Neuropeptide Y (NPY) exerts a powerful orexigenic effect in the hypothalamus. However, extra-hypothalamic nuclei also produce NPY, but its influence on energy homeostasis is unclear. Here we uncover a previously unknown feeding stimulatory pathway that is activated under conditions of stress in combination with calorie-dense food; NPY neurons in the central amygdala are responsible for an exacerbated response to a combined stress and high-fat-diet intervention. Central amygdala NPY neuron-specific Npy overexpression mimics the obese phenotype seen in a combined stress and high-fat-diet model, which is prevented by the selective ablation of Npy. Using food intake and energy expenditure as readouts, we demonstrate that selective activation of central amygdala NPY neurons results in increased food intake and decreased energy expenditure. Mechanistically, it is the diminished insulin signaling capacity on central amygdala NPY neurons under combined stress and high-fat-diet conditions that leads to the exaggerated development of obesity.

The connection between stress and eating patterns is complex. While stress often leads to people eating less, the combination of stress and access to high-calorie food leads to higher food consumption and obesity. A team of researchers from the Garvan Institute in Australia show that chronically stressed mice, which were kept isolated from other mice, became fatter than those kept in a stress-free environment because they ate more of the high caloric food and used less energy to produce heat. The researchers go on to identify the brain circuitry involved in increased food intake and decreased energy burning. Their results highlight the importance of healthy food choices, especially under conditions of prolonged stress.

It is well established that NPY is an anxiolytic peptide that is widely expressed in different amygdala subnuclei where its expression leads to decreased anxiety, particularly in the central (CeA) and basolateral amygdala (BLA) (). NPY has also been described as a regulator of “emotional eating” because of its role in the response to stress in psychiatric disorders (). For example, low-serum NPY concentrations have been found in subjects with post-traumatic stress disorder and depression (), psychiatric conditions classically associated with a loss of appetite. Conversely, increased NPY is associated with stress resilience in subjects that have been exposed to traumatic experiences (). Therefore, dynamic changes in Npy expression levels in the amygdala in response to stress may be an important biochemical signal underlying stress-dependent eating. Even though the role of amygdala-derived NPY in regulating fear and anxiety has been well studied, the part it plays in regard to the regulation of feeding and energy homeostasis is largely unknown. In order to understand the potential interaction between stress-related NPY expression in the CeA and its influence on feeding, we utilized a set of novel mouse models that allow for NPY neuron-specific deletion or overexpression of Npy in an inducible adult-onset fashion, as well as the selective activation of insulin-responsive amygdala NPY neurons, to identify the network linking NPY neurons in the amygdala to critical areas in the hypothalamus that control energy homeostasis.

The hypothalamic-pituitary-adrenal (HPA) axis has been implicated in this process and is tightly intertwined with endocrine parameters that regulate appetitive behaviors. In addition to regulation by the circadian rhythm, studies have suggested that energy balance is also regulated by feedback loops involving glucocorticoids, insulin, leptin, and neuropeptide Y (NPY) under acute HPA activation (). The interactions between these hormones facilitate the storage, distribution, and release of energy according to needs and contribute to the initiation and termination of a meal. Under normal, unstressed circumstances insulin and glucocorticoids have antagonistic effects on metabolism in the periphery (), creating a finely balanced system in order to provide sufficient fuel for the organism proportionate to demands. Importantly, however, glucocorticoid response elements are also located within the promoter of the Npy gene itself (), suggesting a critical direct control of Npy expression under stress conditions. Furthermore, there is also evidence that in addition to glucocorticoids, insulin can directly control Npy expression particularly in the amygdala, opening up an additional major pathway for NPY to influence energy homeostasis under stress conditions ().

