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Differential leptin access into the brain — A hierarchical organization of hypothalamic leptin target sites?
Physiology & Behavior 94 (2008) 664–669
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Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b
Review
Differential leptin access into the brain — A hierarchical organization of hypothalamic leptin target sites? Heike Münzberg ⁎ Division of Metabolism, Endocrinology and Diabetes, Departments of Internal Medicine, University of Michigan Medical School, 1150 W. Medical Center Dr., 5510D MSRB 1, Ann Arbor, MI 48109, USA
A R T I C L E
I N F O
Article history: Received 5 March 2008 Accepted 2 April 2008
A B S T R A C T Leptin target sites are found in several hypothalamic areas with dense populations of long form leptin receptor (LepRb) expressing neurons which mediate important leptin actions. Leptin action has been most intensely investigated in the arcuate nucleus of the hypothalamus (ARC), which represents an important leptin target site. Recent data have shown that non-ARC leptin target sites mediate important aspects of leptin action, however, including the regulation of energy balance. Therefore, the investigation of discrete leptin signaling systems and their interactions will be an important step to understand the homeostatic action of leptin. In this review I discuss our recent data investigating important differences in leptin accessibility to ARC neurons in contrast to other hypothalamic sites like the dorsomedial hypothalamus (DMH) and discuss their importance for the leptin signaling system. © 2008 Elsevier Inc. All rights reserved.
Contents 1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Leptin signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Changes in leptin sensitivity . . . . . . . . . . . . . . . . . . . . . 1.2.1. Leptin resistance . . . . . . . . . . . . . . . . . . . . . . . 1.2.2. ARC-specific leptin resistance. . . . . . . . . . . . . . . . . 1.2.3. ARC-specific increases in leptin sensitivity at low leptin levels . 1.3. How does leptin reach its target cells? . . . . . . . . . . . . . . . . 1.4. Endogenous leptin and signaling capacity . . . . . . . . . . . . . . . 1.5. Two mechanisms regulate leptin access into the brain . . . . . . . . . 1.6. How important is ARC LepRb signaling compared to other hypothalamic 2. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Leptin is a hormone produced and secreted primarily from adipocytes, but which acts predominantly within the central nervous system. The severe hyperphagic, obese and diabetic phenotype of human patients and rodent models with loss of leptin action (due to lack of the hormone leptin itself or its long form leptin receptor (LepRb)) demonstrates the importance of leptin action in energy homeostasis [1,2].
⁎ Tel.: +1 734 615 3497; fax: +1 734 936 6684. E-mail address: [email protected]. 0031-9384/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2008.04.020
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Whereas the regulation of energy homeostasis is the most obvious phenotype associated with leptin action, defects in leptin signaling also produce dysfunction in numerous endocrine outputs, including the reproductive, thyroid, immune and adrenal axes as well as the autonomic system (e.g. thermoregulation, energy expenditure and blood pressure) [3–6]. Indeed, the pattern of LepRb expression in the brain, with the majority of LepRb-expressing neurons within the hypothalamus [7], is consistent with the role for leptin in regulating diverse homeostatic processes. LepRb-expressing sites in the hypothalamus include the arcuate nucleus (ARC), dorsomedial hypothalamus (DMH), ventromedial hypothalamus (VMH) and lateral hypothalamic area (LHA) (Fig. 1); nonhypothalamic LepRb neurons are also found in important autonomic
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activates anorexigenic POMC neurons to promote melanocortin action, and simultaneously suppresses neuronal activity in orexigenic AgRP neurons, decreasing their antagonism of melanocortin action and leading to a net anorexigenic effect [19,20]. These changes in neuronal activity alter the release of neurotransmitters including classical neurotransmitters like gamma amino butyrate acid (GABA) and specific neuropeptides like α-melanocyte stimulating hormone (α-MSH), AgRP, galanin etc. These neuropeptides are released onto 2nd order neurons in different target tissues including the PVN and the DMH which are strongly innervated by arcuate POMC and AgRP neurons ([21,22] and own observations). 1.2. Changes in leptin sensitivity Fig. 1. Immunohistochemical detection of LepRb-expressing neurons within the hypothalamus in reporter mice that express green fluorescence protein (GFP) from the LepRb locus. Discrete populations of LepRb neurons are found in the arcuate nucleus (ARC), ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH) and lateral hypothalamic area (LHA). 3V, 3rd ventricle; ME, median eminence; IHC, immunohistochemistry.
