Fourth-ventricle leptin infusions dose-dependently activate hypothalamic signal transducer and activator of transcription 3

Ruth B S Harris; Bhavna N Desai

doi:10.1152/ajpendo.00343.2016

. 2016 Nov 1;311(6):E939–E948. doi: 10.1152/ajpendo.00343.2016

Fourth-ventricle leptin infusions dose-dependently activate hypothalamic signal transducer and activator of transcription 3

Ruth B S Harris ^1,^✉, Bhavna N Desai ¹

PMCID: PMC5183885 PMID: 27802966

Abstract

Previous studies have shown that very low-dose infusions of leptin into the third or the fourth ventricle alone have little effect on energy balance, but simultaneous low-dose infusions cause rapid weight loss and increased phosphorylation of STAT3 (p-STAT3) in hypothalamic sites that express leptin receptors. Other studies show that injecting high doses of leptin into the fourth ventricle inhibits food intake and weight gain. Therefore, we tested whether fourth-ventricle leptin infusions that cause weight loss are associated with increased leptin signaling in the hypothalamus. In a dose response study 14-day infusions of increasing doses of leptin showed significant hypophagia, weight loss, and increased hypothalamic p-STAT3 in rats receiving at least 0.9 μg leptin/day. In a second study 0.6 μg leptin/day transiently inhibited food intake and reduced carcass fat, but had no significant effect on energy expenditure. In a final study, we identified the localization of STAT3 activation in the hypothalamus of rats receiving 0, 0.3, or 1.2 μg leptin/day. The high dose of leptin, which caused weight loss in the first experiment, increased p-STAT3 in the ventromedial, dorsomedial, and arcuate nuclei of the hypothalamus. The low dose that increased brown fat UCP1 but did not affect body composition in the first experiment had little effect on hypothalamic p-STAT3. We propose that hindbrain leptin increases the precision of control of energy balance by lowering the threshold for leptin signaling in the forebrain. Further studies are needed to directly test this hypothesis.

Keywords: rats, food intake, energy expenditure, body composition

previously, we have reported that rats receiving low-dose infusions of leptin into either the third (0.1 μg/day) or the fourth (0.6 μg/day) ventricle do not lose weight, but when these two infusions are applied simultaneously the rats show a substantial reduction in food intake and lose both lean and fat tissue (5). The weight loss is associated with a significant increase in phosphorylation of signal transducer and activator of transcription 3 (p-STAT3) in specific hypothalamic nuclei, each of which is known to express leptin receptors (6). Because this hypothalamic activation occurs only when there are simultaneous infusions into the third and fourth ventricles, we have proposed that stimulation of leptin receptors in the hindbrain lowers the threshold for activation of hypothalamic leptin receptors (6). In this scenario, if leptin is present in the hindbrain, then forebrain levels of leptin that would normally be subthreshold would activate hypothalamic leptin signaling pathways and modify energy balance. Thus, there is a potential to enhance the precision of control of energy balance because, in a state of positive energy balance, a rise in leptin concentrations reaching the hindbrain would facilitate inhibition of food intake even if leptin concentrations in forebrain remained relatively low.

In contrast to the effects of low-dose infusions of leptin, we and others have shown that infusion or injection of much higher doses of leptin into either the third (1.5 μg) or the fourth (10 μg) ventricle alone can suppress food intake and cause weight loss (23, 28). In these studies it was not determined whether there was an increase in p-STAT3 in brain areas distant from the site of administration. One exception to this was a report by Ruiter et al. (25), who found that 3 μg of leptin injected into the fourth ventricle or 50 ng injected directly into the NTS not only inhibited 24-h food intake and caused weight loss but also increased arcuate and ventromedial hypothalamic p-STAT3 expression 90 min after injection. The experiments described here infused leptin into the fourth ventricle to determine whether chronic elevation of leptin in the hindbrain produced a similar but sustained activation of hypothalamic STAT3 and to test whether inhibition of food intake by leptin delivered to the hindbrain occurred only when STAT3 was activated in both the forebrain and hindbrain.

METHODS

All animal procedures were approved by the Institutional Animal Care and Use Committee at Augusta University, and animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (3). The rats used in these studies were male Sprague-Dawley rats (Envigo, Prattville, AL) weighing 330–350 g at the start of a study. They were individually housed in wire mesh cages, except for those that were placed in calorimetry cages. They had continuous free access to chow and water and a Nylabone for enrichment, and the room was maintained at 70–72°C and ~50% humidity, with the lights on for 12 h/day from 7 AM. Rats were weighed daily, and in most instances, daily food intake, corrected for spillage, was recorded throughout the experiment.

Experiment 1: fourth-ventricle leptin dose response.

Sixty-four rats were fitted with fourth-ventricle infusion cannula, as described previously (20). Daily body weights and food intakes were recorded for 1 wk, and then the rats were divided into seven weight-matched groups. The rats were anesthetized with isoflurane, and miniosmotic pumps were attached to their infusion cannulas. The pumps delivered 0, 0.1, 0.3, 0.6, 0.9, 1.2, or 2.0 μg leptin/day (Rat Recombinant Leptin; R & D Systems, Minneapolis, MN) in a volume of 0.25 μl/h (model 1002; Durect Corporation, Cupertino, CA). At the same time as the pump was attached, an iButton (iButtonLink, Whitewater, WI) was placed on the surface of intrascapular brown adipose tissue (iBAT). The iButtons were programmed to record temperature every 30 min with a precision of 0.06°C until the end of the experiment. Daily measurements of food intake and body weight continued for 13 days.

On day 13, food was removed from the cages at 7 AM. Starting at 10 AM, rats were decapitated, and blood was collected for measurement of serum leptin (Rat Leptin RIA kit; Millipore), insulin (Rat Insulin RIA kit; Millipore), and glucose (EasyGluco Plus blood glucose monitoring system; US Diagnostics, New York, NY). The brain was rapidly removed, and tissue blocks that included the hypothalamus or the NTS were dissected as described previously (16). They were snap-frozen and stored at −80°C until protein was extracted for Western blot detection of p-STAT3, suppressor of cytokine signaling 3 (SOCS3), protein-tyrosine phosphatase 1B (PTP1B), phosphorylation of phosphoinositide 3-kinase (p-PI3K), and p-ERK1/2, as described previously (32). Inguinal, epididymal, retroperitoneal, and mesenteric fat depots, intrascapular brown fat (iBAT), and liver were dissected and weighed. iBAT uncoupling protein 1 (UCP1) was measured by Western blot, as described previously (5). The remaining tissues were returned to the carcass, the gastrointestinal tract was removed, and carcass composition was measured as described previously (10).