The body constantly adapts to both psychological and physiological stressors to maintain homeostasis. While in acute stress this is adaptive, chronic stress has adverse consequences on many organ systems as well as on a variety of physiological processes including eating behavior, adiposity, and fat distribution. Animal studies have shown that stress can lead to an increase, but mostly to a decrease, in food intake (). In rats, for example, the dose response relationship between stress and reduced food intake suggests that decreased food intake and weight loss can serve as reliable markers of stress severity (). Importantly, however, when rats have access to palatable food, high in fat or sugar, stress significantly increases the intake of the palatable food (). In humans, stress also affects eating in a bidirectional way; while some individuals decrease their food intake and lose weight during or after stress, the majority of people actually increase their food intake during stress (). Furthermore, during times of stress, most people report an increase in the intake of highly palatable foods, independent of overall hyperphagia or hypophagia ().

To prove more definitively that the impairment of insulin signaling in CeA NPY neurons is responsible for the food over-consummatory behavior observed in mice under HFDS condition, we generated a new mouse model, Npy;Insr, in which the Insr can be deleted specifically in the CeA NPY neurons after the stereotaxic injection of 4-hydroxytamoxifen (4-H-TAM). The successful deletion of the insulin receptor gene (Insr) in the CeM was verified by RNAscope ( Figure 7 A) and qRT-PCR ( Figure 7 B). Consistently, Npy expression was also significantly higher in the 4-H-TAM-injected Npy;Insrmice than saline-injected Npy;Insrcontrol mice ( Figure 7 C). For functional evaluation, Npy;Insrmice injected with 4-H-TAM were allowed to recover for 2 weeks before entering the HFDS paradigm. Interestingly, specific deletion of Insr only in CeA NPY neurons also led to increased body weight gain ( Figure 7 D) and overall body weight ( Figure 7 E). Serum corticosterone was not changed ( Figure 7 F), which is consistent with the NPY overexpression model. Body fat mass determined by DXA was significantly increased after 2 weeks of HFDS ( Figures 7 G and 7H), while there were no significant changes in lean mass or BMD ( Figures 7 I and 7J). BMC was increased after 2 weeks of treatment but was not different between 4-H-TAM-injected and saline-injected Npy;Insrmice ( Figure 7 K). Consistent with the DXA results, and similar to the CeA NPY overexpression experiment, the weights of most of the dissected individual fat depots (i, e, and m) were significantly elevated in the 4-H-TAM-injected Npy;Insrmice ( Figures 7 L and S7 F), while the weights of other peripheral organs were not changed ( Figures S7 E and S7G). Similar to Npy overexpression, deletion of Insr specifically in NPY neurons of the CeA also increased HFD consumption. Metabolic phenotyping revealed that the obese phenotype was predominantly due to a combination of increased caloric intake and decreased EE ( Figures 7 M and 7N). RER was slightly lower ( Figure 7 O), while no difference was found in physical activity in the 4-H-TAM-injected Npy;Insrmice ( Figure 7 P). Together, our data suggest that the loss of regulatory function of insulin signaling specifically on CeA Npy expression is a key contributor to the accelerated obese phenotype seen under chronic stress.

(B and C) Quantification of the expression of Insr (B) and Npy (C) mRNA in dissected CeA of Npy Cre/+ ;Insr lox/lox injected with 4-H-TAM or saline. Data are means ± SEM, 4–6 mice per group.

To test whether stress combined with HFD also triggers such regulatory effects of insulin in the CeA NPY neurons, we first measured serum insulin levels in the four different diet groups. While there was no major difference in basal insulin levels between the Chow and ChowS group, the HFD group showed an expected elevation of insulin ( Figure 6 D). Importantly, this increase was further enhanced when HFD was combined with chronic stress ( Figure 6 D). To further investigate whether CeA Npy is regulated by insulin, we performed a dose-response curve and time course experiment by injecting insulin directly into the CeA followed by qRT-PCR ( Figure 6 E) and found that Npy expression was most significantly downregulated by a dose of 5 mU insulin 4 h after injection ( Figures 6 F and 6G). To also investigate the responsiveness of Npy expression to insulin in the CeA under chronic stress, a set of WT mice was exposed to our stress paradigm and then injected with insulin into the CeA at the end of the study ( Figure 6 H). Interestingly, after insulin infusion, CeA NPY was significantly downregulated in the Chow, ChowS, and HFD mice ( Figure 6 I). Importantly, however, insulin failed to affect Npy mRNA levels in the HFDS mice in which Npy remained highly upregulated when compared to all the other experimental groups, demonstrating that even high levels of insulin were no longer able to control these NPY neurons ( Figure 6 I). In addition, while baseline GTT was not different between groups ( Figure 6 J), this central insulin resistance phenotype was also reflected by an impaired glucose metabolism in the HFDS group compared to the HFD group with a reduced glucose clearance rate during GTT ( Figure 6 K). Consistent with this, after 4 weeks of the treatment, the HFDS mice still exhibited the most impaired glucose clearance rate when compared to all the other treatment groups ( Figure 6 L).