control sites (e.g. parabrachial nucleus, nucleus of the solitary tract), as well as the limbic system (e.g. ventral tegmental area and lateral septum), which is important for the motivational component of homeostatic regulation [8,9]. The relative importance and specific functions for discrete populations of LepRb-expressing neurons remains obscure, and the majority of leptin related research has focused on the ARC as the major site of leptin signaling. However, recent data have found that ARC-specific LepRb deletions do not recapitulate the severe obesity and other defects of ob/ob or db/db mice [10,11]. Additionally, deletion of LepRb from other hypothalamic areas, such as the VMH, produce increased body weight and adiposity that are similar in their magnitude to that mediated by ARC deletion of LepRb — i.e. modest compared to that observed in db/db mice [12]. Therefore, it is crucial to understand the neural and physiologic functions for each LepRb-expressing site. In this review, I will outline and discuss our recent research demonstrating fundamental differences in leptin accessibility to ARC LepRb neurons in comparison with LepRbexpressing neurons in the DMH, and with a special emphasis on the development of ARC-specific leptin resistance. These differences in hypothalamic, LepRb-expressing sites might be an important part in understanding the hierarchical organization of LepRb neurons in diverse hypothalamic sites. 1.1. Leptin signaling Leptin binding to LepRb on target neurons activates specific signaling pathways [13]. One well understood LepRb signaling pathway involves the binding and activation of janus kinase-2 (JAK2), which phosphorylates tyrosine residues of LepRb and subsequently leads to the activation of several signaling pathways (e.g. phosphorylation of signal-tranducer and -activator of transcription-3 (P-STAT3)) [2]. Leptin-induced phosphorylation of STAT3 is a robust signal and has been used by us and other as a marker for functional LepRb-expressing neurons in the brain [14,15,2]. Phosphorylated STAT3 dimerizes and translocates into the cell nucleus, where it acts as a transcription factor to induce expression of several genes including pro-opiomelanocortin (POMC) [15] in the ARC and suppressor-of-cytokine-signaling-3 (SOCS-3) [16]. SOCS-3 is broadly up-regulated in response to leptin stimulation and binds to LepRb to inhibit leptin signaling [17]. This signaling pathway through SOCS-3 is thought to play an important role in the feedback inhibition of LepRb signaling and in the induction of leptin resistance [18]. While the STAT3→SOCS3 signaling pathway is well described, leptin also modulates neuronal activity via less well understood signaling pathways. In the ARC, leptin differently modulates neuronal activity in anorexigenic and orexigenic neuronal populations. In this system, leptin
Drastic changes in leptin sensitivity are seen in the severe cases of leptin or LepRb deficiency as well as in mouse models with significant changes in circulating leptin levels. Leptin sensitivity can be measured by physiological responses to leptin (e.g. food intake or body weight change) or by signaling responses like leptin-induced P-STAT3 which has been widely used as a quantitative unit for leptin signaling action [23–25]. Whereas a decrease in leptin levels is generally associated with increased leptin sensitivity [26,1], increased leptin levels induce a reduction in leptin sensitivity [27]. 1.2.1. Leptin resistance While leptin clearly plays a central role in the regulation of energy homeostasis, leptin has not performed well as an anti-obesity drug in clinical trials with “garden variety” obese patients. Indeed, the vast majority of obese humans have increased circulating leptin levels commensurate with their adiposity, and demonstrate reduced sensitivity towards exogenous leptin — this is commonly referred to as “leptin resistance”. The mechanisms underlying the development and maintenance of leptin resistance are thus crucial to understand as we seek potential therapies for the treatment of obesity. A number of animal models of leptin resistance have facilitated the investigation of how leptin resistance is triggered and how it influences body weight. The most commonly utilized model is probably the rodent (rat or mouse) model of high fat diet (HFD)-feeding, which rapidly triggers increased caloric intake and body weight [27,23]. Increased body weight in HFD fed mice is mainly due to increased fat depots and therefore circulating leptin levels increase in proportion to the fat mass [28]. We and other have shown that increased circulating leptin levels are associated with a defect in leptin signaling — demonstrated by a dramatic decrease in leptin-induced P-STAT3 [27,23]. Other laboratories have shown that leptin itself, when infused centrally, is sufficient to induce leptin resistance [24]. Indeed, leptin has been demonstrated to induce SOCS-3 expression which suppresses leptin signaling [18,29]. Furthermore, heterozygous deletion of SOCS-3 (homozygous deletion results in prenatal death) as well as POMC-specific homozygous deletion of SOCS-3 increases leptin sensitivity and attenuates weight gain and hyperphagia when fed a HFD [30–32]. Other potential mechanisms that might interfere with leptin signaling include the protein tyrosine phosphatase 1B, another inhibitor of LepRb signaling [33,34]. The availability of LepRb at the cell membrane could also affect leptin action. Leptin resistant rodents show inconsistent alterations in hypothalamic LepRb mRNA expression [27,23,35], however, no data are available to show whether LepRb is correctly integrated into the cell membrane in leptin resistance. 1.2.2. ARC-specific leptin resistance Using an immunohistochemical approach, we were able to investigate central leptin resistance in a neuroanatomical manner and found that HFD induced leptin resistance in mice is only observed in the ARC, but not other hypothalamic or non-hypothalamic structures. Leptin-induced P-STAT3-immunoreactivity (ir) and the number of pSTAT3-ir neurons was dramatically decreased in the ARC of HFD mice compared to chow fed controls (Fig. 2), whereas no significant
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Fig. 2. Immunohistochemical detection of leptin-induced P-STAT3 in different mouse models with different degrees of leptin resistance. Differences in leptin sensitivity can be seen in the intensity of P-STAT3-ir within the ARC, whereas P-STAT3-ir in the VMH remains comparable between mice. All mouse models were on a C57/B6 background and were fed a regular chow diet (17% fat content); except HFD indicates that mice were fed a high fat diet (58% fat content).
change was seen in the DMH and VMH or other sites. Consistently, we found that SOCS-3 mRNA levels were increased in the ARC of HFD mice, but not in the DMH and VMH, suggesting that SOCS3-induced leptin resistance in HFD fed mice is restricted to the ARC [23]. Interestingly, in seasonal rodents with natural cycles of body fat mass changes, seasonally increased levels of circulating leptin were also associated with decreased leptin sensitivity and increased SOCS-3 expression specifically in the ARC, but not other central sites [36]. The ARC-specificity of this leptin resistance is also consistent with the fact that leptin resistance in HFD fed rodents and seasonal animals mainly affects food intake and body weight, while other leptin actions are preserved (e.g. regulation of reproductive axis, and sympathetic nervous system) [37,38]. 1.2.3. ARC-specific increases in leptin sensitivity at low leptin levels Increased leptin sensitivity and leptin signaling has been shown in leptin deficient ob/ob mice. When treated with exogenous leptin, ob/ob mice show a more pronounced decrease in food intake and increases in leptin-induced P-STAT3 (specifically in the ARC) compared to leptin treated wild-type mice [39]. Overall, changes in leptin sensitivity can be best visualized by leptin-induced P-STAT3 in the ARC, as seen in Fig. 2. Within this scheme, db/db mice represent the least leptin-responsive mouse model, although their complete lack of functional leptin receptors represents a special and extreme case of leptin resistance. Their lack of leptin sensitivity is followed by HFD mice, with wild-type mice and ob/ob mice as the most leptin sensitive. Along these same lines, mice with a targeted mutation of LepRb tyrosine 985 (an important binding site for SOCS-3 and feedback inhibition [40]), are lean with decreased leptin levels compared to wild-type mice and show increased leptin sensitivity as well as increased ARC leptin signaling [26]. Altogether, this suggests that leptin sensitivity and leptin resistance is most obviously identified in the ARC. Increased leptin sensitivity associated with low circulating leptin levels is also the basis for the common experimental procedure to fast animals before leptin treatment, in order to reduce endogenous leptin levels and increase leptin signaling. Therefore, the ARC must exhibit certain properties which allow ARC neurons to sense changes in leptin levels more sensitively and directly than other hypothalamic sites and will likely be an important piece in understanding the hierarchical organization of hypothalamic leptin accessibility and signaling.
