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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2013 Feb 5;110(8):E697–E706. doi: 10.1073/pnas.1218284110

Modulation of AgRP-neuronal function by SOCS3 as an initiating event in diet-induced hypothalamic leptin resistance

Louise E Olofsson a, Elizabeth K Unger a, Clement C Cheung b, Allison W Xu a,1
PMCID: PMC3581908  PMID: 23386726

Significance

Multiple neuronal subtypes are involved in metabolic regulation, but little is known about the temporal dysregulation of neuronal functions upon acute consumption of fat-rich diets. We show that AgRP neurons are the predominant cell type situated outside the blood-brain barrier in the mediobasal hypothalamus. AgRP neurons are able to sense slight changes in plasma metabolic signals, such as leptin, but they also more quickly develop cellular leptin resistance compared with other hypothalamic neurons. We further show that modulation of SOCS3 expression in AgRP neurons plays a dynamic role in metabolic fine tuning in response to acute change of nutritional status.

Abstract

Chronic consumption of a fat-rich diet leads to attenuation of leptin signaling in hypothalamic neurons, a hallmark feature of cellular leptin resistance. To date, little is known about the temporal and spatial dysregulation of neuronal function under conditions of nutrient excess. We show that agouti-related protein (AgRP)-expressing neurons precede proopiomelanocortin neurons in developing diet-induced cellular leptin resistance. High-fat diet-induced up-regulation of suppressor of cytokine signaling-3 (SOCS3) occurs in AgRP neurons before proopiomelanocortin and other hypothalamic neurons. SOCS3 expression in AgRP neurons increases after 2 d of high-fat feeding, but reduces after switching to a low-fat diet for 1 d. Consistently, transgenic overexpression of SOCS3 in AgRP neurons produces metabolic phenotypes resembling those observed after short-term high-fat feeding. We further show that AgRP neurons are the predominant cell type situated outside the blood-brain barrier in the mediobasal hypothalamus. AgRP neurons are more responsive to low levels of circulating leptin, but they are also more prone to development of leptin resistance in response to a small increase in blood leptin concentrations. Collectively, these results suggest that AgRP neurons are able to sense slight changes in plasma metabolic signals, allowing them to serve as first-line responders to fluctuation of energy intake. Furthermore, modulation of SOCS3 expression in AgRP neurons may play a dynamic and physiological role in metabolic fine tuning in response to short-term changes of nutritional status.

Most common forms of obesity, including diet-induced obesity, are associated with hyperleptinemia and impairment of leptin signaling in hypothalamic neurons, the hallmark feature of cellular leptin resistance. Suppressor of cytokine signaling-3 (SOCS3), a direct transcriptional product of STAT3, is up-regulated in the hypothalamus of diet-induced obese animals (1, 2). Mice with heterozygous mutation of the Socs3 gene, neuronal, or proopiomelanocortin (POMC)-specific deletion of the Socs3 gene are hypersensitive to leptin and resistant to diet-induced obesity (3–5). Conversely, up-regulation of SOCS3 in POMC neurons of chow-fed mice leads to increased body adiposity (6). In addition, wide-spread up-regulation of SOCS3 has been shown to be associated with neuronal inflammation in diet-induced obese animals (7). Thus SOCS3, which is up-regulated in chronic obesity, is commonly thought to play a pathophysiological role in obesity-associated leptin resistance.

Multiple neuronal subtypes in several regions of the hypothalamus, including the arcuate nucleus, ventromedial hypothalamus, dorsomedial hypothalamus, and lateral hypothalamic area, have been implicated in the regulation of energy balance and leptin action (8, 9). A number of hypothalamic neurons and extrahypothalamic neurons express functional leptin receptor (10, 11). Among these neurons, POMC and agouti-related protein (AgRP) neurons are two key arcuate neuronal subtypes. POMC and AgRP neurons promote negative and positive energy balance, respectively, and they are regulated by leptin in opposite ways. Thus, these two neuronal subtypes are often considered to play equal but reciprocal roles in regulation of energy balance.

Diet-induced obesity is a progressive process. Short-term consumption of a high-fat diet leads to increased feeding and caloric intake, but at the same time results in elevation of energy expenditure, which likely serves as an adaptive response to restore energy balance (12–15). Concurrent to that, however, is the rapid induction of insulin resistance and hepatic steatosis, even in the absence of an apparent weight change (16, 17). Consumption of a fat-rich diet, when allowed to persist, ultimately leads to obesity. Although disruption of leptin signaling and cellular leptin resistance are observed in many hypothalamic neurons in established diet-induced obese animals, little is known about the temporal and spatial dysregulation of neuronal functions during the development and progression of diet-induced obesity. In this study, we provide evidence that AgRP neurons are unique among hypothalamic neurons by being the predominant neuronal subtype situated outside the blood-brain barrier (BBB), and they are more responsive to small changes of circulating leptin. In turn, these neurons are more susceptible to dysregulation of leptin signaling by SOCS3, which may act as an early effector in causing metabolic perturbations after short-term consumption of a fat-rich diet.

Results

AgRP Neurons Precede POMC Neurons in the Development of Cellular Leptin Resistance After Short-Term Consumption of a High-Fat Diet.

To gain understanding of the temporal and spatial regulation of diet-induced cellular leptin resistance, we first evaluated whether responsiveness of POMC and AgRP neurons to leptin is concurrently impaired after exposure to a high-fat diet for various lengths of time. Because Npy and Agrp are coexpressed in the same neurons within the arcuate nucleus (18), AgRP neurons can be identified by presence of GFP fluorescence in mice in which GFP expression is driven by the Npy promoter. The specificity of GFP expression was verified by complete colocalization of Npy mRNA and GFP fluorescent signals in cells within the arcuate nucleus (Fig. S1). Impairment of leptin-induced phosphorylation of STAT3 (pSTAT3) is a hallmark feature of diet-induced obesity (19, 20). To this end, pSTAT3 was analyzed in the hypothalamus of C57BL/6 mice carrying the Npy-GFP reporter after exposure to a high-fat diet for 2 d, 2 wk, or 10 wk, and compared with mice fed with a low-fat diet matched for other macronutrients. To minimize experimental variations, POMC and AgRP neurons from the same brain sections were examined for pSTAT3 by using triple-color immunofluorescence analysis. Two days of high-fat feeding markedly impaired leptin-induced pSTAT3 in the AgRP neurons, but not in the POMC (Fig. 1 and Fig. S2). However, prolonged consumption (2 or 10 wk) of the high-fat diet caused an increase in basal pSTAT3 immunoreactivity in the POMC neurons, whereas leptin’s ability to induce pSTAT3 signaling was progressively attenuated in these neurons (Fig. 1B). This result indicates that AgRP neurons precede POMC neurons in the development of cellular leptin resistance upon exposure to a high-fat diet.

Fig. 1.

Fig. 1.

