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Endocrinology Vol. 148, No. 11 5414-5423
Copyright © 2007 by The Endocrine Society

Differential Accessibility of Circulating Leptin to Individual Hypothalamic Sites

Miro Faouzi, Rebecca Leshan, Marie Björnholm, Thomas Hennessey, Justin Jones and Heike Münzberg

Division of Metabolism, Endocrinology, and Diabetes (M.F., R.L., M.B., T.H., J.J., H.M.), Departments of Medicine and Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109; and Section of Integrative Physiology (M.B.), Department of Molecular Medicine and Surgery, Karolinska Institute, 171 77 Stockholm, Sweden

Address all correspondence and requests for reprints to: Heike Münzberg, Ph.D., Division of Metabolism, Endocrinology, and Diabetes, Department of Internal Medicine, University of Michigan Medical School, 1150 West Medical Center Drive, 5510D MSRB 1, Ann Arbor, Michigan 48109. E-mail: hmuenzbe{at}umich.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hypothalamic neurons expressing the long form of the leptin receptor (LRb) mediate important leptin actions. Although it has been suggested that leptin crosses the blood-brain barrier (BBB) via a specific transport system, we hypothesized the existence of a population of hypothalamic arcuate nucleus (ARC) neurons that senses leptin independently of this transport system. Indeed, endogenous circulating leptin results in detectable levels of baseline activated signal transducer and activator of transcription 3 (STAT3) phosphorylation in a population of ARC/LRb neurons, consistent with increased sensing of circulating leptin in these neurons compared with other LRb neurons. Furthermore, a population of ARC/LRb neurons that responds more rapidly and sensitively to circulating leptin compared with other hypothalamic LRb neurons detected by leptin activated phosphorylated STAT3. In addition, peripheral application of the BBB-impermeant retrograde tracer fluorogold revealed a population of ARC/LRb neurons that directly contact the circulation (e.g. via neuronal processes reaching outside the BBB). Taken together, these data suggest that a population of ARC/LRb neurons directly contacts the circulation and displays increased sensitivity to circulating leptin compared with neurons residing entirely behind the BBB elsewhere in the hypothalamus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEPTIN IS AN adipocyte-derived hormone that acts via a specific long-form leptin receptor (LRb) in the brain to reduce food intake and regulate energy expenditure. Several hypothalamic sites, including the dorsomedial and ventromedial hypothalamus (DMH and VMH), lateral hypothalamus (LH), and arcuate nucleus (ARC), express LRb mRNA and respond to leptin stimulation with LRb-dependent activation/accumulation of signal transducer and activator of transcription 3 (STAT3) (1, 2), suppressor of cytokine signaling 3 (SOCS-3) (3), and/or c-Fos (4). In addition to its anorexigenic effects, leptin regulates neuroendocrine function, including permissive effects upon the reproductive axis, thyroid axis, growth, and immune function (5, 6). Although a role for direct leptin action outside of the central nervous system remains obscure, it is clear that the brain is a major site of leptin action (7, 8). The relative contribution of each of the many leptin-responsive hypothalamic sites to the overall regulation of physiology by leptin remains unclear, however.

Leptin, a 16-kDa protein, is too large to cross the blood-brain barrier (BBB) by simple diffusion, but an active, dose-dependent transport across the BBB via a saturable transport system has been described (9, 10, 11). A variety of data suggest that leptin uptake is not uniform throughout the brain, however; areas displaying increased uptake of labeled leptin include the pineal gland, median eminence (ME) (both are circumventricular organs, which lack a typical BBB), and the ARC, which lies adjacent to the ME (12). Whether the ARC is completely secured by a BBB or whether it might be partially open to circulating substances (including leptin) has been a matter of some debate (13, 14).