1.3. How does leptin reach its target cells? Leptin, a 16 kDa protein, has to reach its target cells in the brain, to function, but is too large to cross the blood brain barrier (BBB) by simple diffusion. It has been suggested that a specific transport system exists, which transports leptin across the BBB in a dose-dependent and saturable manner [41,42]. Systemically injected radiolabeled leptin accumulates more rapidly in the median eminence (ME) and ARC compared to other hypothalamic sites [43], however, suggesting clear differences in leptin accessibility to the ARC compared to nonARC sites. The ME is a specialized structure of the brain that belongs to the circumventricular organs; it fenestrated capillaries play an important part in the neuroendocrine system by providing the connection of hypothalamic neurons to the pituitary or portal vein [44]. It has been intensely discussed whether neuronal cell bodies in the ARC lay outside or behind the BBB. On the one hand, the ME contains a specialized type of glial cells, tanycytes, which are thought to build a specific seal between fenestrated endothelium of the ME and the ARC, to protect the ARC neurons from direct access to circulating substances [45]. On the other hand, leptin and other BBB-impermeable substances rapidly enter the ARC from the circulation, similar to the ME [43,46], suggesting a possible leakage from fenestrated capillaries in the ME to the ARC. Regardless of whether neuronal cell bodies of the ARC reside within the BBB or not, it is generally accepted that many axonal processes of ARC neurons reach into the portal vein to release neuropeptides [19]. This also implies direct accessibility to circulating levels of leptin via these neuronal processes within the circulation. In contrast, other hypothalamic structures like the VMH and DMH that do not contain neuroendocrine cells are thought to reside completely behind the BBB [47]. Therefore, LepRb neurons in the DMH would dependent on a specific leptin transport mechanisms across the BBB to gain access and respond to circulating leptin. 1.4. Endogenous leptin and signaling capacity The notion that, in contrast to non-ARC LepRb neurons, ARC LepRb neurons might directly access circulating leptin, predicts that the ARC LepRb system might be more sensitive to low leptin levels and small changes in circulating leptin, rendering the ARC an excellent sensor of peripheral metabolism. Most leptin studies employ supraphysiologic leptin levels to induce maximum leptin stimulation, commonly resulting
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in circulating leptin levels as high as 3000-fold elevated compared to normal endogenous leptin levels. Whereas, maximal leptin stimulation facilitates the study and understanding of leptin action in general, it is also obvious that low, physiological leptin levels suffice to maintain homeostatic equilibrium in mammals, and pharmacologic leptin levels may poorly reflect some aspects of the physiologic leptin response. Interestingly, leptin signaling induced by endogenous leptin levels can be visualized in LepRb neurons within the ARC of untreated mice. Consistent with the ARC being more sensitive to low circulating leptin levels we found a robust level of P-STAT3-immunoreactivity (ir) specifically in ARC neurons of normal, fed and untreated mice; this baseline P-STAT3-ir was not detectable in other hypothalamic sites. In contrast, leptin- or LepRbdeficient ob/ob and db/db mice lack detectable ARC P-STAT3 in the baseline, while basal P-STAT3-ir remains intact in Ay mice (another obese and diabetic mouse model with intact leptin signaling) [48]. This demonstrates that endogenous leptin action promotes these basal levels of ARC P-STAT3-ir. Additionally, ARC neurons with basal P-STAT3-ir express LepRb, as detected by the expression of green fluorescence protein in LepRb reporter mice. The finding that many of these basal PSTAT3-ir neurons in the ARC take up fluorogold (FG, a BBB-impermeable retrograde tracer) from the circulation (Fig. 3A and B) suggests the direct contact of these leptin-responsive neurons with circulating factors outside of the BBB. FG has been repeatedly used in the literature to label neuroendocrine cells which access the circulation via neuronal processes [49,50]. Furthermore, the external layer of the ME contains a dense network of LepRb-expressing processes, consistent with processes contacting the portal circulation in the ME which lay outside the BBB (Fig. 3C). The fact that only a subpopulation of LepRb neurons in the ARC accumulate peripheral FG could reflect incomplete FG uptake or might indicate that only a subpopulation of ARC neurons is in direct contact to the circulation; this distinction will require further investigation.
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Importantly, the demonstration that ARC LepRb neurons directly access circulating leptin (and presumably other circulating factors) also suggests that the increased leptin sensitivity of the ARC LepRb neurons compared to LepRb neurons at other sites may reflect direct access of the ARC neurons to the circulation. 1.5. Two mechanisms regulate leptin access into the brain We also showed that the ARC responds more rapidly and sensitively to exogenous leptin than non-ARC sites, but these differences were only apparent after peripheral leptin application, which mimics the physiological route of leptin to the brain. In contrast, central injections of leptin, which circumvent the BBB, revealed similar dose- and time-dependencies of leptin-induced P-STAT3 in both the ARC and non-ARC sites (such as the DMH) (Fig. 4). These data suggest that differences in leptin access into non-ARC sites compared to the ARC are BBB-dependent and support the importance of a saturable, dose-dependent leptin transporter system as described by Banks et al. [43] in mediating leptin access to non-ARC sites. This is also consistent with a delayed leptin response in the DMH (compared to the ARC, see Fig. 4B), because leptin availability would be limited by the kinetics of the transporter system. Furthermore, the DMH (and other non-ARC sites) might be relatively protected from increased leptin levels in obesity, as the leptin transport system could be relatively saturated. The leptin transport rate would therefore reach a steady state, independent of further increases in circulating leptin levels. Interestingly, leptin concentrations in the cerebrospinal fluid of obese rodents is proportionally low in relation to their high circulating leptin levels [51], demonstrating that the leptin transport rate cannot translate the high peripheral leptin levels into the brain. The presumably limited
Fig. 3. A. Immunohistochemical detection and cell counts of fluorogold filled neurons (bright, granular stain in the cytoplasm) in the ARC, activated by endogenous leptin levels (positive for P-STAT3, nuclear black stain). B. Examples of ARC neurons that are immunohistochemically positive for GFP/LepRb, P-STAT3 and fluorogold. C. High magnification of the ME (×100 oil) in an LepRbGFP reporter mouse stained for GFP. LepRb projections into the external zone of the ME can be seen, consistent with processes from neuroendocrine cells that reach into the portal circulation of the ME. E, ependymal zone; Ze, external zone; Zi, internal zone.