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AgRP neurons precede POMC neurons in the development of cellular leptin resistance upon exposure to a high-fat diet. Mice carrying the Npy-GFP reporter were fed high-fat diet (HFD) for various lengths of time (2 d, 2 wk, or 10 wk), and compared with mice fed with a matched low-fat diet (LFD). Mice were injected with leptin (3 mg/kg) or saline and perfused 45 min later. (A) Leptin-induced STAT3 phosphorylation in POMC and AgRP neurons were analyzed by triple-color immunofluorescence using the Npy-GFP reporter (blue) and antibodies against Tyrosine-705-STAT3 (red, pSTAT3) and ACTH (green). (B) Quantification of POMC-, AgRP-, and pSTAT3-positive cells shows that 2 d of high-fat feeding did not alter leptin-mediated pSTAT3 signaling in the POMC and other extra-arcuate neurons, but markedly impaired leptin signaling in the AgRP neurons. n = 3–6 mice per group. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 as determined by a two-way ANOVA with a Bonferroni post hoc test. Groups being compared are indicated on the graph. n.s., nonsignificant. Data represent mean ± SEM. 3V: third ventricle.

High-Fat Diet-Induced Up-Regulation of SOCS3 Occurs in AgRP Neurons Before POMC and Other Hypothalamic Neurons.

SOCS3, a negative regulator of JAK-STAT3 signaling, has been implicated in the negative feedback regulation of leptin signaling (21). In light of the above findings, we investigated whether expression of SOCS3 is up-regulated in AgRP and POMC neurons after acute exposure to a high-fat diet. To this end, SOCS3 immunoreactivity was examined in hypothalamic neurons from mice that were fed with a high-fat diet for either 2 d or 2 wk, and compared with mice fed with a low-fat diet matched for other macronutrients. Specificity of the SOCS3 antibody was validated by diminished immunoreactivity after preincubation of the SOCS3 antibody with a full-length SOCS3 protein (Fig. S3). In animals fed with a low-fat diet, SOCS3 immunoreactivity was detected only in the mediobasal hypothalamus (Fig. 2A). Consumption of a high-fat diet for 2 d did not change the number of AgRP neurons that were positive for SOCS3, but caused elevation of SOCS3 intensity in the AgRP neurons by about twofold (Fig. 2 A–C). Neither the number of SOCS3-positive POMC neurons nor SOCS3 protein intensity in POMC neurons changed after 2 d of high-fat feeding (Fig. 2 A–C). However, after 2 wk of high-fat feeding, numbers of SOCS3-positive POMC neurons and extra-arcuate cells were significantly increased (Fig. 2 A, C, and D). Taken together, these results indicate that SOCS3 expression increases in the AgRP neurons before POMC neurons and other hypothalamic cells during the course of high-fat feeding, suggesting that diet-induced SOCS3 up-regulation in the hypothalamus follows a distinct temporal and spatial pattern. In contrast to SOCS3, no significant increase of heat-shock protein 72 (HSP72), a marker for neuronal injury (22), or phosphorylation of eukaryotic initiation factor 2α (eIF2α), a marker for endoplasmic retictulum stress (23), was detected in AgRP neurons after 2-d high-fat feeding (Figs. S4 and S5).

Fig. 2.

Fig. 2.

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HFD-induced SOCS3 up-regulation occurs in AgRP neurons before POMC and other hypothalamic cells. (A) Mice were fed with a LFD or HFD for 2 d or 2 wk. Fed animals were perfused and immunofluorescence analysis was performed to examine SOCS3 expression in the hypothalamus. POMC neurons are identified by ACTH immunoreactivity, and AgRP neurons are identified by GFP signals. (B) SOCS3 intensity was increased in AgRP neurons, but not in POMC neurons, in mice after 2 d of HFD compared with mice fed with a LFD. n = 4–5 mice per group. (C) The numbers of SOCS3-positive AgRP or POMC neurons were not altered in mice fed with a HFD for 2 d compared with mice fed LFD. However, the number of SOCS3-positive POMC neurons was increased significantly after 2 wk of HFD. n = 4–5 mice per group. (D) Number of SOCS3-positive cells outside the arcuate nucleus (ARC) was increased in mice fed with a HFD for 2 wk. n = 4–5 mice per group. *P ≤ 0.05 and **P ≤ 0.01 as determined by a one-way ANOVA with a Bonferroni post hoc test in C and D, and a Student t test was used to compare LFD and 2-d HFD in B. Data represent mean ± SEM. 3V: third ventricle.

Expression of SOCS3 in AgRP Neurons Is Dynamically Modulated in Response to Short-Term Changes of Dietary Conditions.

The above result shows that SOCS3 expression increases rapidly in response to increased consumption of dietary fat. To determine whether SOCS3 expression is reversible upon change of nutritional status, a separate cohort of mice were fed with a high-fat diet for 2 d, followed by low-fat-diet feeding for 1 d. Two-day high-fat feeding increased SOCS3 expression in AgRP neurons, which was significantly reduced 1 d after switching to a low-fat diet (Fig. 3). This result suggests that the increase in SOCS3 expression after short-term consumption of a high-fat diet is reversible, indicating that SOCS3 expression in AgRP neurons is dynamically modulated in response to short-term changes of dietary conditions.

Fig. 3.

Fig. 3.

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Expression of SOCS3 is reversibly regulated in the AgRP neurons in response to acute changes of dietary conditions. (A) Female mice with Npy-GFP reporter were fed with a LFD, a HFD for 2 d, or a HFD for 2 d followed by a LFD for 1 d. Fed animals were perfused and immunofluorescence analysis was performed to examine SOCS3 expression in the hypothalamus. AgRP neurons are identified by GFP signals. (B) SOCS3 intensity was increased in AgRP neurons in mice fed a HFD for 2 d compared with mice fed a LFD. In mice fed a HFD for 2 d followed by 1 d of LFD, the SOCS3 intensity returned to levels comparable to in mice fed a LFD. n = 3–4 mice per group.*P ≤ 0.05 as determined by a one-way ANOVA with a Bonferroni post hoc test. n.s., not significant. Data represent mean ± SEM. 3V: third ventricle.

Up-Regulation of SOCS3 in AgRP Neurons Results in Metabolic Phenotypes Mimicking Those Seen After Short-Term Consumption of a High-Fat Diet.

We have previously shown that transgenic up-regulation of SOCS3 in POMC neurons causes increased adiposity and obesity-induced glucose intolerance (6). We set out to investigate the metabolic phenotypes mediated by SOCS3 up-regulation in AgRP neurons. Mice carrying a Cre-activatable allele of wild-type Socs3 (6) were crossed with mice expressing Cre recombinase specifically in the AgRP neurons (24), such that SOCS3 would be overexpressed specifically in the AgRP neurons. We have previously shown that SOCS3 expression is Cre-dependent, and its expression is up-regulated approximately twofold in Cre-expressing cells upon recombination (6). The Tg.Agrp-Cre mice have been used and validated in a number of independent studies (7, 25–31). The resultant mutant mice, termed AgRP-SOCS3-OE, were born in Mendelian ratio with no detectable gross abnormalities. SOCS3 immunoreactivity increased by approximately twofold in the AgRP neurons of AgRP-SOCS3-OE mice compared with control mice, confirming overexpression of SOCS3 in AgRP neurons (Fig. S6). The number and size of AgRP neurons and their projection patterns were indistinguishable between control and mutant mice (Fig. S7), suggesting that SOCS3 up-regulation did not affect the development of these neurons.