Experiment 2: energy expenditure of rats receiving fourth-ventricle leptin infusions.

The objective of this experiment was to determine whether increased energy expenditure contributed to weight loss in rats receiving fourth-ventricle infusions of leptin. In this study, we tested rats infused with 0.6 μg leptin/day. This dose of leptin did not cause a significant inhibition of food intake during the first 10 days of infusion but inhibited weight gain and caused a loss of body fat with retention of lean body mass in experiment 1, suggesting an effect of leptin on energy expenditure. A total of 18 rats in two cohorts with the treatments equally represented in each cohort were fitted with fourth-ventricle, cannula and baseline measurements of food intake and body weight were recorded for 5 days. The rats were divided into two weight-matched groups, and miniosmotic pumps were attached to the cannulas to infuse either 0 or 0.6 μg leptin/day. On days 1–3 and 9–11 of infusion, the rats were housed in the calorimeter (TSE LabMaster, Metabolic Research Platform; TSE Systems International, Chesterfield, MO). Oxygen consumption, carbon dioxide production, and activity were sampled for 3 min from each cage every 39 min. Values measured during the last of the 3 min were used to calculate energy expenditure expressed both as kcal·h⁻¹·rat⁻¹ and per unit metabolic body weight (kcal·hr⁻¹·weight ^0.75) and respiratory exchange ratio (RER) as an index of macronutrient oxidation. Total activity was measured by Inframot, food intake was recorded every 39 min, and body weight was recorded manually at 7:30 AM each morning when food hoppers and water bottles were refilled and cage bedding changed as necessary. Daily calorimetry measures were initiated at 8 AM and stopped at 7:20 AM the next day so that only one cycle of measurement was lost each day. Only the data from days 3 and 11 were used to compare treatment groups. On day 14, the rats were food deprived from 7 to 10:30 AM. Trunk blood was collected for measurement of serum leptin, insulin, and glucose, and tissue blocks were collected for measurement of hypothalamic and brain stem p-STAT3, SOCS3, and p-ERK1/2.

Experiment 3: hypothalamic and hindbrain p-STAT3 and ERK1/2 in rats receiving fourth-ventricle leptin infusions.

Rats receiving the three highest doses of leptin in experiment 1 lost weight. Therefore, the objective of this experiment was to determine whether a dose of leptin that caused weight loss also caused site-specific increases in hypothalamic p-STAT3. Twenty-one rats were fitted with fourth-ventricle cannulas. Baseline measurements of food intake and body weight were made for 5 days. Miniosmotic pumps were attached to the cannulas to deliver 0, 0.3, or 1.2 μg leptin/24 h. These doses were chosen because the lower dose of leptin did not produce significant inhibition of food intake or loss of body weight or body fat in experiment 1, whereas the higher dose caused loss of weight, fat, and lean tissue. On day 7 of infusion, the rats were food deprived for 5 h before they were anesthetized with ketamine-xylazine (90:10 mg/kg) and perfused pericardially with ice-cold 0.9% saline containing 100 μU/ml heparin, followed by 4% paraformaldehyde. The brains were collected, immersed in paraformaldehyde, and held at 4°C overnight. They were then transferred to 30% sucrose and 0.1% sodium azide and stored at 4°C. Thirty-micrometer sections were made through the hindbrain and hypothalamus. Every fourth section was used for immunohistochemical detection of p-STAT3, as described previously (9). p-STAT3 was reliably detected and quantified by manually counting stained nuclei. Using designations from Paxinos and Watson (22), p-STAT3 was quantified in the medial (mArc) and lateral arcuate nucleus (lARC), compact (DMC) and dorsal dorsomedial hypothalamus (DMD), ventromedial hypothalamus (VMH), and posterior hypothalamic area (PH) in sections that extended from bregma (−3.14 to −3.60 in the rostral arcuate nucleus at bregma −4.70) and medial nucleus of the solitary tract (mNTS; bregma −13.68 to −14.30). In addition, ERK1/2 was detected in hindbrain sections using methods described by Sutton et al. (29) and was quantified in mNTS, dorsal motor nucleus of the vagus, and hypoglossal nucleus in sections representing bregma (−13.68 to −14.30) (22).

Statistical analysis.

Daily food intake, body weight, iBAT temperature, and calorimetry measurements were analyzed by repeated-measures analysis of variance. Comparisons at individual time points were performed by one-way analysis of variance and post hoc Tukey’s test for experiments 1 and 3 or by unpaired t-test for experiment 2. Statistical comparisons were made using Statistica Version 9.0 (StatSoft, Tulsa, OK), and differences with a probability value of P < 0.05 were considered significant.

RESULTS

Experiment 1: fourth-ventricle leptin dose response.

There was a dose-dependent inhibition of food intake in rats infused with increasing doses of leptin into the fourth ventricle (leptin: P < 0.0001; time: P < 0.0001; interaction: P < 0.0001; Fig. 1A). From day 4 until the end of the experiment, rats receiving 0.9, 1.2, or 2.0 μg leptin/day ate less than those receiving 0, 0.1, or 0.3 μg leptin/day. By the end of the experiment the intake of these groups started to increase, even though it remained significantly lower than that of the lower-dose rats. The intake of rats receiving 0.6 μg leptin/day was not different from any other group until day 10, when it became significantly lower than that of rats infused with 0 or 0.1 μg leptin/day. Total food intake during the experimental period (Fig. 1C) was significantly lower in rats receiving the three highest doses of leptin than in those receiving 0, 0.1 or 0.3 μg leptin/day. There was a dose-dependent weight loss in rats receiving leptin infusions (leptin: P < 0.0006; time: P < 0.0001; interaction: P < 0.0001 Figs. 1B and 2). From day 6, the rats receiving the three highest doses of leptin (0.9, 1.2, or 2.0 μg leptin/day) weighed significantly less than those receiving the lower doses (0, 0.1, 0.3, or 0.6 μg leptin/day). Weight change during the experimental period was dose dependent (Fig. 1D). Rats receiving 0, 0.1, or 0.3 μg leptin/day gained weight, whereas those receiving 0.6 μg leptin/day stopped gaining weight, and the rats receiving the three highest doses of leptin lost weight. Weight loss did not increase once the leptin dose exceeded 0.9 μg/day.