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Fig. 4. A. Dose response experiment after peripheral (upper panels) or central (lower panels) leptin application (30 min) in various hypothalamic nuclei. B. Time course after peripheral application of leptin (5 mg/kg body weight). Representative images of hypothalamic level Bregma − 2.8 mm are shown. Scale bars, 200 μm.
transport of leptin to non-ARC sites in the brain may represent a mechanism of leptin resistance, but is also consistent with the notion that this saturable and therefore limited leptin transport system [43] could prevent the leptin-mediated increase in SOCS-3 levels within non-ARC sites, maintaining relative leptin sensitivity outside of the ARC during diet induced obesity. Furthermore, these data predict that central leptin injections in obese rodents would improve leptin signaling due to the increased accessibility of leptin to non-ARC sites (with retained leptin signaling capacities). In contrast, ARC leptin resistance should be independent of the route of leptin application and should persist despite central leptin injections. Indeed, it has been shown that leptin resistance and impaired STAT3 signaling was improved by central leptin administration, but leptin-induced activation of STAT3 remained significantly impaired compared to lean control mice [27]. 1.6. How important is ARC LepRb signaling compared to other hypothalamic sites? It is important to emphasis that despite the increased leptin sensitivity of the ARC and the more rapid temporal response of leptin signaling in the ARC, a variety of data suggest the importance of nonARC LepRb-expressing sites to regulate body weight [12]. Furthermore, the absence of baseline detectable P-STAT3-ir in non-ARC regions does not exclude the important signaling function of endogenous leptin in these sites, but rather reflects our inability to detect the more modest levels of P-STAT3-ir in these neurons in the face of endogenous leptin levels. Therefore, the important question is why the ARC is designed differently than other hypothalamic sites with regard to accessibility and sensitivity to circulating leptin levels. An ARC LepRb system that is directly accessible to the circulation does likely function as an important sensory system for the detection of smaller and subacute
changes in leptin levels. It also opens the possibility that other circulating substances like insulin, estrogen or nutrients (e.g. glucose, fatty acids, ketones) might be more readily sensed by the ARC LepRb neurons, which therefore function as sensors of the peripheral endocrine and nutritional environment. Indeed, the mediobasal hypothalamus responds to a variety of these factors [52–54] and LepRb AgRP/neuropeptide Y expressing neurons in the ARC (which contain detectable basal P-STAT3-ir (unpublished observation)) may play an important role in glucosensing [55]. These peripheral inputs to the ARC LepRb system are further processed by other hypothalamic sites (e.g., the DMH and PVN, which represent two major projection sites for the ARC POMC and AgRP neurons) [22]. While homeostatic changes in the periphery (e.g. during fasting) are first sensed by the ARC (in addition to the autonomic sensory system in the brainstem), these ARC “input” signals are further integrated by structures like the DMH. In the DMH the information from several hypothalamic sites is processed (presumably in the context of longer-term leptin tone), before being relayed to other structures that stand at the end of the hypothalamic hierarchy [56], and which connect the hypothalamus back to the periphery via neuroendocrine and autonomic premotor neurons [57]. 2. Concluding remarks Leptin gains access to LepRb-expressing neurons in the ARC and DMH via fundamentally different mechanisms. ARC LepRb neurons project outside the BBB and display increased accessibility to circulating substances such as leptin, whereas the DMH and other non-ARC LepRb neurons are dependent on a leptin transport across the BBB. ARC LepRb neurons display increased sensitivity towards peripheral leptin (compared to non-ARC hypothalamic nuclei) and likely serve to more rapidly sense changes in circulating leptin. The
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increased access of elevated leptin levels to the ARC may also be crucial in the development of ARC-specific leptin resistance, although the physiological importance of site-specific leptin resistance is not well understood. The role of circulating leptin levels in the establishment of ARC leptin resistance makes sense in this context, but also remains unproven; other circulating factors, e.g. cytokines or hormones, could also induce SOCS3 via direct access to ARC neurons. Future investigations (including understanding the many non-ARC LepRb-expressing neural populations) will be required to understand the significance of site-specific leptin access and resistance. Acknowledgements These studies were supported by grants from the American Heart Association and the Michigan Diabetes Research and Training Center. References [1] Halaas JL, Boozer C, Blair-West J, Fidahusein N, Denton DA, Friedman JM. 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[31] Kievit P, Howard JK, Badman MK, Balthasar N, Coppari R, Mori H, et al. Enhanced leptin sensitivity and improved glucose homeostasis in mice lacking suppressor of cytokine signaling-3 in POMC-expressing cells. Cell Metab 2006;4:123–32. [32] Mori H, Hanada R, Hanada T, Aki D, Mashima R, Nishinakamura H, et al. Socs3 deficiency in the brain elevates leptin sensitivity and confers resistance to dietinduced obesity. Nat Med 2004;10:739–43. [33] Bence KK, Delibegovic M, Xue B, Gorgun CZ, Hotamisligil GS, Neel BG, et al. Neuronal PTP1B regulates body weight, adiposity and leptin action. Nat Med 2006;12:917–24. [34] Zabolotny JM, Bence-Hanulec KK, Stricker-Krongrad A, Haj F, Wang Y, Minokoshi Y, et al. PTP1B regulates leptin signal transduction in vivo. Dev Cell 2002;2:489–95. [35] Wilsey J, Scarpace PJ. Caloric restriction reverses the deficits in leptin receptor protein and leptin signaling 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. [36] Tups A, Ellis C, Moar KM, Logie TJ, Adam CL, Mercer JG, et al. Photoperiodic regulation of leptin sensitivity in the Siberian hamster, Phodopus sungorus, is reflected in arcuate nucleus