Of the known peripheral factors that signal energy status to the brain, leptin and insulin are the most important ones. Both are known to influence NPY neurons in the hypothalamus by directly signaling through their respective receptors (). However, their influence on neurons within the amygdala complex is less clear. It has been suggested that peripheral circulating hormones could also provide homeostatic feedback to the brain in extra-hypothalamic sites such as the CeA (). Our TRAP-seq data revealed that the expression of the insulin receptor (Insr) is far higher than that of the leptin receptor (Lepr), which was not detectable in NPY neurons of the amygdala complex ( Figure 6 A). To further determine whether Insr or Lepr is actually co-localized with Npy in the CeA, we employed RNAscope. Consistent with the previous finding, we found that both Lepr and Insr were co-localized with Npy in the Arc ( Figure 6 B). Interestingly, a high level of Insr mRNA can be found in CeA Npy-expressing cells, while Lepr mRNA was completely absent from CeA NPY neurons ( Figure 6 C), suggesting that insulin is most likely the primary signal communicating with these NPY neurons. This is supported by results from previous work showing that insulin infusion into the CeA downregulates Npy expression, thereby subsequently reducing food intake ().

(J–L) Glucose tolerance tests (GTTs) were performed in Chow, ChowS, HFD, and HFDS mice at baseline (J), 2 weeks (K), and 4 weeks (L) after the treatment. Data are means ± SEM, 5–8 mice per group. Results of the GTT were also expressed as area under the curve. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

(G) Expression of Npy (relative to Actb expression ± SEM, 4–6 mice per group) in response to stereotaxic injection of 5 mU insulin in the CeA at different time points (relative to Actb expression ± SEM, 4–8 mice per group).

(C) RNAscope images of Npy and Insr mRNA or Npy and Lepr mRNA at single-cell resolution in the central amygdala (CeA). Npy mRNA, red; InsR and lepR, dark blue. Higher magnification images of neurons in white boxed areas. White arrows point to cells with Npy and Insr coexpression, and black arrows point to neurons with Npy expression only.

(B) RNAscope images of Npy and Insr mRNA or Npy and Lepr mRNA at single-cell resolution in the arcuate nucleus (Arc). Npy mRNA, red; InsR and lepR, dark blue. Higher magnification images of neurons in white and black boxes. White arrow points to neurons with NPY and Insr coexpression or NPY and Lepr coexpression. Black arrow points to neurons that only express Insr or Lepr, but not NPY.