We previously demonstrated that leptin resistance in diet-induced obese (DIO) mice is characterized by decreased leptin signaling in the ARC but not in other leptin-responsive sites of the brain. In addition, this ARC-specific attenuation of leptin signaling in DIO mice coincides with an ARC-specific increase in mRNA expression for SOCS3, an inhibitor of leptin signaling (15). These data suggest that certain properties unique to the ARC must enable these ARC-specific responses to overnutrition. We hypothesized that unique anatomical properties may allow ARC/LRb neurons direct access to the circulation (e.g. via neuronal processes reaching outside the BBB) and to circulating substances like leptin independently of transport mechanisms. Here, we show that endogenous leptin mediates immunohistochemically detectable STAT3 activation in ARC neurons but not elsewhere in the hypothalamus. Furthermore, we compared leptin responsiveness of ARC neurons to LRb neurons elsewhere in the hypothalamus, demonstrating increased celerity and sensitivity of ARC neurons. In addition, a population of LRb-expressing ARC neurons directly contacts the circulation, and thus, this group of LRb-expressing neurons in the ARC is uniquely poised to be regulated by perturbations in circulating factors such as leptin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Fluorogold (FG) was purchased from Biotium (Hayward, CA), rabbit anti-FG from Chemicon (Temecula, CA), leptin from National Hormone and Peptide Program (Torrance, CA) or Sigma Chemical Co. (St. Louis, MO), rabbit anti-P(Y705)STAT3 and rabbit anti-STAT3 from Cell Signaling Technology, Inc. (Beverly, MA), chicken anti-GFP from Abcam (Cambridge, MA), normal donkey serum and biotinylated donkey antirabbit from Jackson ImmunoReseach Laboratories, Inc. (West Grove, PA), Alexa-488- and -568-conjugated secondary antibodies from Molecular Probes, Inc. (Eugene, OR), and ABC Vectastain Elite kit from Vector Laboratories (Burlingame, CA). For immunoblotting procedures HRP-conjugated antirabbit antibodies and ECL Plus detection reagent and Hyperfilm were from Amersham (Piscataway, NJ). All other chemical supplies were purchased from Sigma.

Animals
Ten-week-old C57BL/6J, Ay mice and some ob/ob and db/db mice were purchased from Charles River Laboratories (Wilmington, MA) either with or without intracerebroventricular (icv) cannulae in the lateral ventricle. All other mice were bred within our University of Michigan specific pathogen free facility and were used at 9–11 wk of age. Mice with a homologously targeted leptin receptor allele containing an internal ribosome entry site plus cre-recombinase within the 3'-untranslated region of the LRb-specific exon (LRbcre) were generated in our laboratory (16). Mice containing a cre-inducible green fluorescent protein (GFP) reporter [Gt(ROSA)26Sortm2Sho; Jackson Laboratories, Bar Harbor, ME] were crossed with LRbcre mice until double-homozygous mice (LRbGFP) were generated. Additional, C57BL/6-ob/ob and -db/db mice were bred within our facility using heterozygous animals from Jackson Laboratories. All animal use was in accordance with the guidelines and approval of the University of Michigan Committee on the Use and Care of Animals. Mice were housed three to four mice per cage (or individually for icv cannulated mice) with ad libitum access to food and water.

Baseline levels of phosphorylated STAT3 (P-STAT3)
We compared leptin signaling-deficient mouse models (ob/ob and db/db mice) to wild-type and Ay mice (additional control for an obese and diabetic mouse model, with intact leptin signaling) (17). Mice were either untreated or received a high dose of ip (5 mg/kg) or icv (100 µg/kg) injection of leptin before perfusion and P-STAT3 immunohistochemistry (IHC). Leptin signaling deficiency increases the risk of infection (18) as seen in some aged ob/ob and db/db mice with high meningeal P-STAT3-immunoreactivity (-ir) (consistent with an inflammatory process); hence, only younger animals (9–11 wk) were used for this study.

Leptin dose-response and time-course experiments
For dose-response and time-course experiments (Tables 1Go and 2Go) with ip or icv leptin administration, four to five independent experiments were performed for IHC analysis, and immunoblotting (IB) analysis was done with n = 3–4.