SOCS-3 (suppressor of cytokine signaling) gene expression. Endocrinology 2004;145:1185–93. [37] Mark AL, Correia ML, Rahmouni K, Haynes WG. Selective leptin resistance: a new concept in leptin physiology with cardiovascular implications. J Hypertens 2002;20: 1245–50. [38] Rahmouni K, Haynes WG, Morgan DA, Mark AL. Selective resistance to central neural administration of leptin in agouti obese mice. Hypertension 2002;39:486–90. [39] Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, et al. Weightreducing effects of the plasma protein encoded by the obese gene. Science 1995;269: 543–6. [40] Myers Jr MG. Leptin receptor signaling and the regulation of mammalian physiology. Recent Prog Horm Res 2004;59:287–304. [41] Banks WA, Niehoff ML, Martin D, Farrell CL. Leptin transport across the blood-brain barrier of the Koletsky rat is not mediated by a product of the leptin receptor gene. Brain Res 2002;950:130–6. [42] Burguera B, Couce ME. Leptin access into the brain: a saturated transport mechanism in obesity. Physiol Behav 2001;74:717–20. [43] Banks WA, Kastin AJ, Huang W, Jaspan JB, Maness LM. Leptin enters the brain by a saturable system independent of insulin. Peptides 1996;17:305–11. [44] Kiss A. Morphological aspects of the median eminence—place of accumulation and secretion of regulatory neurohormones and neuropeptides. Gen Physiol Biophys 1997;16:301–9. [45] Krisch B, Leonhardt H. The functional and structural border of the neurohemal region of the median eminence. 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Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b
Review
Differential leptin access into the brain — A hierarchical organization of hypothalamic leptin target sites? Heike Münzberg ⁎ Division of Metabolism, Endocrinology and Diabetes, Departments of Internal Medicine, University of Michigan Medical School, 1150 W. Medical Center Dr., 5510D MSRB 1, Ann Arbor, MI 48109, USA
A R T I C L E
I N F O
Article history: Received 5 March 2008 Accepted 2 April 2008
A B S T R A C T Leptin target sites are found in several hypothalamic areas with dense populations of long form leptin receptor (LepRb) expressing neurons which mediate important leptin actions. Leptin action has been most intensely investigated in the arcuate nucleus of the hypothalamus (ARC), which represents an important leptin target site. Recent data have shown that non-ARC leptin target sites mediate important aspects of leptin action, however, including the regulation of energy balance. Therefore, the investigation of discrete leptin signaling systems and their interactions will be an important step to understand the homeostatic action of leptin. In this review I discuss our recent data investigating important differences in leptin accessibility to ARC neurons in contrast to other hypothalamic sites like the dorsomedial hypothalamus (DMH) and discuss their importance for the leptin signaling system. © 2008 Elsevier Inc. All rights reserved.
Contents 1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Leptin signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Changes in leptin sensitivity . . . . . . . . . . . . . . . . . . . . . 1.2.1. Leptin resistance . . . . . . . . . . . . . . . . . . . . . . . 1.2.2. ARC-specific leptin resistance. . . . . . . . . . . . . . . . . 1.2.3. ARC-specific increases in leptin sensitivity at low leptin levels . 1.3. How does leptin reach its target cells? . . . . . . . . . . . . . . . . 1.4. Endogenous leptin and signaling capacity . . . . . . . . . . . . . . . 1.5. Two mechanisms regulate leptin access into the brain . . . . . . . . . 1.6. How important is ARC LepRb signaling compared to other hypothalamic 2. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Leptin is a hormone produced and secreted primarily from adipocytes, but which acts predominantly within the central nervous system. The severe hyperphagic, obese and diabetic phenotype of human patients and rodent models with loss of leptin action (due to lack of the hormone leptin itself or its long form leptin receptor (LepRb)) demonstrates the importance of leptin action in energy homeostasis [1,2].
⁎ Tel.: +1 734 615 3497; fax: +1 734 936 6684. E-mail address: [email protected]. 0031-9384/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2008.04.020
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Whereas the regulation of energy homeostasis is the most obvious phenotype associated with leptin action, defects in leptin signaling also produce dysfunction in numerous endocrine outputs, including the reproductive, thyroid, immune and adrenal axes as well as the autonomic system (e.g. thermoregulation, energy expenditure and blood pressure) [3–6]. Indeed, the pattern of LepRb expression in the brain, with the majority of LepRb-expressing neurons within the hypothalamus [7], is consistent with the role for leptin in regulating diverse homeostatic processes. LepRb-expressing sites in the hypothalamus include the arcuate nucleus (ARC), dorsomedial hypothalamus (DMH), ventromedial hypothalamus (VMH) and lateral hypothalamic area (LHA) (Fig. 1); nonhypothalamic LepRb neurons are also found in important autonomic
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activates anorexigenic POMC neurons to promote melanocortin action, and simultaneously suppresses neuronal activity in orexigenic AgRP neurons, decreasing their antagonism of melanocortin action and leading to a net anorexigenic effect [19,20]. These changes in neuronal activity alter the release of neurotransmitters including classical neurotransmitters like gamma amino butyrate acid (GABA) and specific neuropeptides like α-melanocyte stimulating hormone (α-MSH), AgRP, galanin etc. These neuropeptides are released onto 2nd order neurons in different target tissues including the PVN and the DMH which are strongly innervated by arcuate POMC and AgRP neurons ([21,22] and own observations). 1.2. Changes in leptin sensitivity Fig. 1. Immunohistochemical detection of LepRb-expressing neurons within the hypothalamus in reporter mice that express green fluorescence protein (GFP) from the LepRb locus. Discrete populations of LepRb neurons are found in the arcuate nucleus (ARC), ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH) and lateral hypothalamic area (LHA). 3V, 3rd ventricle; ME, median eminence; IHC, immunohistochemistry.