Fig. 1. — A: daily food intakes of rats receiving 4th-ventricle infusions of increasing doses of leptin in *experiment 1*. Data are means ± SE for groups of 7–10 rats. *Rats infused with 0, 0.1, or 0.3 μg leptin/day ate significantly more than rats infused with 0.9, 1.2, or 2.0 μg leptin/day; #rats infused with 0.6 μg leptin/day ate significantly more than those infused with 1.2 or 2.0 μg leptin/day; ^Ψrats infused with 0.6, 0.9, 1.2, or 2.0 μg leptin/day ate significantly less than those infused with 0 or 0.1 μg leptin/day; ^δrats infused with 2.0 μg leptin/day ate less than controls. B: daily body weight of rats. *Rats infused with 0, 0.1, or 0.3 μg leptin/day were significantly heavier than rats infused with 0.9, 1.2, or 2.0 μg leptin/day. C: total food intake of the rats during the experimental period. Means that do not share a common letter are significantly different. D: weight change of the rats during the experimental period. Means that do not share a common letter are significantly different.

Fig. 2. — A: uncoupling protein 1 (UCP1) protein expression in intrascapular brown adipose tissue (iBAT) from rats receiving 4th infusions of increasing doses of leptin for 12 days in *experiment 1*. B: hypothalamic phosphorylated STAT3 (p-STAT3) protein expression. C: hypothalamic suppressor of cytokine signaling 3 (SOCS3) protein expression. Data are means + SE for 7–10 rats. Values that do not share a common letter are significantly different.

The daily nadir in iBAT temperature averaged over the 4 h between 10:30 AM and 2:30 PM and the daily peak averaged between 10:30 PM and 2:30 AM was determined for the last 9 days of infusion for each rat. There were no differences between treatment groups for temperature at either time of day (data not shown). iBAT UCP1, measured by Western blot, showed a dose-dependent increase in expression (P < 0.01) that was significant for rats infused with 0.3 μg leptin/day or more compared with controls (Fig. 2A).

At the end of the experiment, the carcass weights of rats infused with the two highest doses of leptin were significantly lower than those of rats infused with 0, 0.1, 0.3, or 0.6 μg leptin/day (Table 1). Carcass analysis indicated that carcass fat was significantly reduced in rats that received >0.3 μg leptin/day, with a maximal effect achieved with 0.9 μg leptin/day. There were site-specific differences in leptin sensitivity of fat depots. Mesenteric was the most sensitive and was 50% smaller in rats infused with 0.3 μg leptin/day than controls. Epididymal was the least sensitive and was reduced only in rats receiving the three highest doses of leptin. Leptin caused a significant reduction in lean body mass of rats receiving the three highest doses of leptin, which were also the doses that significantly inhibited food intake.

Table 1.

Body composition and serum measurements in rats from experiment 1

	μg/Day
	0	0.1	0.3	0.6	0.9	1.2	2.0
Body composition
Carcass weight, g	344 ± 6^a	340 ± 3^a	332 ± 6^a,c	324 ± 4^a,c	302 ± 11^b,c	294 ± 8^b	295 ± 8^b
Fat, g	23 ± 1^a	21 ± 1^a,c	17 ± 2^a,c	14 ± 2^b,c	8 ± 2^b	7 ± 1^b	9 ± 2^b
Lean mass, g	308 ± 4^a	307 ± 3^a	301 ± 5^a	297 ± 3^a,c	281 ± 10^b,c	274 ± 7^b,c	274 ± 7^b,c
Tissue weights
Liver, g	12.7 ± 0.4^a	12.9 ± 0.7^a	11.7 ± 0.4^a,c	11.00 ± 0.5^a	10.3 ± 0.8^b,c	8.6 ± 0.8^b	8.6 ± 0.6^b
Liver lipid, g	0.41 ± 0.01^a,d	0.42 ± 0.02^a,d	0.39 ± 0.01^a,c	0.36 ± 0.02^a,d	0.32 ± 0.03^a,d	0.30 ± 0.03^b,c,d	0.28 ± 0.02^b,d
Inguinal fat, g	5.8 ± 0.3^a	5.5 ± 0.3^a	4.6 ± 0.2^a,c	3.4 ± 0.4^b,c	3.1 ± 0.7^b	1.7 ± 0.3^b	2.2 ± 0.5^b
Epididymal fat, g	3.9 ± 0.2^a	4.0 ± 0.3^a	3.7 ± 0.2^a	2.9 ± 0.4^a,c	2.4 ± 0.5^b,c	1.5 ± 0.4^b,c	1.7 ± 0.4^b,c
Retroperitoneal fat, g	1.8 ± 0.2^a	1.4 ± 0.2^a,d	1.1 ± 0.2^a,c	0.8 ± 0.2^c,d	0.6 ± 0.2^b,c	0.3 ± 0.1^b	0.4 ± 0.2^b
Mesenteric fat, g	1.6 ± 0.1^a	1.0 ± 0.1^a	0.8 ± 0.1^c	0.8 ± 0.2^b,c	0.8 ± 0.2^b,c	0.4 ± 0.1^b	0.6 ± 0.1^b,c
iBAT, mg	262 ± 22^a	308 ± 31^a	232 ± 10^a,b	203 ± 26^b	196 ± 14^b	168 ± 14^b	162 ± 24^b
Serum measurements
Glucose, mg/dl	143 ± 6^a	141 ± 9^a	133 ± 10^a,c	125 ± 0.7^a,b	113 ± 13^a,b	85 ± 15^b,c	73 ± 15^b
Insulin, ng/ml	0.77 ± 0.09	0.76 ± 0.05	0.48 ± 0.12	0.56 ± 0.10	0.57 ± 0.13	0.60 ± 0.14	0.51 ± 0.11
Leptin, ng/ml	3.5 ± 0.2^a	3.1 ± 0.2^a,b	2.6 ± 0.4^a,b	2.6 ± 0.7^a,b	1.9 ± 0.3^b	1.8 ± 0.3^b	2.0 ± 0.2^b

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Data are means ± SE for groups of 7–10 rats. iBAT, intrascapular brown adipose tissue. Values that do not share a common superscripted letter are significantly different. Rats were infused with increasing doses of leptin for 13 days before the end of the experiment. Blood and tissue were collected following 3 h of food deprivation during the light period.

Hypothalamic p-STAT3 was increased only in the rats receiving the two highest doses of leptin, whereas SOCS3 expression was inhibited in rats receiving ≥0.6 μg leptin/day (Fig. 2, B and C). There were no differences in p-ERK1, p-ERK2, PTP1B, or PI3K (data not shown). There were no treatment effects on expression of any of these proteins in the brainstem (data not shown).

Experiment 2.