RNA-seq analysis of the RNA isolated from the amygdala of the Npy;TRAPmice identified a total of 23,983 genes that were mapped to the mm10 mouse reference genome (UCSC). In the immunoprecipitated (IP) sample, 4,295 genes were enriched significantly with at least a 1.5-fold (or 0.58-fold with logscale) higher FPKM value compared to the corresponding input control ( Figure S6 D). A total of 2,320 genes were depleted in the IP samples, indicating that these genes are not highly expressed in the NPY neurons. Volcano plot of the data revealed that the majority of the enriched genes have between 0.58- and 2-fold enrichment (logscale), while a subset of 132 genes were found to be enriched over 2-fold (logscale; Figures S6 E, S7 A, and S7B; Table S1 ). As expected, Npy is among one of the highest enriched genes with a 14-fold higher FPKM value compared to the input control ( Figure S6 F). Importantly, a group of genes that have previously been identified () as marking other cell types in the brain such as astrocytes, oligodendrocytes, microglia, and endothelial cells were all deriched, and consistently, neuron-specific marker genes were significantly enriched in the IP sample compared to input control ( Figure S7 C). Interestingly, both Npy1r and Npy5r were significantly depleted in the IP sample, while there was no significant difference in the abundance of the Npy2r ( Figures S6 F and S7 D). These results are consistent with the notion that Npy2r are auto-receptors on NPY neurons () but may also have a distinct functional role in non-NPY neurons (), while Npy1r and Npy5r are mostly found post-synaptically. Our results also demonstrated that these NPY neurons are GABAergic (), with both Gad1 (encoding for GAD65) and Gad2 (encoding for GAD67) genes highly enriched in the IP samples ( Figures S6 F and S7 A). Furthermore, a number of genes that have a function in neuronal communication (Sst, Cartpt, Cort, Nts, Ngf, and Pnoc), gene regulation (Lhx6), and stress response (Crh and Crhbp), as well as a gene, Htr2a, that marks orexigenic neurons () were also significantly enriched ( Figures S6 F, S7 A, and S7B). Importantly, Prkcd, which is the gene encoding for PKCδ and has been linked to anorexigenic neurons in the CeA (), was significantly reduced in NPY neurons ( Figures S6 F and S7 D), supporting the notion that NPY neurons in the CeA play a key function in promoting food intake.

To get a clearer insight into the nature of the NPY neurons in the amygdala, we investigated their translational profile by employing TRAP technology and next-generation RNA sequencing (RNA-seq). For this, we utilized our Npy;TRAPmice ( Figure S6 A). For RNA-seq, amygdala tissue was isolated from these mice and processed via immunoprecipitation with an anti-GFP antibody for subsequent isolation of the actively translating RNA bound by the ribosomes ( Figure S6 B). The corresponding input samples were used as the baseline control to reflect the endogenous expression of each gene. To confirm the efficiency of our immunoprecipitation method and the specificity toward NPY neurons, we first performed qRT-PCR to show that both Npy and GFP transcripts were significantly enriched only in the amygdala of the Npy;TRAPmice, but not in the amygdala of the WT mice or the cerebellum of Npy;TRAPmice where NPY is not normally expressed ( Figure S6 C). As an additional control, no Npy or GFP enrichment was found when we immunoprecipitated the tissue without the GFP antibody ( Figure S6 C).

To test the possibility of whether deletion of Npy in the CeA may alter the electrical properties of these neurons, we made patch-clamp recordings in brain slices ( Figure S4 G) prepared from NPY-GFP and Npy-mCherry mice ( Figures S4 G and S4H). Comparison of NPY-GFP (n = 15) and Npy-mCherry (n = 14) recordings showed that the input resistance (340 ± 34 M∧ versus 281 ± 23 M∧, p = 0.17) and resting membrane potential (−70.00 ± 1.13 mV versus −68.02 ± 1.92, p = 0.41) of these neurons did not differ in the absence of Npy expression. Furthermore, the active properties of neurons were also similar between genotypes, with no difference in rheobase current (66.7 ± 14.4 pA versus 47.1 ± 8.3 pA, p = 0.26) or frequency current (F/I) relationship of action potential discharge during step current injections ( Figures S4 I–S4K). Finally, the properties of individual action potentials (APs) did not differ between recordings, with AP threshold (−38.5 ± 0.9 mV versus −37.0 ± 1.2 mV, p = 0.32), AP amplitude (60.9 ± 1.6 mV versus 65.8 ± 2.4 mV, p = 0.10), and AP half-width (2.19 ± 0.14 ms versus 2.31 ± 0.14 ms, p = 0.55) all similar in NPY-GFP and Npy-mCherry neurons. Thus, we conclude that the behavioral consequences of Npy deletion in CeA neurons cannot be explained by compensatory changes to the electrical properties of this population.