control sites (e.g. parabrachial nucleus, nucleus of the solitary tract), as well as the limbic system (e.g. ventral tegmental area and lateral septum), which is important for the motivational component of homeostatic regulation [8,9]. The relative importance and specific functions for discrete populations of LepRb-expressing neurons remains obscure, and the majority of leptin related research has focused on the ARC as the major site of leptin signaling. However, recent data have found that ARC-specific LepRb deletions do not recapitulate the severe obesity and other defects of ob/ob or db/db mice [10,11]. Additionally, deletion of LepRb from other hypothalamic areas, such as the VMH, produce increased body weight and adiposity that are similar in their magnitude to that mediated by ARC deletion of LepRb — i.e. modest compared to that observed in db/db mice [12]. Therefore, it is crucial to understand the neural and physiologic functions for each LepRb-expressing site. In this review, I will outline and discuss our recent research demonstrating fundamental differences in leptin accessibility to ARC LepRb neurons in comparison with LepRbexpressing neurons in the DMH, and with a special emphasis on the development of ARC-specific leptin resistance. These differences in hypothalamic, LepRb-expressing sites might be an important part in understanding the hierarchical organization of LepRb neurons in diverse hypothalamic sites. 1.1. Leptin signaling Leptin binding to LepRb on target neurons activates specific signaling pathways [13]. One well understood LepRb signaling pathway involves the binding and activation of janus kinase-2 (JAK2), which phosphorylates tyrosine residues of LepRb and subsequently leads to the activation of several signaling pathways (e.g. phosphorylation of signal-tranducer and -activator of transcription-3 (P-STAT3)) [2]. Leptin-induced phosphorylation of STAT3 is a robust signal and has been used by us and other as a marker for functional LepRb-expressing neurons in the brain [14,15,2]. Phosphorylated STAT3 dimerizes and translocates into the cell nucleus, where it acts as a transcription factor to induce expression of several genes including pro-opiomelanocortin (POMC) [15] in the ARC and suppressor-of-cytokine-signaling-3 (SOCS-3) [16]. SOCS-3 is broadly up-regulated in response to leptin stimulation and binds to LepRb to inhibit leptin signaling [17]. This signaling pathway through SOCS-3 is thought to play an important role in the feedback inhibition of LepRb signaling and in the induction of leptin resistance [18]. While the STAT3→SOCS3 signaling pathway is well described, leptin also modulates neuronal activity via less well understood signaling pathways. In the ARC, leptin differently modulates neuronal activity in anorexigenic and orexigenic neuronal populations. In this system, leptin
Drastic changes in leptin sensitivity are seen in the severe cases of leptin or LepRb deficiency as well as in mouse models with significant changes in circulating leptin levels. Leptin sensitivity can be measured by physiological responses to leptin (e.g. food intake or body weight change) or by signaling responses like leptin-induced P-STAT3 which has been widely used as a quantitative unit for leptin signaling action [23–25]. Whereas a decrease in leptin levels is generally associated with increased leptin sensitivity [26,1], increased leptin levels induce a reduction in leptin sensitivity [27]. 1.2.1. Leptin resistance While leptin clearly plays a central role in the regulation of energy homeostasis, leptin has not performed well as an anti-obesity drug in clinical trials with “garden variety” obese patients. Indeed, the vast majority of obese humans have increased circulating leptin levels commensurate with their adiposity, and demonstrate reduced sensitivity towards exogenous leptin — this is commonly referred to as “leptin resistance”. The mechanisms underlying the development and maintenance of leptin resistance are thus crucial to understand as we seek potential therapies for the treatment of obesity. A number of animal models of leptin resistance have facilitated the investigation of how leptin resistance is triggered and how it influences body weight. The most commonly utilized model is probably the rodent (rat or mouse) model of high fat diet (HFD)-feeding, which rapidly triggers increased caloric intake and body weight [27,23]. Increased body weight in HFD fed mice is mainly due to increased fat depots and therefore circulating leptin levels increase in proportion to the fat mass [28]. We and other have shown that increased circulating leptin levels are associated with a defect in leptin signaling — demonstrated by a dramatic decrease in leptin-induced P-STAT3 [27,23]. Other laboratories have shown that leptin itself, when infused centrally, is sufficient to induce leptin resistance [24]. Indeed, leptin has been demonstrated to induce SOCS-3 expression which suppresses leptin signaling [18,29]. Furthermore, heterozygous deletion of SOCS-3 (homozygous deletion results in prenatal death) as well as POMC-specific homozygous deletion of SOCS-3 increases leptin sensitivity and attenuates weight gain and hyperphagia when fed a HFD [30–32]. Other potential mechanisms that might interfere with leptin signaling include the protein tyrosine phosphatase 1B, another inhibitor of LepRb signaling [33,34]. The availability of LepRb at the cell membrane could also affect leptin action. Leptin resistant rodents show inconsistent alterations in hypothalamic LepRb mRNA expression [27,23,35], however, no data are available to show whether LepRb is correctly integrated into the cell membrane in leptin resistance. 1.2.2. ARC-specific leptin resistance Using an immunohistochemical approach, we were able to investigate central leptin resistance in a neuroanatomical manner and found that HFD induced leptin resistance in mice is only observed in the ARC, but not other hypothalamic or non-hypothalamic structures. Leptin-induced P-STAT3-immunoreactivity (ir) and the number of pSTAT3-ir neurons was dramatically decreased in the ARC of HFD mice compared to chow fed controls (Fig. 2), whereas no significant
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Fig. 2. Immunohistochemical detection of leptin-induced P-STAT3 in different mouse models with different degrees of leptin resistance. Differences in leptin sensitivity can be seen in the intensity of P-STAT3-ir within the ARC, whereas P-STAT3-ir in the VMH remains comparable between mice. All mouse models were on a C57/B6 background and were fed a regular chow diet (17% fat content); except HFD indicates that mice were fed a high fat diet (58% fat content).