Rats infused with 0.6 μg leptin/day did not gain any weight during the study (0 ± 4 g), whereas the saline-infused animals gained 15 ± 5 g during the experimental period [leptin: not significant (NS); time: P < 0.0001; interaction: P < 0.0003; Fig. 3A]. Leptin inhibited food intake on the first 3 days of infusion and on days 6 and 8 (leptin: P < 0.005; time: P < 0.0001; interaction: P < 0.0001; Fig. 3B). There was no evidence of overeating to compensate for the initial hypophagia, and total intake during the experimental period was significantly lower in the leptin-infused rats than in the controls (251 ± 9 vs. 285 ± 7 g/13 days, P < 0.003). Energy expenditure of the leptin-infused and control rats was the same both when food intake was suppressed in the leptin group (day 3; Fig. 3C) and when food intake was the same for the two groups (day 11; Fig. 2D). By contrast, RER was suppressed by leptin on day 3 (leptin: P < 0.02; time: P < 0.001; interaction: NS; Fig. 3E) but not on day 11 (Fig. 3F). The difference between the two groups was greatest during the last 2 h of the light period and the first 2 h of the dark period (5–9 PM). Activity was higher in leptin-infused than control rats (day 3: leptin, NS; time, P < 0.0001; interaction, P < 0.003; day 11: leptin, P < 0.04; time, P < 0.001; interaction, NS), but post hoc analysis showed few time points when this difference reached significance (Fig. 3, G and H).

Fig. 3. — Data from *experiment 2* in which rats received 4th-ventricle infusions of 0 or 0.6 μg leptin/day for 14 days. Rats were housed in the calorimeter from *days 1* to 3 and *days 9* to 11 of the experiment. Calorimetry data from *days 3* and 11 were used to compare groups. Data are means ± SE for groups of 9 rats. *Significant differences between treatment groups.

At the end of the experiment the leptin-infused rats had significantly less carcass fat than their controls, but there were no differences in lean tissue mass. The loss of fat was apparent in all fat depots weighed, except the epididymal fat (Table 2). There were no significant differences in serum glucose, insulin, or leptin at the end of the experiment (Table 2). UCP1 expression in iBAT, an index of thermogenesis, was not different between groups, and there were no differences in hypothalamic or brainstem p-STAT3, SOCS3, p-ERK1, or p-ERK2 expression measured by Western blot (data not shown).

Table 2.

Body composition and serum measurements from rats in experiment 2

	Controls	Leptin
Body composition
Carcass weight, g	332 ± 3	325 ± 5
Fat, g	24 ± 1	18 ± 2^*
Lean mass, g	294 ± 5	290 ± 4
Tissue weights
Liver, g	12.1 ± 0.9	11.9 ± 0.3
Inguinal fat, g	5.6 ± 0.3	4.3 ± 0.4^*
Epididymal fat, g	3.2 ± 0.2	2.7 ± 0.3
Retroperitoneal fat, g	1.4 ± 0.1	0.9 ± 0.2^*
Mesenteric fat, g	1.4 ± 0.2	0.8 ± 0.1^*
iBAT, mg	293 ± 32	261 ± 22^*
Serum measurements
Glucose, mg/dl	191 ± 8	184 ± 6
Insulin, ng/ml	2.9 ± 0.8	1.6 ± 0.2
Leptin, ng/ml	3.1 ± 0.8	4.2 ± 1.0

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Data are means ± SE for groups of 9 rats. Rats were infused with saline or 0.6 μg/24 h leptin for 13 days before the end of the experiment. Blood and tissue were collected following 3 h of food deprivation during the light period.

Significant difference between treatment groups.

Experiment 3.

Immunohistochemistry showed a significant increase in activation of STAT3 in the mNTS of rats receiving fourth-ventricle infusions of 1.2 μg leptin/day. This was true for sections that spanned from −13.68 to −14.30 mm from the bregma (Fig. 4). These data contrast with those from experiment 1, in which Western blot did not detect differences in hindbrain p-STAT3, possibly because the site-specific response was diluted out by analysis of protein from a tissue block. There was no effect of leptin on p-ERK1/2 in any of the areas evaluated at these levels of the brainstem (data not shown). Infusing 1.2 μg of leptin activated STAT3 in the mArc, DMC or DMD, VMH, and PH in sections at −3.14 and −3.60 mm from bregma (Fig. 5). There was no effect of leptin on p-STAT3 in the lArc at any level, and 0.3 μg of leptin had no effect on p-STAT3 in any area on these sections. By contrast, at bregma −3.30 mm there was a dose response effect of leptin on p-STAT3 in the DMC and VMH, with no effect in either the mArc or lArc. The low dose of leptin inhibited p-STAT3 in the PH, whereas 1.2 μg leptin/day had no effect. In the rostral mArc, 0.3 and 1.2 μg leptin/day caused similar increases in p-STAT3 (Fig. 5).

Fig. 4. — Quantification of p-STAT3-positive nuclei in the medial nucleus of the solitary tract (mNTS) of rats that received 4th-ventricle infusions of 0, 0.3, or 1.2 μg/24 h leptin for 7 days in *experiment 3*. Representative images, adjusted for brightness, contrast and sharpness, are from bregma −14.80 mm. Values for a specific level of the bregma that do not share a common letter are significantly different. AP, area postrema; CC, central canal.

Fig. 5. — Quantification of p-STAT3 in the hypothalamus of rats receiving fourth-ventricle infusions of 0, 0.3, or 1.2 μg/24 h leptin for 7 days in *experiment 3*. Representative images, adjusted for brightness, contrast, and sharpness, are from bregma −3.60. Values for p-STAT3 in a specific area at a specific level of the hypothalamus that do not share a common letter are significantly different. ArcM, medial arcuate nucleus; ArcL, lateral arcuate nucleus; DMC, compact dorsomedial nucleus; VMH, ventromedial nucleus; PH, posterior hypothalamic area; DMD, dorsal dorsomedial nucleus; 3V, 3rd ventricle.

DISCUSSION

The objective of these experiments was to determine whether weight loss in rats receiving fourth-ventricle infusions of leptin was associated with activation of p-STAT3 in the hypothalamus. Only the highest doses of leptin increased STAT3 activation in hypothalamic nuclei, and these doses also caused hypophagia, weight loss, and a reduction of both fat and lean body mass. It is likely that the loss of lean mass was secondary to negative energy balance rather than a specific effect of leptin on muscle, because food-restricted and starved rats lose both fat and lean mass (12), and the rats treated with the higher doses of leptin were in a state of chronic voluntary food restriction.