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  		TABLE 1. Dose-response experiments

 

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  		TABLE 2. Time-course experiments

 
Leptin was applied peripherally (ip) or centrally (icv) using different doses (0.1–5 mg/kg body weight ip; 0.01–1 µg/kg body weight icv in 2 µl vol) for 30 min for dose-response experiments. For time-course experiments, leptin was applied for different durations (15 min or 1 or 4 h with ip leptin 5 mg/kg body weight and icv leptin 0.6 µg/kg body weight). After treatment, mice were anesthetized with on overdose of sodium pentobarbital and perfused or processed for hypothalamic microdissection as described (2).

For icv time courses, our goal was to use a low leptin dose, which would result in leptin cerebrospinal fluid concentrations comparable to ip injections. Therefore, we used a dose slightly lower than the lowest dose resulting in significant P-STAT3 induction in our icv dose-response experiments.

Peripheral retrograde tracing with FG
Animals received a bolus injection of 50 µl 1% FG solution via the tail vein, and 24 h or 5 d later, they were treated with vehicle or leptin (5 mg/kg) followed by anesthesia and perfusion as described (2).

Leptin ELISA
Whole blood was collected by cardiac puncture or from the trunk after decapitation. Serum leptin was quantified by ELISA (Chrystalchem, Downers Grove, IL) performed following the manufacturer’s guidelines.

Perfusion and IHC
Perfusion and IHC were performed as described earlier (2). Antibodies were diluted 1:2000 (rabbit anti-P-STAT3), 1:1000 (chicken anti-GFP), or 1:3000 (rabbit-anti-FG) and were incubated for 48 h at 4 C. Primary antibodies were detected using secondary antibodies (1:200) conjugated to Alexa-488, Alexa-568, or biotin followed by further labeling with avidin-biotin complex and development in diaminobenzidine solution.

Microscopy and cell counts
Results were visualized and recorded by fluorescence or bright-field microscopy (Olympus BX51; Olympus, Melville, NY) and a digital camera (Olympus DP30BW). Cell counts were performed as described earlier (15). All contrast levels were adjusted manually using identical adjustment settings for images that were assembled together in one figure. In addition, all photographs per experimental set-up were captured using fixed exposure times to preserve comparable signal intensities among sections and brains. To compare the ration of FG/P-STAT3-ir in PBS vs. leptin-treated mice, we counted cells in ARC slices of two brains per group (a total of 17 slices) to calculate the percentage of FG/P-STAT3 double-labeled neurons. Statistical significance was analyzed using a Student’s t test. Note that in graphs showing no error bars, only n = 1 was analyzed, which is meant to give a qualitative result, but is not meant to obtain accurate quantitative data.

Microdissection of hypothalamic nuclei and IB
Microdissections and IB of hypothalamic nuclei were performed as described elsewhere (15). ARC- and VMH/DMH-enriched tissue pieces were collected, and 15 µg (ARC) or 35 µg (VMH/DMH) of lysate was loaded per lane onto polyacrylamide gels. The Western blot membranes were probed first for P-STAT3 and then stripped (ReBlot Plus mild stripping solution; Chemicon) and reprobed for total STAT3.

