellence Initiativeby German Federal and State Governments.

Conflicts of interest

The authors declare no conflicts of interest.

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