The lowest dose of leptin used in this study, 0.1 μg/day, could be considered subthreshold because it had no effect on food intake, weight gain, UCP1 expression, or body composition. The dose of 0.3 μg leptin/day caused a 26% reduction in body fat that was associated with a <10% reduction in total food intake. Neither of these changes was significant, but there was a significant increase in iBAT UCP1 expression, which implies increased thermogenesis. The 0.6 μg leptin/day rats showed a consistent trend to consume less than controls in experiment 1. In experiment 2, when there were only two groups of animals to compare and a larger number of animals per group, intake was inhibited for the first 3 days of leptin infusion. Because the rats did not overeat during the rest of the experiment, the initial hypophagia may have been enough to cause the loss of body fat that was measured after 12 days of infusion. RER was low on the days that food intake was inhibited, consistent with the rats mobilizing body fat for energy rather than consuming the high-carbohydrate chow that was freely available. Although leptin increased iBAT UCP1 in experiment 1, there was no effect of leptin on energy expenditure in experiment 2. There also was no effect of 0.6 μg leptin/day on iBAT UCP1 expression in experiment 2, possibly because these rats were housed in bedding in calorimetry shoebox cages and were less thermogenically stressed than rats housed in wire mesh cages without bedding. We have found previously that leptin-infused rats maintain energy expenditure, even when they have lost a significant amount of weight, including lean mass (5), and, therefore, are maintaining an inappropriately high level of expenditure for the size of their body energy stores. UCP1 protein expression is an indicator only of the maximal potential capacity for thermogenesis, rather than actual heat production, but it is possible that there was a subtle change in heat loss even with the lowest doses of leptin and that this made some contribution to the change in body composition of the rats. It has been reported that restoration of normal body temperature in leptin-treated ob/ob mice is due to a reduction in heat loss rather than an increase in thermogenesis (8). There is no effect of exogenous leptin on body temperature of leptin sufficient, normothermic wild-type mice, and tail heat loss has not been measured (8), but it is possible that a relative pyrexia contributed to the reduction in body fat of rats receiving the higher doses of leptin in our experiment.

Leptin injections into the mNTS reduce food-seeking behavior and the willingness of rats to work for sucrose solution (15). We did not measure any motivational behavior, but it is unlikely that the high doses of leptin inhibited food intake by suppressing motivation because food was freely available inside the cage in experiment 1, and in experiment 2 a minimal amount of work would have been required to obtain the food that was in a hopper hanging inside the cage. Leptin has also been reported to reduce the rewarding aspects of food through activation of dopaminergic neurons in the ventral tegmentum (VTA) (7), but we did not find any p-STAT3 in the VTA of leptin-treated rats in experiment 3. The inhibitory effect of leptin on food intake may be mediated at least in part by exaggerating the response to peripheral satiation signals. Others have shown that hindbrain leptin enhances the satiation effect of gastric distention (14) and of peripheral injections of CCK without shortening meal size (18, 19). Matson et al. (17) found that daily central injections of leptin and peripheral injections of CCK for 9 days caused continuous weight loss in rats, even though food intake was not any different from that of rats injected only with CCK. Therefore, the synergy between central leptin and vagal signals initiated in the periphery is not limited to food intake, and it would be of interest to determine whether constant infusion of leptin enhanced the response to meal-related satiety signals. Whatever mechanism was primarily responsible for inhibiting food intake in leptin-infused rats, there was a dose-related effect on duration of hypophagia. In experiment 2, 0.6 μg leptin/day inhibited intake for 3 days, whereas 1.2 or 2.0 μg leptin/day in experiment 1 inhibited intake for 10 days. When the inhibition of food intake was relieved there was no rebound overeating to compensate for the initial hypophagia, suggesting that some other mechanism is in place to drive down body fat mass and that food intake is inhibited only until the desired equilibrium is achieved.

All of the fat pads that were weighed responded to leptin, but there were site-specific differences in sensitivity. Retroperitoneal and mesenteric fat responded to lower concentrations of leptin than epididymal or inguinal fat. These differences were unlikely to be related to sympathetic drive to the fat depots because we have previously shown that loss of white fat from rats receiving peripheral infusions of leptin is independent of sympathetic innervation of the fat depot (24), that norepinephrine turnover does not correlate with the degree of response from a specific fat depot in rats receiving central injections of leptin, and that some fat depots will decrease in size in the absence of a change in norepinephrine turnover (23). In addition, the responsiveness of different fat depots to leptin did not correlate with the ability of sympathetic stimulation to induce the transformation of white adipocytes into thermogenic, UCP1-expressing brown adipocytes (beigeing) (2). Cold exposure causes a much greater induction of beigeing in inguinal than visceral fat of mice (2). By contrast, we found that visceral fat was more responsive to fourth-ventricle infusion of leptin than inguinal fat. We have reported previously that peripheral infusions of leptin induce release of unidentified circulating factors that contribute to the loss of fat (11), and it is possible that central leptin also promotes release of these factors.

The increase in hypothalamic p-STAT3 in rats receiving the higher doses of leptin in experiments 1 and 3 is consistent with our previous observations that infusion of leptin into the fourth ventricle enhances leptin signaling and response to a very-low-dose infusion of leptin into the third ventricle. It also confirms the previous report from Ruiter et al. (25) that leptin injection into either the fourth ventricle or the NTS induced phosphorylation of STAT3 in the hypothalamus. We found that the areas of the hypothalamus that showed increased p-STAT3 in experiment 1 were identical to those activated by simultaneous subthreshold dose infusions into the third and fourth ventricle (6). Although we have not confirmed the coexpression of p-STAT3 with ObRb, the activated areas have all been reported to express ObRb (26). We also have confirmed that there is not a generalized activation of the hypothalamus because expression of δ-FosB is limited almost exclusively to the sites that show an increase in p-STAT3 (6). In experiment 3 the high dose infusion of 1.2 μg leptin/day increased p-STAT3 in the mArc, which has been associated with leptin-induced inhibition of food intake (21) and stimulation of BAT thermogenesis (27), the DMH, which has been associated with leptin-induced stimulation of thermogenesis (31), and the VMH, which is involved in leptin-induced glucose utilization (30). These observations are consistent with the phenotype of rats receiving fourth-ventricle infusions of the three highest high doses of leptin in experiment 1. There was a much more limited effect of infusing 0.3 μg leptin/day on hypothalamic p-STAT3, consistent with the minimal physiological impact of this dose of leptin. These rats had increased levels of expression of iBAT UCP1, which correlated with increased p-STAT3 in the DMC and VMH at the level of bregma −3.30 mm and the mArc at bregma −4.70. Zhang et al. (31) have shown colocalization of the retrograde transsynaptic tract tracer pseudorabies virus injected into brown adipose tissue and leptin receptors in neurons in the DMH.