As expected, leptin-induced pSTAT3 was significantly diminished in the AgRP neurons of mutant animals compared with weight-matched control mice (Fig. S8A). Fos expression was significantly elevated in AgRP neurons in the mutant animals (Fig. S8B), consistent with increased firing rate of AgRP neurons in diet-induced obese animals (32, 33). Phosphorylation of S6, a downstream component of the mammalian target of rapamycin complex 1 pathway, was increased in the mutant mice (Fig. S8C), consistent with the notion that pS6 signals positively correlate with AgRP neuronal activity (34). In contrast, Agrp and Npy expression were not significantly altered (Fig. S8D). When analyzed using the comprehensive laboratory animal monitoring system (CLAMS), AgRP-SOCS3-OE mice exhibited elevation of nighttime feeding, which was accompanied by significant increase in oxygen consumption (Fig. 4 A and B). Locomotor activity was not different between control and mutant mice (Fig. S8E). No significant changes in body weight, lean mass, fat mass, and circulating leptin levels were detected in AgRP-SOCS3-OE mutant mice throughout adulthood (Fig. 4 C–F). Mutant animals showed reduced response to leptin’s anorectic effects (Fig. 4G), consistent with the rapid attenuation of leptin’s anorectic effects in wild-type animals after short-term high-fat feeding (35). Liver triglyceride levels were elevated in the mutant animals (Fig. 4H). Blood glucose and insulin levels were not significantly different, although the insulin levels trended toward an increase in the mutant mice (Fig. 4 I and J). Despite having normal body adiposity, weight-matched mutant mice exhibited impaired glucose tolerance and reduced systemic insulin sensitivity, as shown by insulin tolerance test (Fig. 4 K and L). Together, these results indicate that transgenic SOCS3 up-regulation in AgRP neurons results in metabolic phenotypes mimicking those seen after short-term consumption of a high-fat diet (12–14, 16, 17).

Fig. 4.

Fig. 4.

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Transgenic SOCS3 up-regulation in AgRP neurons results in metabolic phenotypes mimicking those seen after short-term consumption of a high-fat diet. (A) Caloric intake during the light and dark cycle in 12-wk-old control mice (n = 16) and AgRP-SOCS3-OE mutant mice (n = 5) as measured by CLAMS. (B) Oxygen consumption was measured in 12-wk-old mice and the values were normalized to lean body mass of each mouse. Control mice n = 16, mutant mice n = 5. (C) Body weight of control mice (n = 20–74) and AgRP-SOCS3-OE mutant mice (n = 14–39) on chow diet at indicated ages. (D) Lean mass and fat mass as analyzed by DEXA in 25- to 35-wk-old control mice (n = 36) and AgRP-SOCS3-OE mutant mice (n = 21). (E) Weight of dissected fat pads from 30- to 40-wk-old control mice (n = 18) and AgRP-SOCS3-OE mutant mice (n = 9). (F) Plasma leptin levels under fed conditions for 30- to 42-wk-old control mice (n = 11) and AgRP-SOCS3-OE mutant mice (n = 9). (G) Eight-week-old control (n = 7) and mutant (n = 5) mice were injected with vehicle (saline) or leptin (2.5 mg/kg body weight), and 24-h food intake was measured. (H) Liver triglyceride in 30- to 40-wk-old control mice (n = 8) and AgRP-SOCS3-OE mutant mice (n = 8). (I and J) Blood glucose and plasma insulin after 6 h fast in 20- to 30-wk-old control mice (n = 9) and AgRP-SOCS3-OE mutant mice (n = 6). (K) AgRP-SOCS3-OE (n = 11) and control mice (n = 22) at 12 wk of age were fasted for 6 h, and injected with 2.5 g/kg glucose. Glucose levels were measured from tail blood using a glucometer. (L) Thirteen-week-old control mice (n = 17) and AgRP-SOCS3-OE mutant mice (n = 11) were fasted overnight, and injected with 1 U/kg insulin. Glucose levels were measured from tail blood using a glucometer. Data represent mean± SEM *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001 between control and mutant mice as determined by two-way repeated measures ANOVA (A–C, K, and L) and Student t tests (D–J).

AgRP Neurons Are the Predominant Cell Type Outside the BBB in the Mediobasal Hypothalamus.

Fenestrated capillaries are present in the median eminence, a circumventricular organ, and considerable vascular permeability is observed in the arcuate nucleus (36–38). Given that AgRP and POMC neurons are both located in the arcuate nucleus of the hypothalamus, adjacent to the median eminence, we investigated the anatomical relationship of AgRP and POMC neurons with the BBB. It has been previously shown that a subset of neurons within the mediobasal hypothalamus readily take up fluorogold, a dye that does not penetrate the BBB, indicating that these neurons are outside the BBB (39). In the present study, we used Evans blue, a commonly used fluorescent dye for assessing BBB permeability, to visualize hypothalamic cells that are in direct contact with the circulation. Evans blue is not accessible to cells inside the BBB under normal condition, but readily penetrates neurons and other cells when the BBB is damaged under pathologic conditions (37, 40). Evans blue was administered peripherally into normal mice carrying the Npy-GFP reporter. Direct fluorescence emitted by Evans blue was observed in blood vessels throughout the brain, as well as in a population of cells in the arcuate nucleus of the hypothalamus (Fig. 5A). AgRP neurons made up close to 70% of Evans blue-positive cells in the anterior, medial and posterior arcuate nucleus, but POMC neurons constituted about 10% of Evans blue-positive cells in these regions (Fig. 5 B–D). Using an independent method, immunoreactivity of albumin, which does not penetrate the BBB, was found to be present in most AgRP neurons, further confirming that AgRP neurons are directly exposed to blood-borne substances (Fig. 5E). Taken together, our result indicates that AgRP neurons represent the predominant cell type situated outside the BBB in the mediobasal hypothalamus.

Fig. 5.

Fig. 5.

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AgRP neurons are the predominant cell type situated outside the BBB in the mediobasal hypothalamus. Transgenic mice expressing Npy-GFP were transcardially injected with 1% Evans blue solution and perfused 10 min later. Hypothalamic sections were prepared and immunofluorescence was carried out to examine the relationship between the BBB and AgRP and POMC neurons. (A) Image shows direct fluorescence emitted by Evans blue in blood vessels throughout the brain as well as in the median eminence and the ARC in 500-µm-thick brain explants. (B) Percentages of Evans blue-positive cells in the mediobasal hypothalamus that are either AgRP or POMC neurons or unknown cells. Posterior, medial, and anterior positions refer to Bregma −2.30 to −2.06, −2.06 to −1.82, and −1.82 to −1.46, respectively. n = 3 mice. Errors bar present SEM. (C) Representative images (10-µm sections) showing extensive colocalization of Npy-GFP (green) and Evans blue (red) in the ARC. (D) Representative images (10-µm sections) showing limited colocalization of ACTH (green) and Evans blue (red) in the arcuate nucleus. (E) Albumin immunoreactivity (red) in AgRP neurons (green). 3V: third ventricle. ME, median eminence.

Cells Outside the BBB Are More Sensitive to Basal Level of Leptin Signaling.

To further determine whether neurons that are directly exposed to the circulation are more sensitive to blood-borne metabolic signals, we first examined the distribution of pSTAT3 signals in the hypothalamus from wild-type mice under free-fed conditions on regular chow. Consistent with what was described previously (39), basal pSTAT3 signal was only detected in a small population of cells in the mediobasal hypothalamus. Importantly, about 90% of these basal pSTAT3-positive cells are also positive for Evans blue (Fig. 6). Because the presence of the basal pSTAT3 signal is leptin-dependent (39), our result suggests that cells located outside the BBB in the mediobasal hypothalamus are more sensitive to basal level of leptin signaling.