IB quantification and statistical analysis
Results from IB were scanned into digital tif images, and pixel intensity of the P-STAT3 signal was quantified with Quantity One software (Bio-Rad, Hercules, CA). Statistical analysis was done separately for ip, icv, dose-response, and time-course experiments with a one way ANOVA to analyze statistical significance between groups, followed by a Fisher least significant differences test for statistical analysis within groups unless otherwise noted. Significant differences were accepted if the P value was ≤0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Endogenous leptin mediates the prominent activation of STAT3 in a population of ARC neurons
Endogenous leptin levels are very low (about 2–3 ng/ml serum) in comparison with the supraphysiological leptin levels commonly employed in leptin research (e.g. 3000 ng/ml after 5 mg/kg body weight leptin dose). These endogenous leptin levels must mediate meaningful LRb signals, however, because these levels prevent the physiological abnormalities observed in rodents and humans with pathologically low leptin levels that result from a variety of genetic and nutritional perturbations (19, 20, 21). Indeed, we consistently observed a population of P-STAT3-ir neurons in the ARC, but not other hypothalamic sites, of vehicle-treated or untreated wild-type mice and postulated that these baseline ARC/P-STAT3-ir neurons derive from endogenous leptin levels acting via LRb. Even though exogenous leptin treatment clearly leads to a dramatic increase of P-STAT3-ir in the ARC as well as other hypothalamic sites (e.g. VMH, Fig. 1AGo, right), untreated mice show a considerable population of P-STAT3-ir neurons particularly in the ventromedial part of the ARC, adjacent to the ME (Fig. 1AGo, left). In striking contrast, lack of leptin signaling in leptin-deficient ob/ob and LRb-deficient db/db mice (left, Fig. 1Go, B and C, respectively) leads to a loss of these baseline ARC/P-STAT3-ir neurons, demonstrating that leptin signaling is required for the induction of P-STAT3-ir in these neurons. To prove that P-STAT3-ir is indeed specific to LRb signaling, we applied a high central leptin dose (100 µg/kg) for 2 h to ob/ob and db/db mice. As expected, ob/ob mice show a strong induction of P-STAT3 in the hypothalamus (Fig. 1BGo, right), whereas db/db mice failed to show any P-STAT3-ir in the hypothalamus comparable to untreated db/db mice (Fig. 1CGo, right). Furthermore, Ay mice (an obese and diabetic mouse model with intact leptin signaling) demonstrate baseline ARC/P-STAT3-ir neurons (Fig. 1DGo, left) comparable to wild-type mice, and leptin stimulation further increases P-STAT3-ir in these mice (Fig. 1DGo, right). These data demonstrate the requirement of LRb signaling for endogenous leptin levels to induce ARC/P-STAT3-ir. Furthermore, although endogenous leptin levels likely act in all LRb-expressing neurons throughout the brain (although below detection levels), IHC-detectable P-STAT3-ir from endogenous leptin levels is found solely in the ARC, supporting the notion that the ARC more sensitively responds to circulating leptin than do other hypothalamic sites.


Figure 1
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  		FIG. 1. P-STAT3 IHC for wild-type (A), ob/ob (B), db/db (C), and Ay (D) mice either with no treatment (left panels) or after ip or icv leptin stimulation for 2 h (Bregma level, –2.3 mm). 3V, Third ventricle. Scale bars, 100 µm.

 
Baseline ARC/P-STAT3-ir neurons express LRb
To gain further evidence for leptin signaling by endogenous leptin levels, we used LRbGFP reporter mice to visualize LRb-expressing neurons via GFP-IHC. As shown in Fig. 2Go, the distribution of GFP-ir in LRbGFP mice is consistent with earlier data using in situ hybridization to detect LRb transcripts (3, 22). We also validated the use of cellular GFP expression in LRbGFP as a marker of LRb expression by demonstrating that essentially all GFP-expressing neurons colocalize with P-STAT3-ir after leptin treatment (Fig. 2Go, C and D). Note that not all leptin-induced P-STAT3-ir neurons express detectable GFP, however, suggesting that efficient excision of the transcription blocker and/or expression of GFP from the ROSA26 promoter does not occur in a small percentage of LRb neurons.


Figure 2
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  		FIG. 2. A, Schematic drawing for the generation of LRbCre mice targeting expression of cre recombinase specifically into LRb-expressing cells. LRbCre mice were further crossed with Gt(ROSA)26Sortm2Sho reporter mice resulting in the generation of LRbGFP mice. B, Distribution of LRbGFP neurons within the hypothalamus of a homozygous LRbGFP mouse. C and D, IHC for P-STAT3/GFP in the hypothalamus (C) and ARC (D) of a leptin-treated LRbGFP mouse. E, IHC for the astrocyte marker glial fibrillary acidic protein (GFAP) and GFP in the ARC/restrochiasmatic area (RCh) of an LRbGFP mouse. 3V, Third ventricle. Scale bars, 200 µm (B and C) and 50 µm (D).