To assess whether these effects on increasing food intake and reducing EE upon activation of CeA neurons actually require the presence of NPY, we tested a further model. In this model, we utilized our NPY-Cre knockin mice that carry the Cre-recombinase gene instead of Npy under the endogenous NPY promoter, which in the homozygote state (Npy) represent Npymice. Importantly, CNO treatment of Npy;AAV-hM3Dq mice did not show any effect on food intake ( Figure 5 M), EE ( Figure 5 N), or physical activity ( Figure 5 O), similar to the same mice being injected with saline. These data unambiguously prove that NPY within these CeA neurons is the critical neurotransmitter that mediates the increase in food intake and the decrease in EE.

Importantly, food intake during the first 5 h after CNO injection was significantly increased in the Npy;AAV-hM3Dq mice compared with that of the same mice treated with saline ( Figure 5 G). As an additional negative control, we injected our Npymice with an AAV empty vector to generate Npy;AAV-Empty mice. Importantly, the CNO-induced feeding response seen in the Npy;AAV-hM3Dq mice was absent in the control mice ( Figure 5 J). Interestingly, CNO injection into the Npy;AAV-hM3Dq mice also led to a significant reduction in EE ( Figure 5 H), which again was not seen in the Npy;AAV-Empty control mice ( Figure 5 K). Physical activity, on the other hand, was not altered in response to CNO in either the Npy;AAV-hM3Dq or the Npy;AAV-Empty control mice ( Figures 5 I and 5L). Together, this suggests that activation of CeA NPY neurons drives food consummatory behavior and at the same time prevents the normally seen activation of diet-induced EE.

Since both hypothalamic areas identified are known to be important in feeding and energy homeostasis regulation, we performed additional functional studies to investigate the physiological consequences immediately following CeA NPY neuronal activation. For this, Npy Cre/+ mice injected with the stimulatory DREADD hM3Dq into the CeA and mice injected with an empty AAV control vector were allowed to recover for 14 days and then entered into our metabolic phenotyping system (Promethion) for a detailed evaluation of energy homeostatic parameters. Mice were injected with either CNO or saline at the start of the dark phase, and food intake and EE were recorded. The next day, the experiment was repeated but in the reverse order so that each mouse acted as its own control.

To verify whether NPY neurons in the CeA directly project to cells residing in the Arc and PVN, we employed an AAV-FLEX construct that expresses the synaptic protein synaptophysin fused with an EGFP (EGFP-synaptophysin; AAV-FLEX-tdTomato-SynEGFP;). Virus functionality and selectivity were confirmed by unilateral injection into the CeM of the Npy;TRAPmice and subsequent visualization of both GFP and RFP signals in the same cell in the CeM ( Figure S5 A). For the neuron tracing experiment, we injected the construct into the CeM of Npymice and verified the selectivity of the expression in the CeM and not in the CeL ( Figure S5 B). The EGFP-synaptophysin fusion protein was found in synapses of both RFP-positive neurons and RFP-negative neurons, suggesting that there are direct projections from NPY-expressing neurons to surrounding non-NPY neurons in the CeA. Consistent with the cFos activation experiments, EGFP-synaptophysin-labeled synapses were found in both PVN and Arc neurons of the CeA-injected brains, but not in the control-injected brains ( Figures S5 C and S5D).