Fig. 6.

Fig. 6.

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Basal expression of phosphorylation of STAT3 is restricted to cells outside the BBB. (A) Mice carrying the Npy-GFP reporter were transcardially injected with 1% Evans blue solution and perfused 10 min later. Hypothalamic sections were prepared and immunofluorescence analysis using antibody against Tyrosine-705-STAT3 (green, pSTAT3) was performed to determine the relationship between basal STAT3 phosphorylation and the BBB in the ARC. Cells outside the BBB are identified by Evans blue staining (red). (B) The vast majority of the basal pSTAT3-positive cells are situated outside the BBB. n = 3 mice. ***P ≤ 0.001 as determined by Student t tests. Data represent mean ± SEM. 3V: third ventricle.

AgRP Neurons Situated Outside the BBB Are Primary Targets of SOCS3 Up-Regulation After Short-Term Consumption of a High-Fat Diet.

We next investigated whether AgRP neurons situated outside the BBB displayed more SOCS3 expression after short-term consumption of a high-fat diet. C57BL/6 mice carrying Npy-GFP were fed with a high-fat diet or a matching low-fat diet for 2 d. AgRP neurons were identified by GFP expression. Because of technical considerations related to antibody compatibility, cells outside the BBB were revealed by positive immunoreactivity of albumin instead of using Evans blue in this experiment. After 2 d of high-fat diet feeding, SOCS3 intensity increased in the AgRP neurons that were albumin-positive, but not in AgRP neurons that were albumin negative (Fig. 7), suggesting that the AgRP neurons outside the BBB are more sensitive to SOCS3 up-regulation after short-term consumption of a high-fat diet.

Fig. 7.

Fig. 7.

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AgRP neurons situated outside the BBB are primary targets of SOCS3 up-regulation after short-term consumption of a HFD. (A) Transgenic mice expressing Npy-GFP were fed a HFD for 2 d or a matched LFD. Fed animals were perfused and immunofluorescence analysis was performed to examine SOCS3 (green) and albumin (red) expression in the hypothalamus. Npy-GFP (blue) was used to identify AgRP neurons, and albumin was used to identify cells outside the BBB. (B) Two days of HFD increased the SOCS3 intensity in AgRP neurons situated outside the BBB. In contrast, feeding a HFD for 2 d did not increase the SOCS3 intensity in AgRP neurons situated inside the BBB. n = 4–5 mice per group. *P ≤ 0.05 between LFD and 2 d HFD as determined by a two-way ANOVA with a Bonferroni post hoc test. n.s., not significant. Data represent mean ± SEM. 3V: third ventricle.

Low Dose of Peripheral Leptin Treatment Induces Cellular Leptin Resistance Specifically in AgRP Neurons Situated Outside the BBB.

The heightened sensitivity of AgRP neurons, defined by their permeability to the circulation, to basal pSTAT3, and increased SOCS3 expression suggests that AgRP neurons outside the BBB are more sensitive to blood-borne signals, the circulating levels of which change after short-term consumption of a high-fat diet. Leptin is commonly considered an adiposity signal for long-term regulation of energy balance because of the close association between its circulating levels and fat mass. However, plasma leptin is known to exhibit a meal-entrained diurnal rhythm, and leptin levels are influenced by short-term overfeeding or underfeeding in humans and rodents without changes in fat mass (12, 41–46). These findings indicate that leptin levels are modulated dynamically in response to short-term changes of nutritional status. In particular, a small but significant increase in circulating leptin levels is observed after 2 d of high-fat feeding (12). Thus, we speculated that the heightened sensitivity of AgRP neurons outside the BBB to leptin may also render these neurons more susceptible to the development of cellular leptin resistance under conditions of acute nutrient excess. To test this possibility, we investigated whether a low dose of peripheral leptin administration induced cellular leptin resistance primarily in AgRP neurons outside the BBB. C57BL/6 mice carrying the Npy-GFP reporter were given an intraperitoneal injection of leptin (0.01 mg/kg) or vehicle four times at 2-h intervals, and then injected with a large dose of leptin (3 mg/kg) and killed 45 min later. Intraperitoneal injection of 0.01 mg/kg leptin has been shown to raise blood leptin levels approximately twofold (39), similar in magnitude to the small increase of leptin levels after 2 d of high-fat feeding (12). Compared with mice that received vehicle pretreatment, low-dose (0.01 mg/kg) leptin pretreatment caused impairment of leptin-induced pSTAT3 signaling specifically in AgRP neurons that were Evans blue-positive, but not in those AgRP neurons that were Evans blue-negative (Fig. 8). Taken together, our results suggest that AgRP neurons situated outside the BBB are more sensitive to leptin, and in turn they are more vulnerable to the development of cellular leptin resistance.

Fig. 8.

Fig. 8.

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Low dose of peripheral leptin treatment induces cellular leptin resistance specifically in AgRP neurons situated outside the BBB. (A) Mice carrying the Npy-GFP reporter were fasted from 0900 hours and intraperitoneally injected with either saline or 0.01 mg/kg leptin at 0900 hours, 1100 hours, 1300 hours, and 1500 hours. All mice were injected with 3 mg/kg leptin intraperitoneally at 1700 hours, transcardially injected with 1% Evans blue solution at 1735 hours, and perfused 10 min later. Hypothalamic sections were prepared and immunofluorescence analysis was performed to compare leptin-induced pSTAT3 signaling in hypothalamus from mice pretreated with leptin or saline. Cells outside the BBB were identified by Evans blue staining (blue), and AgRP neurons were identified by GFP signal (green). Antibody against Tyrosine-705-STAT3 was used to detect pSTAT3 (red). (B) Evans blue-positive AgRP neurons show diminished response to leptin (3 mg/kg)-induced pSTAT3 after pretreatment with low-dose (0.01 mg/kg) leptin, but Evans blue-negative AgRP neurons were not affected by this pretreatment. n = 3 mice per group. *P ≤ 0.05 between saline and leptin pretreatments as determined by a two-way ANOVA with a Bonferroni post hoc test. n.s., not significant. Data represent mean ± SEM. 3V: third ventricle.

Discussion

Development and progression of diet-induced obesity are mediated by a number of neurological changes that progressively impair hypothalamic neuronal functions. However, very little is known about the temporal and spatial regulation of these changes during the course of high-fat feeding. In this study, we show that AgRP neurons develop cellular leptin resistance before POMC and other hypothalamic neurons. Upon acute exposure to a high-fat diet, SOCS3 is up-regulated in AgRP neurons and this is followed by wide-spread SOCS3 up-regulation in a broader hypothalamic area after prolonged high-fat feeding. Transgenic up-regulation of SOCS3 in AgRP neurons causes several key phenotypes reminiscent of those observed after short-term consumption of fat-rich diets; this includes hyperphagia, elevation of energy expenditure, development of insulin resistance, and increased hepatic lipid content without significant changes in body adiposity (12–17). This result suggests that modulation of SOCS3 expression may play a dynamic role in metabolic fine tuning in response to short-term changes of nutritional status.