 
In untreated LRbGFP mice, approximately 60% of baseline ARC/P-STAT3-ir neurons colocalized with LRbGFP neurons throughout the ARC (Fig. 3Go, A and B). In addition, we noted that the majority of GFP/P-STAT3-ir neurons in untreated mice were located in the ventromedial ARC close to the ME (Fig. 3Go, C and D). We therefore concluded that baseline ARC/P-STAT3 neurons represent LRb-expressing neurons that are stimulated by endogenous leptin levels.


Figure 3
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  		FIG. 3. A and B, Double IHC for P-STAT3/GFP in LRbGFP mice showing the total number of P-STAT3/GFP-ir neurons in the ARC (A) or the rostral to caudal distribution of P-STAT3/GFP-ir neurons throughout the ARC (B) (n = 1). C, Representative photograph of LRb-expressing/GFP-positive neurons (left), P-STAT3 (middle), and overlay (right) demonstrating colocalization of P-STAT3 in GFP-expressing neurons (white arrows). Scale bar, 100 µm. D, Drawing of a representative section showing single GFP-labeled (green triangle), single P-STAT3-labeled (x), and double-labeled (red circles) neurons. Note that the majority of double-labeled neurons are localized in the ventromedial part of the ARC in close proximity to the ME.

 
Increased leptin responsiveness in the ARC compared with other hypothalamic sites in response to peripherally but not centrally applied leptin
Increased leptin responsiveness in ARC neurons could result from enhanced cellular LRb signaling (e.g. increased LRb expression) in these neurons or from enhanced leptin accessibility across the BBB into the ARC compared with other hypothalamic sites. In the case of enhanced cellular LRb signaling, leptin action in these ARC neurons should be present after peripheral or central leptin treatment, whereas differences due to accessibility should be overcome by central leptin administration.

We therefore examined STAT3 phosphorylation in various hypothalamic nuclei in response to different doses of peripheral and central leptin (Fig. 4Go). We found increased leptin responsiveness in ARC neurons compared with other hypothalamic sites after peripheral leptin treatment (Fig. 4AGo, top panels). Importantly, this difference in responsiveness was overcome by central leptin administration (Fig. 4AGo, lower panels): Although no peripheral leptin dose fully induced leptin signaling in the VMH, DMH, and LH, central application of leptin similarly activated P-STAT3-ir in ARC and non-ARC hypothalamic sites even with low leptin doses (Fig. 4AGo). In striking contrast, the ARC showed robust stimulation after both peripheral and central leptin stimulation. We confirmed these IHC results by P-STAT3 IB analysis of microdissected ARC- and VMH/DMH-enriched tissue lysates (Fig. 4Go, B and C). As in the IHC analysis, peripherally applied leptin robustly induced P-STAT3 within the ARC, whereas we were not able to detect P-STAT3 in VMH/DMH tissues under these conditions. In contrast, central leptin injection equally activated P-STAT3 in ARC and non-ARC hypothalamic tissue (Fig. 4CGo).


Figure 4
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  		FIG. 4. Dose response for peripheral and central leptin-induced P-STAT3 in various hypothalamic nuclei. A, P-STAT3 IHC after peripheral and central leptin administration (Bregma level, –2.8 mm). 3V, Third ventricle. Scale bars, 200 µm. B and C, P-STAT3 IB from ARC and VMH/DMH after ip (B) and icv (C) leptin injections. PANOVA = 0.05 (B) and <0.0001 (C); *, Pposthoc < 0.03; **, Pposthoc < 0.0001. (For an extended dose-response experiment see supplemental Fig. 1Go, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org.)

