Lateral hypothalamic GLP-1 receptors are critical for the control of food reinforcement, ingestive behavior and body weight

Abstract

Increased motivation for highly rewarding food is a major contributing factor to obesity. Most of the literature focuses on the mesolimbic nuclei as the core of reward behavior regulation. However, the lateral hypothalamus (LH) is also a key reward-control locus in the brain. Here we hypothesize that manipulating glucagon-like peptide-1 receptor (GLP-1R) activity selectively in the LH can profoundly affect food reward behavior, ultimately leading to obesity. Progressive ratio operant responding for sucrose was examined in male and female rats, following GLP-1R activation and pharmacological or genetic GLP-1R blockade in the LH. Ingestive behavior and metabolic parameters, as well as molecular and efferent targets, of the LH GLP-1R activation were also evaluated. Food motivation was reduced by activation of LH GLP-1R. Conversely, acute pharmacological blockade of LH GLP-1R increased food motivation but only in male rats. GLP-1R activation also induced a robust reduction in food intake and body weight. Chronic knockdown of LH GLP-1R induced by intraparenchymal delivery of an adeno-associated virus-short hairpin RNA construct was sufficient to markedly and persistently elevate ingestive behavior and body weight and ultimately resulted in a doubling of fat mass in males and females. Interestingly, increased food reinforcement was again found only in males. Our data identify the LH GLP-1R as an indispensable element of normal food reinforcement, food intake and body weight regulation. These findings also show, for we believe the first time, that brain GLP-1R manipulation can result in a robust and chronic body weight gain. The broader implications of these findings are that the LH differs between females and males in its ability to control motivated and ingestive behaviors.

Figure 1

Behavioral, molecular and neuroanatomical consequences of glucagon-like peptide-1 receptor (GLP-1R) activation in the lateral hypothalamus (LH) in male rats. Activation of GLP-1R in the LH reduces food reinforcement and food intake. Intra-LH GLP-1 analogue, exendin-4 (Ex4), microinjection reduced the amount of sucrose rewards earned (a) and the number of lever presses for the rewards (b) in a progressive ratio schedule, without changing activity at the inactive lever (c) in male rats. Food ingestion is also affected, as illustrated by a potent reduction in 1 (d) and 24 h (e) chow intake. Data are expressed as mean±s.e.m. n=14–15 (male rats). Moreover, GLP-1R-expressing LH neurons were found to project to the mesolimbic ventral tegmental area (VTA), a likely neuroanatomical target area for reinforcement control exerted by LH GLP-1R. Representative images showing GLP-1R on LH neurons that project to the VTA. Cholera toxin subunit B (CTB) conjugated to AlexaFluor 488 was injected into the VTA (f, g). Back-labeled cells were present in the rostral LH region dorsal to the fornix (h) where the majority of the LH GLP-1R-containing neurons are localized. Images of GLP-1R in situ hybridization (red; i), back-labeled CTB-positive neurons from the VTA (green; j) and 4,6-diamidino-2-phenylindole (DAPI) nuclear counterstain (blue; k) at × 20 magnification. A merged image showing co-localization of GLP-1R and CTB in the LH (white arrows; l) and a confocal image taken at × 40 magnification of a double-labeled GLP-1R and CTB-positive neuron with DAPI counterstain (m). Intra-LH Ex4 infusion produced a marked increase in anorexic interleukins 1 (IL1) and 6 (IL6) and also reduced orexigenic LH neuropeptide expression (melanin-concentrated hormone (MCH)) in male rats (n). n=14–22 (male rats). GLP-1R activation with Ex4 increased IL6 expression (o) in hypothalamic neuronal cell culture. In order to determine whether endogenous GLP-1 release also leads to similar changes within the LH, three groups of rats were allowed to eat chow, sugar or lard for 1 h. Lard meal did not alter any of the genes measured; however, a consumption of sucrose water increased LH IL6 expression (p). Data were normalized to the housekeeping gene beta-actin and are expressed as mean±s.e.m.. n=7–9 (male rats). ARC: arcuate nucleus of the hypothalamus; DMH: dorsomedial hypothalamus; VMH: ventromedial hypothalamus; NT: neurotensin; ORX: orexin; IL6R: IL6 receptor; Cart: cocaine- and amphetamine-related peptide. *P<0.05, **P<0.01,***P<0.001, ****P<0.0001 compared with vehicle (artificial cerebrospinal fluid).