Because they are the predominant neurons situated outside the BBB, AgRP neurons may possess the unique ability to directly sense blood-borne metabolic signals. This theory is supported by our finding that basal level of pSTAT3 signal, which has been previously shown to be leptin-dependent (39), is present almost exclusively in cells outside the BBB. This finding is further supported by our result that early elevation of SOCS3 expression upon acute exposure to a high-fat diet occurs primarily in AgRP neurons that are situated outside the BBB. It is likely that up-regulation of SOCS3 after short-term high-fat feeding is induced by circulating molecules whose access to the brain is tightly controlled. One of these molecules is leptin, which is known to activate SOCS3 expression. Indeed, arcuate SOCS3 expression is lower in obese mice with inactivation of leptin receptor in all tissues compared with control animals (47). Leptin is transported into the brain via a saturable transport system (48, 49). However, the amount of leptin transported into the brain is not proportional to the plasma leptin level, because of the limited capacity of the transport machinery (49, 50). Leptin is commonly considered an adiposity signal for long-term regulation of energy balance because of the close association between its circulating levels and fat mass. However, plasma leptin levels are influenced by short-term changes in nutritionals status in humans and rodents in the absence of significant changes in body adiposity, and leptin’s diurnal rhythm can be entrained by meal timing (41–46). In particular, plasma leptin levels rise moderately after 2-d consumption of a high-fat diet before significant increase of body adiposity (12). These studies suggest that circulating levels of leptin are more dynamic in nature and can be influenced by short-term changes of nutritional intake or alteration of feeding patterns. Thus, by being situated outside the BBB, AgRP neurons could be more sensitive to the subtle changes of leptin levels compared with neurons situated inside the BBB. Modulation of SOCS3 in AgRP neurons outside the BBB may play a physiological role in adjusting leptin-signaling strengths in response to short-term changes of nutritional status. However, the heightened sensitivity of these neurons to leptin may in turn render them more susceptible to the development of cellular leptin resistance. The early development of cellular leptin resistance in AgRP neurons upon short-term high-fat feeding or by lowdose leptin treatment, as demonstrated in this study, supports this notion. In addition, transgenic up-regulation of SOCS3 in AgRP neurons diminishes leptin’s anorectic effects, consistent with the finding that leptin’s anorectic effects are greatly attenuated in wild-type animals after 3 d of high-fat feeding (35).

AgRP neurons are known to project to multiple regions of the brain, including the arcuate nucleus, the paraventricular nucleus, and neurons in the dorsomedial hypothalamus, lateral hypothalamic area, and the parabrachial nucleus in the hindbrain (51–55). In particular, AgRP neurons are known to project directly onto POMC neurons in the arcuate nucleus, and inhibit POMC neuronal firing (15, 54). Thus, modulation of AgRP neurons by blood-borne metabolic signals could indirectly regulate their downstream neurons in many regions of the brain via neuropeptide or neurotransmitter release from AgRP neurons. The heightened sensitivity of AgRP neurons outside the BBB allows these neurons to sense small changes of circulating leptin levels, and to relay this information to POMC and other downstream target neurons. The functional integrities of components in the higher-order neuronal circuits are likely required for dynamic metabolic fine tuning by blood-borne metabolic signals acting on AgRP neurons outside the BBB.

It should be acknowledged that the expression of SOCS3 in the basal state is found in non-AgRP cells and also in cells behind the BBB. However, after consumption of a high-fat diet for 2 d, SOCS3 is significantly increased predominantly in AgRP neurons outside the BBB, but not in AgRP neurons inside the BBB. SOCS3 expression in POMC neurons, which are mostly situated inside the BBB, does not change after 2 d of high-fat feeding. Taken together, these results suggest that expression of SOCS3 in AgRP neurons outside the BBB is more sensitive to a short-term change of dietary condition. Consistent with the notion that cells outside the BBB are more sensitive to leptin, we show that 90% of pSTAT3-positive cells are found outside the BBB, and these basal pSTAT3 signals have been previously shown to be leptin-dependent (39). Although SOCS3 is a direct transcriptional target of STAT3, SOCS3 has also been shown to be regulated by other signaling pathways such as NF-κB (7) or Epac (56). Thus, it is possible that basal SOCS3 expression in cells behind the BBB is regulated by STAT3-independent mechanisms.

Rapid and reversible regulation of SOCS3 in AgRP neurons upon acute changes of dietary conditions suggests that modulation of SOCS3 expression in AgRP neurons is dynamic in nature. This notion is further supported by the findings that arcuate SOCS3 expression is decreased upon fasting (57). The ability of AgRP neurons to sense slight changes in plasma metabolic signals make them important integrators of peripheral hormonal actions and first-line responders to daily fluctuation of energy intake and expenditure in normal weight subjects. However, under conditions of chronic energy surplus, such as during the persistent consumption of a high-fat diet, SOCS3 expression is up-regulated in broader hypothalamic area, which may be attributed to the development of neuroinflammation (7) and chronic hyperleptinemia. It should be noted that AgRP-SOCS3-OE mice do not develop obesity despite displaying hyperphagia and other metabolic perturbations, suggesting that metabolic effects caused by restricted up-regulation of SOCS3 in the AgRP neurons can be compensated by other hypothalamic neurons or SOCS3-independent pathways in AgRP neurons. This observation further suggests that up-regulation of SOCS3 in POMC and other hypothalamic neuronal subtypes may ultimately impair homeostatic regulation of energy balance, resulting in obesity. This notion is supported by our previous findings that transgenic up-regulation of SOCS3 in POMC neurons causes obesity (6). Collectively, confined up-regulation of SOCS3 in the AgRP neurons may signal short-term changes in dietary condition, but up-regulation of SOCS3 in POMC neurons and additional hypothalamic neurons may signal a steady surplus of energy supply and thus promote weight gain and energy storage as fat.

Experimental Procedures

Mice.

Mice expressing GFP under the control of the mouse Npy promoter were purchased from the Jackson Laboratory [B6.FVB-Tg(NPY-hrGFP)1Lowl/J]. Generation of transgenic mice that express Cre-activatable Socs3 allele (Tg.CMV-Flox-Stop-Socs3 mice) has previously been described and validated (6). Tg.Agrp-Cre mice have been validated by multiple research groups. Tg.Agrp-Cre/+ mice were crossed with the Tg.CMV-Flox-Stop-Socs3 mice. The control cohort contains three different genotypes: +/+;+/+ mice, +/+; Tg.CMV-Flox-Stop-Socs3/+ mice, and Tg.Agrp-Cre/+;+/+ mice. No phenotypic differences were seen between different control groups so they were pooled for analysis. Mutant mice are doubly heterozygous for the Socs3 allele and the Agrp-Cre, and are designated AgRP-SOCS3-OE. For all experiments age-matched males were used with the exception of Figs. 3, 5, and 6, and Fig. S6–S8, where both males and females were used. Mice were housed in barrier facility with a 0700 hour to 1900 hour light-dark cycle. Mice were fed standard mouse chow (21.6% kcal from fat; Purina mouse diet #5058), high-fat diet (60% kcal from fat, Research Diet D12492), or a matching low-fat diet (10% kcal from fat, Research Diet D12450B). All experiments were carried out under a protocol approved by the University of California at San Francisco Institutional Animal Care and Use Committee.