 
The ARC responds more rapidly than other hypothalamic sites to peripherally applied leptin
We further examined the temporal nature of STAT3 activation after presumably maximal (5 mg/kg body weight) (23) ip leptin administration. We found striking differences in the temporal responses to peripheral leptin in the ARC compared with other hypothalamic sites by both IHC and IB (Fig. 5Go, A–C). Although STAT3 phosphorylation in the ARC was visibly increased 15–30 min after peripheral leptin, the DMH/VMH and LH did not attain peak signal intensity until 1–2 h after stimulation (Fig. 5AGo). Additionally, although the onset and decay of the ARC signal closely mirrored serum leptin levels (Table 2Go and Fig. 5Go), non-ARC hypothalamic sites showed maximal responses well after the peak in circulating leptin had passed. In contrast, this difference in the timing of leptin responsiveness was abrogated in centrally injected mice. After icv leptin administration, P-STAT3 was increased after 15 min in the DMH/VMH as well as the ARC, with similar timing of P-STAT3 activation and deactivation (Fig. 5Go, A and C). Thus, these results suggest that differing access of circulating leptin to ARC vs. non-ARC LRb neurons accounts for their differences in sensitivity to circulating leptin.


Figure 5
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  		FIG. 5. Time course of peripherally and centrally leptin-induced P-STAT3 in various hypothalamic nuclei. A, P-STAT3 IHC after peripheral and central leptin administration (Bregma level, –2.8 mm). 3V, Third ventricle. Scale bars, 200 µm. B and C, P-STAT3 IB from ARC and VMH/DMH after ip (B) and icv (C) leptin injections. PANOVA < 0.01 (B) and <0.0001 (C); *, Pt-test < 0.005; **, Pposthoc < 0.03;***, Pposthoc < 0.001.

 
LRb-expressing ARC neurons accumulate the retrograde tracer FG from the blood circulation
To examine whether LRb neurons are expressed in ARC neurons that are accessible to the circulation (acARC neurons), we employed the BBB-impermeant retrograde tracer FG, which has been extensively used to identify neuroendocrine cells in the hypothalamus (24, 25). When given peripherally, FG is taken up by neuronal processes that directly contact the circulation via blood vessels in circumventricular organs including the portal vessels in the ME. Figure 6Go shows a sagittal overview of the hypothalamus demonstrating that the ARC contains numerous FG-filled neurons, whereas few FG-positive neurons lie within the VMH and DMH area. Consistent with the literature, we also found many paraventricular nucleus (PVN) neurons labeled with FG (24, 25), although no notable expression of LRb was observed in these cells by either GFP or STAT3 phosphorylation (data not shown).


Figure 6
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  		FIG. 6. Peripheral (iv) administration of FG. A and B, Sagittal section showing hypothalamic nuclei including PVN, VMH, DMH, and ARC stained for the neuronal marker NeuN (A), to demonstrate the outlining of the VMH, or FG (B). C, Representative sagittal section of a leptin-treated mouse stained for P-STAT3-ir. D, Coronal section overlaying FG and P-STAT3 to demonstrate double-labeled neurons in the ARC (x20 magnification). Insets show high magnification (x100 oil immersion; scale bar, 50 µm) of FG/P-STAT3 colocalized neurons in the ARC.

 
In untreated, vehicle-treated, or leptin-treated C57/BL6 or LRbGFP mice (Figs. 6Go and 7Go), about 30–35% of baseline or leptin-induced ARC/P-STAT3-ir neurons were colocalized with FG (Fig. 7Go), and no statistical difference was found between baseline and leptin-induced groups, demonstrating the direct contact of a high percentage of baseline P-STAT3-ir neurons with the circulation. The fact that exogenous leptin treatment is not able to further increase the number of FG/P-STAT3-ir neurons shows that baseline leptin levels indeed are sufficient to stimulate all LRb neurons that contact the circulation outside the BBB.


Figure 7
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  		FIG. 7. A. Number of baseline double-labeled FG/P-STAT3-ir neurons throughout the ARC. The inset shows an example of double-labeled neurons. Note the granular appearance of the FG signal identifying vital incorporation of FG. B, Double-labeled FG/P-STAT3-ir neurons after PBS or leptin treatment. Shown are means ± SEM of counted sections, and statistical differences were analyzed with the Student’s t test. C and D, FG/P-STAT3-ir neurons also colocalize with LRb-expressing neurons (blue) in the ventromedial ARC; high magnification images of triple-labeled FG/P-STAT3/LRbGFP neurons are shown. E, Colocalization of FG/LRbGFP shown in a confocal image.