change was seen in the DMH and VMH or other sites. Consistently, we found that SOCS-3 mRNA levels were increased in the ARC of HFD mice, but not in the DMH and VMH, suggesting that SOCS3-induced leptin resistance in HFD fed mice is restricted to the ARC [23]. Interestingly, in seasonal rodents with natural cycles of body fat mass changes, seasonally increased levels of circulating leptin were also associated with decreased leptin sensitivity and increased SOCS-3 expression specifically in the ARC, but not other central sites [36]. The ARC-specificity of this leptin resistance is also consistent with the fact that leptin resistance in HFD fed rodents and seasonal animals mainly affects food intake and body weight, while other leptin actions are preserved (e.g. regulation of reproductive axis, and sympathetic nervous system) [37,38]. 1.2.3. ARC-specific increases in leptin sensitivity at low leptin levels Increased leptin sensitivity and leptin signaling has been shown in leptin deficient ob/ob mice. When treated with exogenous leptin, ob/ob mice show a more pronounced decrease in food intake and increases in leptin-induced P-STAT3 (specifically in the ARC) compared to leptin treated wild-type mice [39]. Overall, changes in leptin sensitivity can be best visualized by leptin-induced P-STAT3 in the ARC, as seen in Fig. 2. Within this scheme, db/db mice represent the least leptin-responsive mouse model, although their complete lack of functional leptin receptors represents a special and extreme case of leptin resistance. Their lack of leptin sensitivity is followed by HFD mice, with wild-type mice and ob/ob mice as the most leptin sensitive. Along these same lines, mice with a targeted mutation of LepRb tyrosine 985 (an important binding site for SOCS-3 and feedback inhibition [40]), are lean with decreased leptin levels compared to wild-type mice and show increased leptin sensitivity as well as increased ARC leptin signaling [26]. Altogether, this suggests that leptin sensitivity and leptin resistance is most obviously identified in the ARC. Increased leptin sensitivity associated with low circulating leptin levels is also the basis for the common experimental procedure to fast animals before leptin treatment, in order to reduce endogenous leptin levels and increase leptin signaling. Therefore, the ARC must exhibit certain properties which allow ARC neurons to sense changes in leptin levels more sensitively and directly than other hypothalamic sites and will likely be an important piece in understanding the hierarchical organization of hypothalamic leptin accessibility and signaling.
1.3. How does leptin reach its target cells? Leptin, a 16 kDa protein, has to reach its target cells in the brain, to function, but is too large to cross the blood brain barrier (BBB) by simple diffusion. It has been suggested that a specific transport system exists, which transports leptin across the BBB in a dose-dependent and saturable manner [41,42]. Systemically injected radiolabeled leptin accumulates more rapidly in the median eminence (ME) and ARC compared to other hypothalamic sites [43], however, suggesting clear differences in leptin accessibility to the ARC compared to nonARC sites. The ME is a specialized structure of the brain that belongs to the circumventricular organs; it fenestrated capillaries play an important part in the neuroendocrine system by providing the connection of hypothalamic neurons to the pituitary or portal vein [44]. It has been intensely discussed whether neuronal cell bodies in the ARC lay outside or behind the BBB. On the one hand, the ME contains a specialized type of glial cells, tanycytes, which are thought to build a specific seal between fenestrated endothelium of the ME and the ARC, to protect the ARC neurons from direct access to circulating substances [45]. On the other hand, leptin and other BBB-impermeable substances rapidly enter the ARC from the circulation, similar to the ME [43,46], suggesting a possible leakage from fenestrated capillaries in the ME to the ARC. Regardless of whether neuronal cell bodies of the ARC reside within the BBB or not, it is generally accepted that many axonal processes of ARC neurons reach into the portal vein to release neuropeptides [19]. This also implies direct accessibility to circulating levels of leptin via these neuronal processes within the circulation. In contrast, other hypothalamic structures like the VMH and DMH that do not contain neuroendocrine cells are thought to reside completely behind the BBB [47]. Therefore, LepRb neurons in the DMH would dependent on a specific leptin transport mechanisms across the BBB to gain access and respond to circulating leptin. 1.4. Endogenous leptin and signaling capacity The notion that, in contrast to non-ARC LepRb neurons, ARC LepRb neurons might directly access circulating leptin, predicts that the ARC LepRb system might be more sensitive to low leptin levels and small changes in circulating leptin, rendering the ARC an excellent sensor of peripheral metabolism. Most leptin studies employ supraphysiologic leptin levels to induce maximum leptin stimulation, commonly resulting
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in circulating leptin levels as high as 3000-fold elevated compared to normal endogenous leptin levels. Whereas, maximal leptin stimulation facilitates the study and understanding of leptin action in general, it is also obvious that low, physiological leptin levels suffice to maintain homeostatic equilibrium in mammals, and pharmacologic leptin levels may poorly reflect some aspects of the physiologic leptin response. Interestingly, leptin signaling induced by endogenous leptin levels can be visualized in LepRb neurons within the ARC of untreated mice. Consistent with the ARC being more sensitive to low circulating leptin levels we found a robust level of P-STAT3-immunoreactivity (ir) specifically in ARC neurons of normal, fed and untreated mice; this baseline P-STAT3-ir was not detectable in other hypothalamic sites. In contrast, leptin- or LepRbdeficient ob/ob and db/db mice lack detectable ARC P-STAT3 in the baseline, while basal P-STAT3-ir remains intact in Ay mice (another obese and diabetic mouse model with intact leptin signaling) [48]. This demonstrates that endogenous leptin action promotes these basal levels of ARC P-STAT3-ir. Additionally, ARC neurons with basal P-STAT3-ir express LepRb, as detected by the expression of green fluorescence protein in LepRb reporter mice. The finding that many of these basal PSTAT3-ir neurons in the ARC take up fluorogold (FG, a BBB-impermeable retrograde tracer) from the circulation (Fig. 3A and B) suggests the direct contact of these leptin-responsive neurons with circulating factors outside of the BBB. FG has been repeatedly used in the literature to label neuroendocrine cells which access the circulation via neuronal processes [49,50]. Furthermore, the external layer of the ME contains a dense network of LepRb-expressing processes, consistent with processes contacting the portal circulation in the ME which lay outside the BBB (Fig. 3C). The fact that only a subpopulation of LepRb neurons in the ARC accumulate peripheral FG could reflect incomplete FG uptake or might indicate that only a subpopulation of ARC neurons is in direct contact to the circulation; this distinction will require further investigation.