Immunohistochemistry.

After perfusing mice with 4% (wt/vol) PFA, brains were removed and postfixed in 4% (wt/vol) PFA, and then transferred to 30% (wt/vol) sucrose in PBS overnight at 4 °C. Brains were sectioned using a cryostat. For staining of ACTH, albumin and double staining for ACTH and pSTAT3, coronal brain sections (10 µm) were boiled in a 10 mM citrate solution. For SOCS3, coronal sections (10 µm) were incubated sequentially for 10 min each in 0.3% (wt/vol) glycine and 0.3% (wt/vol) SDS. Sections were incubated with primary antibody against pSTAT3 (1:200; Cell Signaling), ACTH (1:400; National Hormone & Peptide Program), or albumin (1:250; Thermo Scientific Pierce) overnight at 4 °C or against SOCS3 (1:120; Novus Biologicals) for 36 h at 4 °C, washed, and incubated for 1 h at room temperature with a secondary goat anti-rabbit or goat anti-guinea pig IgG antibody (1:200; Invitrogen). Fluorescence images were captured using a Zeiss Axioscope2 imaging system equipped with an AxioCam digital camera or an Olympus BX51WI microscope equipped with a QImaging Retiga 2000R digital camera. Images of the Npy-GFP and the Evans blue signal were taken before staining the sections for ACTH and pSTAT3. Double-immunofluorescence analyses of SOCS3 and ACTH and of SOCS3 and albumin were done by applying the two different antibodies sequentially. Pictures taken before and after immunohistochemistry were merged in Adobe Photoshop software using anatomical landmarks.

Measurement of Intensity of SOCS3 Immunoreactivity.

All sections being compared were processed in the same immunohistochemical experiment. Fluorescent images were captured with identical exposure time without saturation of pixel intensities. By using ImageJ software, Npy-GFP signal in the arcuate nucleus was used to determine the outline of AgRP cell bodies, and ACTH signal was used to determine the outline of POMC cell bodies. To measure SOCS3 protein intensity, background was determined based on average intensity value in several areas without any detectable SOCS3 immunoreactivity. SOCS3 intensity in the AgRP and POMC neurons were determined as signals over the background intensity.

Assessment of Permeability of BBB with Evans Blue.

For studies of the BBB, mice were anesthetized and injected with 1% (wt/vol) Evans blue (Sigma) in 50 µL saline transcardially. Mice were subsequently perfused with 4% (wt/vol) PFA 10 min later. Direct fluorescence emitted by Evans blue was captured from brain explants or 10-µm cryo-sections before being washed and processed for immunohistochemical studies. Pictures taken before and after immunohistochemistry were merged with Adobe Photoshop software using anatomical landmarks.

Cell Counting.

pStat3-, Evans blue-, or SOCS3-positive cells were counted from matched sections ranging from bregma −1.22 to −2.30 (Paxinos Mouse Brain Atlas; ref. 58). To determine the number of pSTAT3-positive cells in the ARC from mice given a high- or low-fat diet, three different methods were used to analyze the data. In the first analysis, intensity of pSTAT3 staining in the cell nucleus was measured and normalized to background intensity using ImageJ software. Cells with an intensity of more than 1.3× above background were considered positive as this threshold defines cells that are clearly pSTAT3-positive by visual inspection. This analysis method was used to generate the graphs in Fig. 1. In the second analysis, cells that visually had a stronger signal than the basal pSTAT3 was considered positive. This analysis method was used to generate the graph in Fig. S2A. In the third analysis, all cells with a visible nuclear staining were counted as positive. Data from this analysis are presented in Fig. S2B. The percentage of the AgRP and POMC neurons that were positive for pSTAT3 was determined. Only cell nuclei that were clearly visible using DAPI and located in the center of the cells were considered in this analysis.

Body Composition Analysis and Metabolic Studies.

Lean and fat mass were determined by dual energy X-ray absorptiometry (DEXA) using a PIXImus II (Lunar Corp.). The DEXA apparatus was calibrated with a phantom mouse with manufacturer-set value of bone density and fat mass. Fat-pad weights were determined by postmortem dissection. To measure oxygen consumption (VO2), food intake, and locomotor activity, mice were analyzed using CLAMS (Columbus Instruments). Mice were allowed 24 h to acclimatize before taking measurements. VO2 is normalized to lean body mass as determined by DEXA analysis.

Measurement of Leptin’s Effects on Food Intake.

To determine the anorectic effect of leptin, mice were singly housed and intraperitoneally injected with saline twice daily (1100 hours and 1900 hours) for approximately 2 wk. Twenty-four–hour food intake was measured at 1100 hours each day. The mice were given 2 d to acclimatize to the procedure. An average 24-h food intake was determined for all of the days of saline injection. Mice were then intraperitoneally injected with 2.5 mg/kg leptin (National Hormone Peptide Program) at 1100 hours and at 1900 hours. Twenty-four–hour food intake was measured at 1100 hours the day after leptin injection and compared with the average 24-h food intake when saline was injected.

Biochemical Assays.

Blood was collected via mandibular vein puncture under either fed (leptin) or 6-h fasted (insulin) conditions. Hormones were measured with a leptin ELISA kit (Crystal Chem) and an insulin ELISA kit (Alpco). For glucose-tolerance tests, mice were fasted for 6 h and injected intraperitoneally with glucose (2.5 g/kg). Blood glucose was measured with a glucometer (Freestyle; Abbott Diabetes Care) at indicated times. For insulin tolerance tests, mice were fasted overnight, and intraperitoneally injected with insulin (1 U/kg, Novolin R). Blood glucose was measured as above. Liver triglycerides were extracted using the Folch method (59) and quantified by colorimetric assay.

Statistics.

Mean values in the text and figures are expressed as mean ± SEM. Groups being compared and the types of statistical analyses are described in each figure legend. Briefly, two-tailed Student t tests were used to compare difference between two independent groups. In cases where multiple independent groups were compared, one-way or two-way ANOVA followed by post hoc test were performed, using the GraphPad Prism analytical software. In cases where the same animals were analyzed over time or by different treatment, repeated-measures ANOVA was used.

Supplementary Material

Supporting Information
supp_110_8_E697__index.html (7KB, html)

Acknowledgments

We thank Dr. Richard Daneman for providing the albumin antibody. This work was supported in part by research Grant R01DK080427 from the National Institute of Diabetes and Digestive and Kidney Diseases (to A.W.X.); by postdoctoral fellowships from the Henning and Johan Throne-Holst's foundation, and the Swedish Research Council (to L.E.O.); by research Grant 1K08DK076721 from the National Institute of Diabetes and Digestive and Kidney Diseases, and the Lawson Wilkins Pediatric Endocrine Society (to C.C.C.); and by core facilities funded by National Institutes of Health Diabetes Endocrinology Research Centers Grant P30 DK063720.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218284110/-/DCSupplemental.