The results from this experiment are similar to those from previous experiments in which we found that low-dose infusions of leptin into the fourth ventricle facilitated STAT3 activation in the hypothalamus (5, 6). We have proposed that an increase in leptin concentration in the hindbrain lowers the threshold for activation of hypothalamic leptin receptors. There is precedence for hindbrain leptin gating the response to thyrotropin-releasing hormone (13), CCK (17), and gastric distention (14). These are all examples of leptin acting locally to enhance signaling within the hindbrain. We are proposing that hindbrain leptin also gates a distant response in the hypothalamus. The enhanced sensitivity to leptin in the forebrain would increase the precision of weight control by allowing changes in circulating leptin concentrations to be sensed in the hindbrain and lead to an integrated central response to restore energy balance. Although it is clear that the hypothalamic response to fourth-ventricle leptin is centrally mediated, we have not determined how the hindbrain and forebrain are communicating. CSF does not flow from the fourth to the third ventricle (4); therefore, if leptin in the fourth ventricle was raising the concentration of leptin in the third ventricle, it would have to be delivered by diffusion through the subarachanoid space. The highest doses of leptin used here and infused constantly over 24 h were lower than those used to test the effect of an acute fourth-ventricle injection on energy balance (25, 28) but likely caused a moderate and sustained increase in fourth-ventricle leptin concentration. If this leptin then diffused through the subarachanoid space, then the increase in leptin concentrations at the hypothalamus would have been minimal. The substantial increase in hypothalamic p-STAT3 in the absence of exogenous leptin administration to the forebrain supports the proposal that neural communication from the hindbrain lowers the threshold for leptin signaling in the forebrain. There are multiple neural connections between the NTS and hypothalamic nuclei (e.g., see Ref. 1), and future studies will test whether any of these originate in leptin receptor-expressing cells.

GRANTS

This work was supported by National Institutes of Health Grant DK-053903 awarded to R. B. S. Harris.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

R.B.H. and B.N.D. performed experiments; R.B.H. and B.N.D. analyzed data; R.B.H. and B.N.D. interpreted results of experiments; R.B.H. prepared figures; R.B.H. drafted manuscript; R.B.H. and B.N.D. edited and revised manuscript; R.B.H. and B.N.D. approved final version of manuscript.

ACKNOWLEDGMENTS

We thank Mary Shaw for running the Western blots in experiment 1.

Present address of B. N. Desai: Beth Israel Deaconess Medical Center, Department of Medicine-Endocrinology, Diabetes, and Metabolism, 330 Brookline Ave., Boston, MA 02215.

REFERENCES

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9.Haring SJ, Harris RB. The relation between dietary fructose, dietary fat and leptin responsiveness in rats. Physiol Behav 104: 914–922, 2011. doi: 10.1016/j.physbeh.2011.05.032. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Harris RB. Growth measurements in Sprague-Dawley rats fed diets of very low fat concentration. J Nutr 121: 1075–1080, 1991. [DOI] [PubMed] [Google Scholar]
11.Harris RB. In vivo evidence for unidentified leptin-induced circulating factors that control white fat mass. Am J Physiol Regul Integr Comp Physiol 309: R1499–R1511, 2015. doi: 10.1152/ajpregu.00335.2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
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13.Hermann GE, Barnes MJ, Rogers RC. Leptin and thyrotropin-releasing hormone: cooperative action in the hindbrain to activate brown adipose thermogenesis. Brain Res 1117: 118–124, 2006. doi: 10.1016/j.brainres.2006.08.018. [DOI] [PubMed] [Google Scholar]
14.Huo L, Maeng L, Bjørbaek C, Grill HJ. Leptin and the control of food intake: neurons in the nucleus of the solitary tract are activated by both gastric distension and leptin. Endocrinology 148: 2189–2197, 2007. doi: 10.1210/en.2006-1572. [DOI] [PubMed] [Google Scholar]
15.Kanoski SE, Alhadeff AL, Fortin SM, Gilbert JR, Grill HJ. Leptin signaling in the medial nucleus tractus solitarius reduces food seeking and willingness to work for food. Neuropsychopharmacology 39: 605–613, 2014. doi: 10.1038/npp.2013.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Kasser TR, Harris RB, Martin RJ. Level of satiety: fatty acid and glucose metabolism in three brain sites associated with feeding. Am J Physiol Regul Integr Comp Physiol 248: R447–R452, 1985. [DOI] [PubMed] [Google Scholar]
17.Matson CA, Reid DF, Cannon TA, Ritter RC. Cholecystokinin and leptin act synergistically to reduce body weight. Am J Physiol Regul Integr Comp Physiol 278: R882–R890, 2000. [DOI] [PubMed] [Google Scholar]
18.Matson CA, Ritter RC. Long-term CCK-leptin synergy suggests a role for CCK in the regulation of body weight. Am J Physiol Regul Integr Comp Physiol 276: R1038–R1045, 1999. [DOI] [PubMed] [Google Scholar]
19.Matson CA, Wiater MF, Kuijper JL, Weigle DS. Synergy between leptin and cholecystokinin (CCK) to control daily caloric intake. Peptides 18: 1275–1278, 1997. doi: 10.1016/S0196-9781(97)00138-1. [DOI] [PubMed] [Google Scholar]
20.Miragaya JR, Harris RB. Antagonism of corticotrophin-releasing factor receptors in the fourth ventricle modifies responses to mild but not restraint stress. Am J Physiol Regul Integr Comp Physiol 295: R404–R416, 2008. doi: 10.1152/ajpregu.00565.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Morton GJ, Niswender KD, Rhodes CJ, Myers MG Jr, Blevins JE, Baskin DG, Schwartz MW. Arcuate nucleus-specific leptin receptor gene therapy attenuates the obesity phenotype of Koletsky (fa(k)/fa(k)) rats. Endocrinology 144: 2016–2024, 2003. doi: 10.1210/en.2002-0115. [DOI] [PubMed] [Google Scholar]
22.Paxinos G, Watson C. The Rat Brain. New York: Academic, 1998. [Google Scholar]
23.Penn DM, Jordan LC, Kelso EW, Davenport JE, Harris RB. Effects of central or peripheral leptin administration on norepinephrine turnover in defined fat depots. Am J Physiol Regul Integr Comp Physiol 291: R1613–R1621, 2006. doi: 10.1152/ajpregu.00368.2006. [DOI] [PubMed] [Google Scholar]
24.Rooks CR, Penn DM, Kelso E, Bowers RR, Bartness TJ, Harris RB. Sympathetic denervation does not prevent a reduction in fat pad size of rats or mice treated with peripherally administered leptin. Am J Physiol Regul Integr Comp Physiol 289: R92–R102, 2005. doi: 10.1152/ajpregu.00858.2004. [DOI] [PubMed] [Google Scholar]
25.Ruiter M, Duffy P, Simasko S, Ritter RC. Increased hypothalamic signal transducer and activator of transcription 3 phosphorylation after hindbrain leptin injection. Endocrinology 151: 1509–1519, 2010. doi: 10.1210/en.2009-0854. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Scott MM, Lachey JL, Sternson SM, Lee CE, Elias CF, Friedman JM, Elmquist JK. Leptin targets in the mouse brain. J Comp Neurol 514: 518–532, 2009. doi: 10.1002/cne.22025. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Shi YC, Lau J, Lin Z, Zhang H, Zhai L, Sperk G, Heilbronn R, Mietzsch M, Weger S, Huang XF, Enriquez RF, Castillo L, Baldock PA, Zhang L, Sainsbury A, Herzog H, Lin S. Arcuate NPY controls sympathetic output and BAT function via a relay of tyrosine hydroxylase neurons in the PVN. Cell Metab 17: 236–248, 2013. doi: 10.1016/j.cmet.2013.01.006. [DOI] [PubMed] [Google Scholar]
28.Skibicka KP, Grill HJ. Hindbrain leptin stimulation induces anorexia and hyperthermia mediated by hindbrain melanocortin receptors. Endocrinology 150: 1705–1711, 2009. doi: 10.1210/en.2008-1316. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Sutton GM, Patterson LM, Berthoud HR. Extracellular signal-regulated kinase 1/2 signaling pathway in solitary nucleus mediates cholecystokinin-induced suppression of food intake in rats. J Neurosci 24: 10240–10247, 2004. doi: 10.1523/JNEUROSCI.2764-04.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Toda C, Shiuchi T, Kageyama H, Okamoto S, Coutinho EA, Sato T, Okamatsu-Ogura Y, Yokota S, Takagi K, Tang L, Saito K, Shioda S, Minokoshi Y. Extracellular signal-regulated kinase in the ventromedial hypothalamus mediates leptin-induced glucose uptake in red-type skeletal muscle. Diabetes 62: 2295–2307, 2013. doi: 10.2337/db12-1629. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Zhang Y, Kerman IA, Laque A, Nguyen P, Faouzi M, Louis GW, Jones JC, Rhodes C, Münzberg H. Leptin-receptor-expressing neurons in the dorsomedial hypothalamus and median preoptic area regulate sympathetic brown adipose tissue circuits. J Neurosci 31: 1873–1884, 2011. doi: 10.1523/JNEUROSCI.3223-10.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Zimmerman AD, Harris RB. In vivo and in vitro evidence that chronic activation of the hexosamine biosynthetic pathway interferes with leptin-dependent STAT3 phosphorylation. Am J Physiol Regul Integr Comp Physiol 308: R543–R555, 2015. doi: 10.1152/ajpregu.00347.2014. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B1] 1.Affleck VS, Coote JH, Pyner S. The projection and synaptic organisation of NTS afferent connections with presympathetic neurons, GABA and nNOS neurons in the paraventricular nucleus of the hypothalamus. Neuroscience 219: 48–61, 2012. doi: 10.1016/j.neuroscience.2012.05.070. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B2] 2.Barbatelli G, Murano I, Madsen L, Hao Q, Jimenez M, Kristiansen K, Giacobino JP, De Matteis R, Cinti S. The emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation. Am J Physiol Endocrinol Metab 298: E1244–E1253, 2010. doi: 10.1152/ajpendo.00600.2009. [DOI] [PubMed] [Google Scholar]