 
Using triple IHC, we also show examples of FG/P-STAT3/LRbGFP-ir neurons (Fig. 7Go, C–E), demonstrating that baseline ARC/P-STAT3-ir neurons indeed represent LRb-expressing acARC neurons. In addition, we found that LRb neuronal cell bodies within the ME are restricted to the ependymal zone of the ME but that a dense projection of LRb neurons is found in the external part of the ME (Fig. 8Go), consistent with LRb neurons contacting the portal circulation of the ME, which lies outside the BBB. Hence, at least a large subpopulation of ARC/LRb-expressing neurons contacts the circulation, and these neurons sensitively respond to endogenous leptin levels.


Figure 8
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  		FIG. 8. A, High magnification of the ARC and ME (x100 oil) in an LRbGFP mouse stained for GFP. B, Highlighted LRb projections to the external zone of the ME. E, Ependymal zone; Ze, external zone; Zi, internal zone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The question of whether ARC neurons may have increased access to circulating factors such as leptin (with attendant functional consequences) has long been debated (13, 14) but never resolved.

Leptin binding to LRb robustly promotes STAT3 phosphorylation to mediate its nuclear translocation and transcriptional activation (26); the detection of leptin-induced P-STAT3 by IHC and IB thus sensitively mirrors LRb action in the central nervous system (1, 15). In our study, we visualize P-STAT3 immunohistochemically to show that endogenous leptin acts on a population of ARC/LRb neurons to induce detectable baseline P-STAT3-ir in wild-type and Ay mice but not in ob/ob and db/db mice, demonstrating that endogenous leptin more robustly activates the ARC than other sites.

Furthermore, our hypothesis that circulating leptin can access some ARC neurons directly (in contrast to other hypothalamic sites, where leptin needs to cross the BBB) predicts that these ARC neurons should respond to peripheral leptin more rapidly and sensitively than other sites, whereas central leptin (circumventing the BBB) should similarly activate LRb signaling in ARC and non-ARC hypothalamic nuclei. In contrast, any intrinsic neuronal properties responsible for differential time and dose responsiveness should persist regardless of the route of leptin application.

Here we demonstrate a more sensitive and rapid leptin signaling in ARC neurons relative to other hypothalamic structures in response to peripheral leptin. Importantly, centrally applied leptin abolished these differences between the ARC and other hypothalamic sites, demonstrating that this divergent response underlies differences in leptin accessibility as opposed to the innate responsiveness of these neurons. These data suggest ARC/LRb neurons do not require leptin transport across the BBB to sense peripheral leptin, although we cannot rule out the possibility of increased leptin transport into the ARC as a potential mechanism for this increased leptin sensitivity in ARC/LRb neurons.

In addition, many baseline ARC/P-STAT3-ir neurons, which also coexpress LRb, are labeled by the BBB-impermanent tracer FG from the periphery, demonstrating that these LRb-expressing acARC neurons directly contact the circulation, likely via processes reaching into sites outside the BBB. Indeed, we could show that LRb neurons densely project into the external zone of the ME where neuroendocrine neurons within the ARC (as well as PVN) terminate and contact the portal circulation outside the BBB.

Therefore, we postulate that acARC soma are likely secured by the BBB but have direct access to circulating leptin via projections into areas outside the BBB, consistent with their ability to accumulate FG from the circulation. This is consistent with the notion that increased sensitivity and rapid response of acARC neurons to peripherally applied leptin is mediated by the direct accessibility of these neurons to the circulation, whereas other hypothalamic sites depend on transport of leptin across the BBB.