References

  • 1.Münzberg H, Björnholm M, Bates SH, Myers MG., Jr Leptin receptor action and mechanisms of leptin resistance. Cell Mol Life Sci. 2005;62(6):642–652. doi: 10.1007/s00018-004-4432-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Enriori PJ, et al. Diet-induced obesity causes severe but reversible leptin resistance in arcuate melanocortin neurons. Cell Metab. 2007;5(3):181–194. doi: 10.1016/j.cmet.2007.02.004. [DOI] [PubMed] [Google Scholar]
  • 3.Mori H, et al. Socs3 deficiency in the brain elevates leptin sensitivity and confers resistance to diet-induced obesity. Nat Med. 2004;10(7):739–743. doi: 10.1038/nm1071. [DOI] [PubMed] [Google Scholar]
  • 4.Howard JK, et al. Enhanced leptin sensitivity and attenuation of diet-induced obesity in mice with haploinsufficiency of Socs3. Nat Med. 2004;10(7):734–738. doi: 10.1038/nm1072. [DOI] [PubMed] [Google Scholar]
  • 5.Kievit P, 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(2):123–132. doi: 10.1016/j.cmet.2006.06.010. [DOI] [PubMed] [Google Scholar]
  • 6.Reed AS, et al. Functional role of suppressor of cytokine signaling 3 upregulation in hypothalamic leptin resistance and long-term energy homeostasis. Diabetes. 2010;59(4):894–906. doi: 10.2337/db09-1024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Zhang X, et al. Hypothalamic IKKbeta/NF-kappaB and ER stress link overnutrition to energy imbalance and obesity. Cell. 2008;135(1):61–73. doi: 10.1016/j.cell.2008.07.043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Morton GJ, Cummings DE, Baskin DG, Barsh GS, Schwartz MW. Central nervous system control of food intake and body weight. Nature. 2006;443(7109):289–295. doi: 10.1038/nature05026. [DOI] [PubMed] [Google Scholar]
  • 9.Myers MG, Jr, Leibel RL, Seeley RJ, Schwartz MW. Obesity and leptin resistance: Distinguishing cause from effect. Trends Endocrinol Metab. 2010;21(11):643–651. doi: 10.1016/j.tem.2010.08.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Leshan RL, Björnholm M, Münzberg H, Myers MG., Jr Leptin receptor signaling and action in the central nervous system. Obesity (Silver Spring) 2006;14(Suppl 5):208S–212S. doi: 10.1038/oby.2006.310. [DOI] [PubMed] [Google Scholar]
  • 11.Scott MM, et al. Leptin targets in the mouse brain. J Comp Neurol. 2009;514(5):518–532. doi: 10.1002/cne.22025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Ziotopoulou M, Mantzoros CS, Hileman SM, Flier JS. Differential expression of hypothalamic neuropeptides in the early phase of diet-induced obesity in mice. Am J Physiol Endocrinol Metab. 2000;279(4):E838–E845. doi: 10.1152/ajpendo.2000.279.4.E838. [DOI] [PubMed] [Google Scholar]
  • 13.Bachman ES, et al. betaAR signaling required for diet-induced thermogenesis and obesity resistance. Science. 2002;297(5582):843–845. doi: 10.1126/science.1073160. [DOI] [PubMed] [Google Scholar]
  • 14.Kim KW, et al. Steroidogenic factor 1 directs programs regulating diet-induced thermogenesis and leptin action in the ventral medial hypothalamic nucleus. Proc Natl Acad Sci USA. 2011;108(26):10673–10678. doi: 10.1073/pnas.1102364108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Tong Q, Ye CP, Jones JE, Elmquist JK, Lowell BB. Synaptic release of GABA by AgRP neurons is required for normal regulation of energy balance. Nat Neurosci. 2008;11(9):998–1000. doi: 10.1038/nn.2167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Ono H, et al. Activation of hypothalamic S6 kinase mediates diet-induced hepatic insulin resistance in rats. J Clin Invest. 2008;118(8):2959–2968. doi: 10.1172/JCI34277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Samuel VT, et al. Mechanism of hepatic insulin resistance in non-alcoholic fatty liver disease. J Biol Chem. 2004;279(31):32345–32353. doi: 10.1074/jbc.M313478200. [DOI] [PubMed] [Google Scholar]
  • 18.Broberger C, Johansen J, Johansson C, Schalling M, Hökfelt T. The neuropeptide Y/agouti gene-related protein (AGRP) brain circuitry in normal, anorectic, and monosodium glutamate-treated mice. Proc Natl Acad Sci USA. 1998;95(25):15043–15048. doi: 10.1073/pnas.95.25.15043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.El-Haschimi K, Pierroz DD, Hileman SM, Bjørbaek C, Flier JS. Two defects contribute to hypothalamic leptin resistance in mice with diet-induced obesity. J Clin Invest. 2000;105(12):1827–1832. doi: 10.1172/JCI9842. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Münzberg H, Flier JS, Bjørbaek C. Region-specific leptin resistance within the hypothalamus of diet-induced obese mice. Endocrinology. 2004;145(11):4880–4889. doi: 10.1210/en.2004-0726. [DOI] [PubMed] [Google Scholar]
  • 21.Bjorbak C, et al. SOCS3 mediates feedback inhibition of the leptin receptor via Tyr985. J Biol Chem. 2000;275(51):40649–40657. doi: 10.1074/jbc.M007577200. [DOI] [PubMed] [Google Scholar]
  • 22.Thaler JP, et al. Obesity is associated with hypothalamic injury in rodents and humans. J Clin Invest. 2012;122(1):153–162. doi: 10.1172/JCI59660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Quan W, et al. Role of hypothalamic proopiomelanocortin neuron autophagy in the control of appetite and leptin response. Endocrinology. 2012;153(4):1817–1826. doi: 10.1210/en.2011-1882. [DOI] [PubMed] [Google Scholar]
  • 24.Xu AW, et al. PI3K integrates the action of insulin and leptin on hypothalamic neurons. J Clin Invest. 2005;115(4):951–958. doi: 10.1172/JCI24301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.van de Wall E, et al. Collective and individual functions of leptin receptor modulated neurons controlling metabolism and ingestion. Endocrinology. 2008;149(4):1773–1785. doi: 10.1210/en.2007-1132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Gropp E, et al. Agouti-related peptide-expressing neurons are mandatory for feeding. Nat Neurosci. 2005;8(10):1289–1291. doi: 10.1038/nn1548. [DOI] [PubMed] [Google Scholar]
  • 27.Xu AW, et al. Effects of hypothalamic neurodegeneration on energy balance. PLoS Biol. 2005;3(12):e415. doi: 10.1371/journal.pbio.0030415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Kitamura T, et al. Forkhead protein FoxO1 mediates Agrp-dependent effects of leptin on food intake. Nat Med. 2006;12(5):534–540. doi: 10.1038/nm1392. [DOI] [PubMed] [Google Scholar]
  • 29.Claret M, et al. AMPK is essential for energy homeostasis regulation and glucose sensing by POMC and AgRP neurons. J Clin Invest. 2007;117(8):2325–2336. doi: 10.1172/JCI31516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Könner AC, et al. Insulin action in AgRP-expressing neurons is required for suppression of hepatic glucose production. Cell Metab. 2007;5(6):438–449. doi: 10.1016/j.cmet.2007.05.004. [DOI] [PubMed] [Google Scholar]