[B3] 3.National Research Council. Guide For the Care and Use of Laboratory Animals. Washington, DC: National Academies, 1996. [Google Scholar]

[B4] 4.Daniels D, Marshall A. Evaluating the potential for rostral diffusion in the cerebral ventricles using angiotensin II-induced drinking in rats. Brain Res 1486: 62–67, 2012. doi: 10.1016/j.brainres.2012.09.040. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B5] 5.Desai BN, Harris RB. Integrated effects of leptin in the forebrain and hindbrain of male rats. Endocrinology 154: 2663–2675, 2013. doi: 10.1210/en.2013-1099. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6.Desai BN, Harris RB. Leptin in the hindbrain facilitates phosphorylation of STAT3 in the hypothalamus. Am J Physiol Endocrinol Metab 308: E351–E361, 2015. doi: 10.1152/ajpendo.00501.2014. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7.Figlewicz DP, Evans SB, Murphy J, Hoen M, Baskin DG. Expression of receptors for insulin and leptin in the ventral tegmental area/substantia nigra (VTA/SN) of the rat. Brain Res 964: 107–115, 2003. doi: 10.1016/S0006-8993(02)04087-8. [DOI] [PubMed] [Google Scholar]

[B8] 8.Fischer AW, Hoefig CS, Abreu-Vieira G, de Jong JM, Petrovic N, Mittag J, Cannon B, Nedergaard J. Leptin raises defended body temperature without activating thermogenesis. Cell Reports 14: 1621–1631, 2016. doi: 10.1016/j.celrep.2016.01.041. [DOI] [PubMed] [Google Scholar]

[B9] 9.Haring SJ, Harris RB. The relation between dietary fructose, dietary fat and leptin responsiveness in rats. Physiol Behav 104: 914–922, 2011. doi: 10.1016/j.physbeh.2011.05.032. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10.Harris RB. Growth measurements in Sprague-Dawley rats fed diets of very low fat concentration. J Nutr 121: 1075–1080, 1991. [DOI] [PubMed] [Google Scholar]

[B11] 11.Harris RB. In vivo evidence for unidentified leptin-induced circulating factors that control white fat mass. Am J Physiol Regul Integr Comp Physiol 309: R1499–R1511, 2015. doi: 10.1152/ajpregu.00335.2015. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12.Harris RB, Kasser TR, Martin RJ. Dynamics of recovery of body composition after overfeeding, food restriction or starvation of mature female rats. J Nutr 116: 2536–2546, 1986. [DOI] [PubMed] [Google Scholar]

[B13] 13.Hermann GE, Barnes MJ, Rogers RC. Leptin and thyrotropin-releasing hormone: cooperative action in the hindbrain to activate brown adipose thermogenesis. Brain Res 1117: 118–124, 2006. doi: 10.1016/j.brainres.2006.08.018. [DOI] [PubMed] [Google Scholar]