Figure 2

Behavioral and molecular consequences of glucagon-like peptide-1 receptor (GLP-1R) activation in the lateral hypothalamus (LH), as well as their interactions with the estrous cycle, in female rats. In females only the higher dose of exendin-4 (Ex4) reduced the amount of sucrose rewards earned (a) and resulted in a trend to reduce the number of lever presses for the rewards (b) in a progressive ratio schedule, without changing activity at the inactive lever (c). Food ingestion was also affected in female rats but only by the higher dose of Ex4, as illustrated by a reduction in 1 (d) and 24 h (e) chow intake (n=9–37). LH GLP-1R activation in females has divergent behavioral and molecular impact in different estrus cycle phases. Intra-LH Ex4 reduced the effort to obtain a reward (f–i) in a sucrose-motivated progressive ratio task in females in the estrus phase (E) but not metestrus or diestrus (MD) phase of their cycle. Performance on the inactive lever or locomotor activity during the task was not altered by Ex4 in any of the cycle phases (j–m). Although Ex4 potently reduced ingestive behavior in all females irrespective of the cycle phase, the reduction was enhanced for females in E (n–q). Data are expressed as mean±s.e.m. For each measured parameter, raw values and values calculated as the percentage of vehicle response are presented in order to minimize the impact of baseline differences across the two cycle groups. n=10–11. Behavioral differences were mirrored by similarly divergent gene expression changes induced by intra-LH Ex4 application, where a potent induction of interleukins was only detected in females in E, and a reduction in orexin or neurotensin expression was only present in E (r). A reduced expression of melanin-concentrating hormone (MCH) was the only mRNA change preserved in MD phases. Data were normalized to the housekeeping gene beta-actin and are expressed as mean±s.e.m. n=19: vehicle in E, n=20: Ex4 in E, n=8: vehicle in MD, n=8: Ex4 in MD. *P<0.05, **P<0.01,***P<0.001, ****P<0.0001.

Figure 3

Acute pharmacological blockade of lateral hypothalamus (LH) glucagon-like peptide-1 receptor (GLP-1R) increases food reinforcement but not intake. Intra-LH microinjection of GLP-1R antagonist, Exendin 9 (Ex9; 10 μg) increased the amount of sucrose rewards earned (a, b) and the number of lever presses for the rewards (c, d) in a progressive ratio schedule, without changing activity at the inactive lever (e, f) in male rats. Similarly food-seeking (number of entries into a food dispenser) was significantly increased by this treatment in male rats (g, h). None of these parameters were altered in female rats. Food ingestion measured as 1 (i, j) and 24 h (k, l) chow intake were unaffected in either sex. Locomotor activity was also not significantly altered (m, n). Data are expressed as mean±s.e.m. n=8–10 (male rats: m) and n=14 (female rats: f). *P<0.05, **P<0.01, ^#P=0.07.