  • 31.Al-Qassab H, et al. Dominant role of the p110beta isoform of PI3K over p110alpha in energy homeostasis regulation by POMC and AgRP neurons. Cell Metab. 2009;10(5):343–354. doi: 10.1016/j.cmet.2009.09.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Diano S, et al. Peroxisome proliferation-associated control of reactive oxygen species sets melanocortin tone and feeding in diet-induced obesity. Nat Med. 2011;17(9):1121–1127. doi: 10.1038/nm.2421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.van den Top M, Lee K, Whyment AD, Blanks AM, Spanswick D. Orexigen-sensitive NPY/AgRP pacemaker neurons in the hypothalamic arcuate nucleus. Nat Neurosci. 2004;7(5):493–494. doi: 10.1038/nn1226. [DOI] [PubMed] [Google Scholar]
  • 34.Villanueva EC, et al. Complex regulation of mammalian target of rapamycin complex 1 in the basomedial hypothalamus by leptin and nutritional status. Endocrinology. 2009;150(10):4541–4551. doi: 10.1210/en.2009-0642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Wang J, et al. Overfeeding rapidly induces leptin and insulin resistance. Diabetes. 2001;50(12):2786–2791. doi: 10.2337/diabetes.50.12.2786. [DOI] [PubMed] [Google Scholar]
  • 36.Morita S, Miyata S. Different vascular permeability between the sensory and secretory circumventricular organs of adult mouse brain. Cell Tissue Res. 2012;349(2):589–603. doi: 10.1007/s00441-012-1421-9. [DOI] [PubMed] [Google Scholar]
  • 37.Mullier A, Bouret SG, Prevot V, Dehouck B. Differential distribution of tight junction proteins suggests a role for tanycytes in blood-hypothalamus barrier regulation in the adult mouse brain. J Comp Neurol. 2010;518(7):943–962. doi: 10.1002/cne.22273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Ciofi P, et al. Brain-endocrine interactions: A microvascular route in the mediobasal hypothalamus. Endocrinology. 2009;150(12):5509–5519. doi: 10.1210/en.2009-0584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Faouzi M, et al. Differential accessibility of circulating leptin to individual hypothalamic sites. Endocrinology. 2007;148(11):5414–5423. doi: 10.1210/en.2007-0655. [DOI] [PubMed] [Google Scholar]
  • 40.van Vliet EA, et al. Blood-brain barrier leakage may lead to progression of temporal lobe epilepsy. Brain. 2007;130(Pt 2):521–534. doi: 10.1093/brain/awl318. [DOI] [PubMed] [Google Scholar]
  • 41.Chin-Chance C, Polonsky KS, Schoeller DA. Twenty-four-hour leptin levels respond to cumulative short-term energy imbalance and predict subsequent intake. J Clin Endocrinol Metab. 2000;85(8):2685–2691. doi: 10.1210/jcem.85.8.6755. [DOI] [PubMed] [Google Scholar]
  • 42.Schoeller DA, Cella LK, Sinha MK, Caro JF. Entrainment of the diurnal rhythm of plasma leptin to meal timing. J Clin Invest. 1997;100(7):1882–1887. doi: 10.1172/JCI119717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Harris RB, Ramsay TG, Smith SR, Bruch RC. Early and late stimulation of ob mRNA expression in meal-fed and overfed rats. J Clin Invest. 1996;97(9):2020–2026. doi: 10.1172/JCI118637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Palou M, et al. Induction of NPY/AgRP orexigenic peptide expression in rat hypothalamus is an early event in fasting: Relationship with circulating leptin, insulin and glucose. Cell Physiol Biochem. 2009;23(1-3):115–124. doi: 10.1159/000204100. [DOI] [PubMed] [Google Scholar]
  • 45.Sánchez J, Oliver P, Picó C, Palou A. Diurnal rhythms of leptin and ghrelin in the systemic circulation and in the gastric mucosa are related to food intake in rats. Pflugers Arch. 2004;448(5):500–506. doi: 10.1007/s00424-004-1283-4. [DOI] [PubMed] [Google Scholar]
  • 46.Ahima RS, Prabakaran D, Flier JS. Postnatal leptin surge and regulation of circadian rhythm of leptin by feeding. Implications for energy homeostasis and neuroendocrine function. J Clin Invest. 1998;101(5):1020–1027. doi: 10.1172/JCI1176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Leshan RL, Greenwald-Yarnell M, Patterson CM, Gonzalez IE, Myers MG., Jr Leptin action through hypothalamic nitric oxide synthase-1-expressing neurons controls energy balance. Nat Med. 2012;18(5):820–823. doi: 10.1038/nm.2724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Banks WA, Kastin AJ, Huang W, Jaspan JB, Maness LM. Leptin enters the brain by a saturable system independent of insulin. Peptides. 1996;17(2):305–311. doi: 10.1016/0196-9781(96)00025-3. [DOI] [PubMed] [Google Scholar]
  • 49.Burguera B, et al. Obesity is associated with a decreased leptin transport across the blood-brain barrier in rats. Diabetes. 2000;49(7):1219–1223. doi: 10.2337/diabetes.49.7.1219. [DOI] [PubMed] [Google Scholar]
  • 50.Banks WA, DiPalma CR, Farrell CL. Impaired transport of leptin across the blood-brain barrier in obesity. Peptides. 1999;20(11):1341–1345. doi: 10.1016/s0196-9781(99)00139-4. [DOI] [PubMed] [Google Scholar]
  • 51.Elias CF, et al. Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area. Neuron. 1999;23(4):775–786. doi: 10.1016/s0896-6273(01)80035-0. [DOI] [PubMed] [Google Scholar]
  • 52.Wilson BD, et al. Physiological and anatomical circuitry between Agouti-related protein and leptin signaling. Endocrinology. 1999;140(5):2387–2397. doi: 10.1210/endo.140.5.6728. [DOI] [PubMed] [Google Scholar]
  • 53.Cowley MA, et al. Integration of NPY, AGRP, and melanocortin signals in the hypothalamic paraventricular nucleus: Evidence of a cellular basis for the adipostat. Neuron. 1999;24(1):155–163. doi: 10.1016/s0896-6273(00)80829-6. [DOI] [PubMed] [Google Scholar]
  • 54.Cowley MA, et al. Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus. Nature. 2001;411(6836):480–484. doi: 10.1038/35078085. [DOI] [PubMed] [Google Scholar]
  • 55.Wu Q, Boyle MP, Palmiter RD. Loss of GABAergic signaling by AgRP neurons to the parabrachial nucleus leads to starvation. Cell. 2009;137(7):1225–1234. doi: 10.1016/j.cell.2009.04.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Fukuda M, Williams KW, Gautron L, Elmquist JK. Induction of leptin resistance by activation of cAMP-Epac signaling. Cell Metab. 2011;13(3):331–339. doi: 10.1016/j.cmet.2011.01.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Baskin DG, Breininger JF, Schwartz MW. SOCS-3 expression in leptin-sensitive neurons of the hypothalamus of fed and fasted rats. Regul Pept. 2000;92(1-3):9–15. doi: 10.1016/s0167-0115(00)00143-9. [DOI] [PubMed] [Google Scholar]
  • 58.Paxinos G, Franklin KBJ. The Mouse Brain in Stereotaxic Coordinates. 2004 (Elsevier, Academic Press, San Diego, CA), 2nd Ed. [Google Scholar]
  • 59.Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957;226(1):497–509. [PubMed] [Google Scholar]

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