[B14] 14.Huo L, Maeng L, Bjørbaek C, Grill HJ. Leptin and the control of food intake: neurons in the nucleus of the solitary tract are activated by both gastric distension and leptin. Endocrinology 148: 2189–2197, 2007. doi: 10.1210/en.2006-1572. [DOI] [PubMed] [Google Scholar]

[B15] 15.Kanoski SE, Alhadeff AL, Fortin SM, Gilbert JR, Grill HJ. Leptin signaling in the medial nucleus tractus solitarius reduces food seeking and willingness to work for food. Neuropsychopharmacology 39: 605–613, 2014. doi: 10.1038/npp.2013.235. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16.Kasser TR, Harris RB, Martin RJ. Level of satiety: fatty acid and glucose metabolism in three brain sites associated with feeding. Am J Physiol Regul Integr Comp Physiol 248: R447–R452, 1985. [DOI] [PubMed] [Google Scholar]

[B17] 17.Matson CA, Reid DF, Cannon TA, Ritter RC. Cholecystokinin and leptin act synergistically to reduce body weight. Am J Physiol Regul Integr Comp Physiol 278: R882–R890, 2000. [DOI] [PubMed] [Google Scholar]

[B18] 18.Matson CA, Ritter RC. Long-term CCK-leptin synergy suggests a role for CCK in the regulation of body weight. Am J Physiol Regul Integr Comp Physiol 276: R1038–R1045, 1999. [DOI] [PubMed] [Google Scholar]

[B19] 19.Matson CA, Wiater MF, Kuijper JL, Weigle DS. Synergy between leptin and cholecystokinin (CCK) to control daily caloric intake. Peptides 18: 1275–1278, 1997. doi: 10.1016/S0196-9781(97)00138-1. [DOI] [PubMed] [Google Scholar]

[B20] 20.Miragaya JR, Harris RB. Antagonism of corticotrophin-releasing factor receptors in the fourth ventricle modifies responses to mild but not restraint stress. Am J Physiol Regul Integr Comp Physiol 295: R404–R416, 2008. doi: 10.1152/ajpregu.00565.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B21] 21.Morton GJ, Niswender KD, Rhodes CJ, Myers MG Jr, Blevins JE, Baskin DG, Schwartz MW. Arcuate nucleus-specific leptin receptor gene therapy attenuates the obesity phenotype of Koletsky (fa(k)/fa(k)) rats. Endocrinology 144: 2016–2024, 2003. doi: 10.1210/en.2002-0115. [DOI] [PubMed] [Google Scholar]

[B22] 22.Paxinos G, Watson C. The Rat Brain. New York: Academic, 1998. [Google Scholar]

[B23] 23.Penn DM, Jordan LC, Kelso EW, Davenport JE, Harris RB. Effects of central or peripheral leptin administration on norepinephrine turnover in defined fat depots. Am J Physiol Regul Integr Comp Physiol 291: R1613–R1621, 2006. doi: 10.1152/ajpregu.00368.2006. [DOI] [PubMed] [Google Scholar]

[B24] 24.Rooks CR, Penn DM, Kelso E, Bowers RR, Bartness TJ, Harris RB. Sympathetic denervation does not prevent a reduction in fat pad size of rats or mice treated with peripherally administered leptin. Am J Physiol Regul Integr Comp Physiol 289: R92–R102, 2005. doi: 10.1152/ajpregu.00858.2004. [DOI] [PubMed] [Google Scholar]

[B25] 25.Ruiter M, Duffy P, Simasko S, Ritter RC. Increased hypothalamic signal transducer and activator of transcription 3 phosphorylation after hindbrain leptin injection. Endocrinology 151: 1509–1519, 2010. doi: 10.1210/en.2009-0854. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B26] 26.Scott MM, Lachey JL, Sternson SM, Lee CE, Elias CF, Friedman JM, Elmquist JK. Leptin targets in the mouse brain. J Comp Neurol 514: 518–532, 2009. doi: 10.1002/cne.22025. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27.Shi YC, Lau J, Lin Z, Zhang H, Zhai L, Sperk G, Heilbronn R, Mietzsch M, Weger S, Huang XF, Enriquez RF, Castillo L, Baldock PA, Zhang L, Sainsbury A, Herzog H, Lin S. Arcuate NPY controls sympathetic output and BAT function via a relay of tyrosine hydroxylase neurons in the PVN. Cell Metab 17: 236–248, 2013. doi: 10.1016/j.cmet.2013.01.006. [DOI] [PubMed] [Google Scholar]

[B28] 28.Skibicka KP, Grill HJ. Hindbrain leptin stimulation induces anorexia and hyperthermia mediated by hindbrain melanocortin receptors. Endocrinology 150: 1705–1711, 2009. doi: 10.1210/en.2008-1316. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B29] 29.Sutton GM, Patterson LM, Berthoud HR. Extracellular signal-regulated kinase 1/2 signaling pathway in solitary nucleus mediates cholecystokinin-induced suppression of food intake in rats. J Neurosci 24: 10240–10247, 2004. doi: 10.1523/JNEUROSCI.2764-04.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B30] 30.Toda C, Shiuchi T, Kageyama H, Okamoto S, Coutinho EA, Sato T, Okamatsu-Ogura Y, Yokota S, Takagi K, Tang L, Saito K, Shioda S, Minokoshi Y. Extracellular signal-regulated kinase in the ventromedial hypothalamus mediates leptin-induced glucose uptake in red-type skeletal muscle. Diabetes 62: 2295–2307, 2013. doi: 10.2337/db12-1629. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B31] 31.Zhang Y, Kerman IA, Laque A, Nguyen P, Faouzi M, Louis GW, Jones JC, Rhodes C, Münzberg H. Leptin-receptor-expressing neurons in the dorsomedial hypothalamus and median preoptic area regulate sympathetic brown adipose tissue circuits. J Neurosci 31: 1873–1884, 2011. doi: 10.1523/JNEUROSCI.3223-10.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B32] 32.Zimmerman AD, Harris RB. In vivo and in vitro evidence that chronic activation of the hexosamine biosynthetic pathway interferes with leptin-dependent STAT3 phosphorylation. Am J Physiol Regul Integr Comp Physiol 308: R543–R555, 2015. doi: 10.1152/ajpregu.00347.2014. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Fourth-ventricle leptin infusions dose-dependently activate hypothalamic signal transducer and activator of transcription 3

Ruth B S Harris

Bhavna N Desai

Abstract