Figure 4

Glucagon-like peptide-1 receptor (GLP-1R) in the lateral hypothalamus (LH) are necessary for normal body weight, food intake and food reinforcement control. Decreased GLP-1R expression in the LH promoted weight gain (a) and hyperphagia (b), along with small impairments in glucose tolerance (c). Knockdown of GLP-1R in the LH led to increased fat mass in gonadal and inguinal adipose tissues (GWAT and IWAT, respectively; d). The hyperphagia and increased body weight gain persisted even after food deprivation/refeeding procedure (e). Data are expressed as mean±s.e.m. n=14–15 (male rats). Knockdown of GLP-1R in the LH resulted in an increased amount of sucrose rewards earned (f) and increased number of lever presses for the rewards (g) in a progressive ratio schedule. In addition, food-seeking (number of entries into a food dispenser) behavior was increased (h) without concurrent changes in locomotor activity (i). Similar potentiation of food reinforcement and seeking behaviors was detected in highly motivated (fasted) rats (j–l), again without significant changes in locomotor activity (m). Data are expressed as mean±s.e.m. n=19–20 (9-week-old male rats). Four weeks after adeno-associated virus (AAV) construct injections, GLP-1R expression was reduced by ~50% in the LH of rats treated with AAV-GLP-1R-shRNA (short hairpin RNA) compared with controls (n). Coronal brain section depicting LH-targeted infusions of control and AAV-GLP-1R-shRNA (o). Nuclear stain, DAPI (4,6-diamidino-2-phenylindole), is shown in blue. Panels (p and q) show green fluorescent protein-expressing cells in the LH. *P<0.05, **P<0.01,***P<0.001, ****P<0.0001; ^#P<0.1. DMH, dorsomedial hypothalamus; KD, knockdown; VMH, ventromedial hypothalamus.

Figure 5

Glucagon-like peptide-1 receptor (GLP-1R) in the lateral hypothalamus (LH) are necessary for normal body weight, body fat and food intake control in males and young females but not older females. Decreased GLP-1R expression in the LH promoted a marked, chronic, hyperphagia and weight gain in male (a, d, g) but not in female (b, e, h) rats (both sexes 13 week old on injection day). In contrast to the 13-week-old females, younger females (9 week old) presented with a massive hyperphagia (c, f) and weight gain after LH GLP-1R KD (i). Likewise, male knockdown rats have double the amount of fat (j) compared with controls, while fat mass in same age females was unaffected by the treatment (k). However, the adipose tissue weight of younger females with LH GLP-1R knockdown was nearly threefold higher compared with control rats (l). Fasted blood glucose levels are not altered by the knockdown in either sex (13 week old; m). Potential compensatory adaptations of the central GLP-1 system were assessed by determining whether the GLP-1 precursor (preproglucagon) expression in the nucleus of the solitary tract (NTS), or GLP-1R expression in the nucleus accumbens (NAc) or in the NTS, were altered in response to the LH GLP-1R knockdown. The expression of preproglucagon was increased in all three knockdown groups tested, thus irrespective of sex, age or weight gain response to the knockdown (n). A compensatory increase in GLP-1R expression was detected in NAc, a nucleus with dense connections to the LH, but interestingly this change was only detected in males (o). This elevation in males seemed to be area specific as GLP-1R expression in the NTS was not altered in males or any of the female groups. Thus chronic loss of LH GLP-1R, induced by an adeno-associated virus (AAV)-shRNA (short hairpin RNA) GLP-1R-mediated knockdown, not only increased food reinforcement behavior but also led to a marked hyperphagia and weight gain, at a level unparalleled to that found by blockade of any other GLP-1R population in rats or mice, despite compensatory changes detected outside of the LH. In conclusion, GLP-1R in the LH is a key component of normal body weight homeostasis and food reward control; LH emerges as one of the most critical sites for the endogenous GLP-1 effect on energy balance in males. Data are expressed as mean±s.e.m. n=10 (male rats, 5 in each treatment group); n=10 (female rats, 5 in each treatment group) for 13-week-old rats and n=16, 8 in each treatment group, for 9-week-old rats. GWAT: gonadal white adipose tissue, IWAT: inguinal (subcutaneous) white adipose tissue mass. M and F, males and females, respectively (13 week old at the time of AAV-shRNA infusion, and 17–18 week old at the time of tissue collection), YF: younger females, 9 week old at the time of infusion, and 15 at the time of tissue collection. Shaded gray area indicates a period of operant testing performed to capture a potential interaction of the knockdown with the estrous cycle. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.