The ARC is a crucial site for leptin-mediated effects on glucose homeostasis, energy homeostasis, and other effects, although the ARC alone is not sufficient to mediate the full leptin effect on feeding (27). Our data also open the possibility that many blood-borne substances such as insulin, cytokines, and nutrients (e.g. glucose, fatty acids, and ketones) may directly act on acARC neurons to communicate peripheral conditions to central sites secured by a BBB. Indeed, the mediobasal hypothalamus is known to respond to a variety of these factors (28, 29, 30), and agouti-related peptide (AgRP)/neuropeptide Y (NPY)-expressing neurons in particular have been suggested to play an important role in glucose sensing (31). The anatomical distribution of AgRP/NPY neurons is restricted to the ventromedial ARC, and they are known to express LRb (32). Therefore, it is possible that acARC neurons represent at least partially AgRP/NPY neurons, which needs to be proven in additional studies.

Furthermore, our results may help to reveal the biological basis for several findings in the field of leptin research. For example, it is well known that central leptin application more potently regulates food intake and body weight than does peripheral leptin (23); our data suggest that this could reflect the more robust recruitment of non-ARC hypothalamic sites of leptin action after central administration of leptin.

Also, in leptin-resistant DIO mice, it was shown that decreased hypothalamic leptin signaling was improved, but not fully corrected, by central administration of leptin (23). However, hypothalamic leptin resistance (decreased leptin-induced P-STAT3-ir) in DIO mice is restricted to the ARC (15); therefore, our data suggest that central application of leptin to DIO mice reflects an increased response of non-ARC hypothalamic neurons, whereas the ARC remains leptin resistant.

The mechanisms and mediators of ARC-specific leptin resistance remain unclear. Possibilities include the induction of negative feedback mechanisms by leptin (e.g. via SOCS3 induction) or the induction of leptin signaling-inhibitory molecules by other obesity-associated cytokines. Increased access of ARC LRb-expressing neurons also opens the possibility that other factors, e.g. cytokines or hormones, may access the ARC equally directly and differentially to other hypothalamic sites. This may render the ARC especially prone to the induction of cellular leptin resistance by these factors. Indeed, whereas DIO animals exhibit ARC-specific resistance, chronic central infusion of leptin (again circumventing the BBB) induces SOCS3 expression and leptin resistance more widely (33).

In conclusion, we show evidence that a population of LRb-expressing ARC neurons project outside the BBB and display increased accessibility to circulating substances such as leptin. These neurons display increased sensitivity toward peripheral leptin compared with non-ARC hypothalamic nuclei. These neurons likely function to sensitively and rapidly sense changes in circulating leptin and may be central to the physiological response to leptin and the development of leptin resistance.


   Acknowledgments
 
We acknowledge Dr. Martin G. Myers, Jr., for providing the LRbGFP mice for this study and for critical discussion of the manuscript.


   Footnotes
 
This project was supported by National Institutes of Health Cellular and Molecular Approaches to Systems and Integrative Biology Training Grant to R.L., the Swedish Foundation for International Cooperation in Research and Higher Education, the AFA Sjukförsäkring Jubilée Foundation for Research in National Diseases to M.B., and American Heart Association Scientist Development Grant AHA0535298N and MDRTC P/F Grant to H.M.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 9, 2007

Abbreviations: acARC, ARC neurons accessible to the circulation; AgRP, agouti-related peptide; ARC, hypothalamic arcuate nucleus; BBB, blood-brain barrier; DIO, diet-induced obese; DMH, dorsomedial hypothalamus; FG, fluorogold; GFP, green fluorescent protein; IB, immunoblotting; icv, intracerebroventricular; IHC, immunohistochemistry; ir, immunoreactivity; LH, lateral hypothalamus; LRb, long-form leptin receptor; ME, median eminence; NPY, neuropeptide Y; P-STAT3, phosphorylated STAT3; PVN, paraventricular nucleus; SOCS-3, suppressor of cytokine signaling 3; STAT3, signal transducer and activator of transcription 3; VMH, ventromedial hypothalamus.

Received May 17, 2007.

Accepted for publication August 1, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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