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Importantly, the demonstration that ARC LepRb neurons directly access circulating leptin (and presumably other circulating factors) also suggests that the increased leptin sensitivity of the ARC LepRb neurons compared to LepRb neurons at other sites may reflect direct access of the ARC neurons to the circulation. 1.5. Two mechanisms regulate leptin access into the brain We also showed that the ARC responds more rapidly and sensitively to exogenous leptin than non-ARC sites, but these differences were only apparent after peripheral leptin application, which mimics the physiological route of leptin to the brain. In contrast, central injections of leptin, which circumvent the BBB, revealed similar dose- and time-dependencies of leptin-induced P-STAT3 in both the ARC and non-ARC sites (such as the DMH) (Fig. 4). These data suggest that differences in leptin access into non-ARC sites compared to the ARC are BBB-dependent and support the importance of a saturable, dose-dependent leptin transporter system as described by Banks et al. [43] in mediating leptin access to non-ARC sites. This is also consistent with a delayed leptin response in the DMH (compared to the ARC, see Fig. 4B), because leptin availability would be limited by the kinetics of the transporter system. Furthermore, the DMH (and other non-ARC sites) might be relatively protected from increased leptin levels in obesity, as the leptin transport system could be relatively saturated. The leptin transport rate would therefore reach a steady state, independent of further increases in circulating leptin levels. Interestingly, leptin concentrations in the cerebrospinal fluid of obese rodents is proportionally low in relation to their high circulating leptin levels [51], demonstrating that the leptin transport rate cannot translate the high peripheral leptin levels into the brain. The presumably limited
Fig. 3. A. Immunohistochemical detection and cell counts of fluorogold filled neurons (bright, granular stain in the cytoplasm) in the ARC, activated by endogenous leptin levels (positive for P-STAT3, nuclear black stain). B. Examples of ARC neurons that are immunohistochemically positive for GFP/LepRb, P-STAT3 and fluorogold. C. High magnification of the ME (×100 oil) in an LepRbGFP reporter mouse stained for GFP. LepRb projections into the external zone of the ME can be seen, consistent with processes from neuroendocrine cells that reach into the portal circulation of the ME. E, ependymal zone; Ze, external zone; Zi, internal zone.
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Fig. 4. A. Dose response experiment after peripheral (upper panels) or central (lower panels) leptin application (30 min) in various hypothalamic nuclei. B. Time course after peripheral application of leptin (5 mg/kg body weight). Representative images of hypothalamic level Bregma − 2.8 mm are shown. Scale bars, 200 μm.
transport of leptin to non-ARC sites in the brain may represent a mechanism of leptin resistance, but is also consistent with the notion that this saturable and therefore limited leptin transport system [43] could prevent the leptin-mediated increase in SOCS-3 levels within non-ARC sites, maintaining relative leptin sensitivity outside of the ARC during diet induced obesity. Furthermore, these data predict that central leptin injections in obese rodents would improve leptin signaling due to the increased accessibility of leptin to non-ARC sites (with retained leptin signaling capacities). In contrast, ARC leptin resistance should be independent of the route of leptin application and should persist despite central leptin injections. Indeed, it has been shown that leptin resistance and impaired STAT3 signaling was improved by central leptin administration, but leptin-induced activation of STAT3 remained significantly impaired compared to lean control mice [27]. 1.6. How important is ARC LepRb signaling compared to other hypothalamic sites? It is important to emphasis that despite the increased leptin sensitivity of the ARC and the more rapid temporal response of leptin signaling in the ARC, a variety of data suggest the importance of nonARC LepRb-expressing sites to regulate body weight [12]. Furthermore, the absence of baseline detectable P-STAT3-ir in non-ARC regions does not exclude the important signaling function of endogenous leptin in these sites, but rather reflects our inability to detect the more modest levels of P-STAT3-ir in these neurons in the face of endogenous leptin levels. Therefore, the important question is why the ARC is designed differently than other hypothalamic sites with regard to accessibility and sensitivity to circulating leptin levels. An ARC LepRb system that is directly accessible to the circulation does likely function as an important sensory system for the detection of smaller and subacute
changes in leptin levels. It also opens the possibility that other circulating substances like insulin, estrogen or nutrients (e.g. glucose, fatty acids, ketones) might be more readily sensed by the ARC LepRb neurons, which therefore function as sensors of the peripheral endocrine and nutritional environment. Indeed, the mediobasal hypothalamus responds to a variety of these factors [52–54] and LepRb AgRP/neuropeptide Y expressing neurons in the ARC (which contain detectable basal P-STAT3-ir (unpublished observation)) may play an important role in glucosensing [55]. These peripheral inputs to the ARC LepRb system are further processed by other hypothalamic sites (e.g., the DMH and PVN, which represent two major projection sites for the ARC POMC and AgRP neurons) [22]. While homeostatic changes in the periphery (e.g. during fasting) are first sensed by the ARC (in addition to the autonomic sensory system in the brainstem), these ARC “input” signals are further integrated by structures like the DMH. In the DMH the information from several hypothalamic sites is processed (presumably in the context of longer-term leptin tone), before being relayed to other structures that stand at the end of the hypothalamic hierarchy [56], and which connect the hypothalamus back to the periphery via neuroendocrine and autonomic premotor neurons [57]. 2. Concluding remarks Leptin gains access to LepRb-expressing neurons in the ARC and DMH via fundamentally different mechanisms. ARC LepRb neurons project outside the BBB and display increased accessibility to circulating substances such as leptin, whereas the DMH and other non-ARC LepRb neurons are dependent on a leptin transport across the BBB. ARC LepRb neurons display increased sensitivity towards peripheral leptin (compared to non-ARC hypothalamic nuclei) and likely serve to more rapidly sense changes in circulating leptin. The
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increased access of elevated leptin levels to the ARC may also be crucial in the development of ARC-specific leptin resistance, although the physiological importance of site-specific leptin resistance is not well understood. The role of circulating leptin levels in the establishment of ARC leptin resistance makes sense in this context, but also remains unproven; other circulating factors, e.g. cytokines or hormones, could also induce SOCS3 via direct access to ARC neurons. Future investigations (including understanding the many non-ARC LepRb-expressing neural populations) will be required to understand the significance of site-specific leptin access and resistance. Acknowledgements These studies were supported by grants from the American Heart Association and the Michigan Diabetes Research and Training Center. References [1] Halaas JL, Boozer C, Blair-West J, Fidahusein N, Denton DA, Friedman JM. 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