To further define the downstream pathways that are influenced by the activation of CeA NPY neurons, we employed DREADD technology. We first injected our Npy;TRAPmice with the AAV-FLEX-hM3Dq-mCherry construct bilaterally into the CeM region, which expresses a high level of Npy ( Figure 5 A). Successful delivery of AAV-FLEX-hM3Dq-mCherry receptors into NPY neurons was confirmed by the visualization of red fluorescence protein (RFP) in the CeM region between the SI and internal capsule juxtaposing the BLA that showed complete overlap with the green fluorescence only produced by NPY neurons ( Figures 5 Bi–5Biv). Importantly, no RFP-only-expressing neuron cell bodies were detected, confirming the NPY neuron-specific expression of the hM3Dq receptor ( Figures 5 Bv–5Bxiii). That our injection of the AAV-FLEX-hM3Dq-mCherry receptors is highly area specific is also shown in consecutive sections spanning across the rostral to caudal CeM, but not into the CeL ( Figures S4 A and S4B). For functional studies, we utilized Npymice without the GFP reporter and visualized the expression of cFos activity as an indicator of neuronal activity using the green channel ( Figure 5 C). Precision of the CeM injection was confirmed by visualizing RFP between SI and internal capsule ( Figure 5 C), and successful activation of cFos activity in the CeA is shown by the increased number of Fos-positive cells in this nuclei 1 h after Clozapine N-oxide (CNO) treatment ( Figure 5 C). The CNO-induced cFos activity was also used to identify areas that may be functionally connected with CeA NPY neurons. From this analysis, two main regions were identified including the Arc ( Figure 5 D) and the paraventricular nucleus (PVN; Figure 5 E), both showing prominent increases in cFos activity in CNO-injected mice compared to saline-injected control mice. Interestingly, we found that the majority of these Fos-positive cells in the Arc resided predominantly in an area that is densely packed with NPY-expressing neurons ( Figure 5 F), suggesting that an NPY-dependent CeA circuitry may modulate Arc NPY neurons.

(F) Visualization of DAPI (blue), NPY (red), and cFos (green) staining in the Arc of Npy Cre/+ ;AAV-FLEX-hM3Dq mice 1 h after CNO injection. Merged image shows co-localization of DAPI, NPY, and cFos staining. SI, substantia innominate; CeL, lateral nuclei of central amygdala; CeM, medial nuclei of central amygdala; BLA, basolateral amygdala; Arc, arcuate nucleus; PVN, paraventricular nucleus; 3 rd V, third ventricle.

NPY mediates its effects by signaling through at least five different Y receptors in the mouse (), and as such the overproduction of NPY per se cannot discriminate the pathway(s) activated. Importantly, NPY acting through Y1 signaling has been demonstrated to exert anxiolytic effects by antagonizing the anxiogenic effect induced by corticotropin-releasing hormone (). To gain more insights into this, we repeated the overexpression experiment by employing a modified AAV-FLEX-NPY viral construct (FLEX-L/P/-NPY), known to have preference for the Y1 receptor (). Importantly, a significant increased body weight gain was observed in mice that received the FLEX-L/P-NPY ( Figure 4 D), accompanied by a significantly higher fat mass after 2 weeks of HFDS treatment ( Figures 4 F and 4J). This increase in body fat mass was also predominantly due to increased caloric intake ( Figure 4 E) and no effect was seen on lean mass, BMD, or BMC ( Figures 4 G–4I). Interestingly, preferential activation of Y1 receptor signaling pathways resulted in significantly lower EE compared to endogenous NPY overexpression ( Figures 4 L and 4M). Similarly, while no differences were found in physical activity or RER between NPY-overexpressing and GFP-expressing mice ( Figures 4 N and 4P), L/P-NPY-overexpressing mice showed a significantly lower RER ( Figure 4 Q) and a slight reduction in physical activity ( Figure 4 O). Serum corticosterone was again not affected by L/P-NPY overexpression ( Figure 4 K). Taken together, use of theNpy variant suggests that Y1 receptor responsive pathways are activated by CeA-derived NPY neurons during stress and contribute to the establishment of the increased obesity in the HFDS model.

To verify the results above as well as to determine the consequences of elevated NPY signaling in the CeA in the context of stress-induced obesity development, we generated a CeA-specific NPY overexpression model utilizing our Npyknockin line and an AAV-FLEX-NPY vector ( Figure 4 A). Male Npymice were bilaterally injected with the AAV-FLEX-NPY vector into the CeA. To confirm the overexpression of Npy, qRT-PCR was performed, which demonstrated significantly higher Npy mRNA levels in the amygdala of the AAV-FLEX-NPY vector injected mice compared to control mice ( Figure 4 B). Overproduction of NPY at the protein level was also confirmed by immunohistochemistry showing strong and specific NPY staining in both the CeM neuron fibers and some synapses juxtaposing the substantia innominate (SI), internal capsule, and BLA ( Figure 4 C) of both brain hemispheres (H1 and H2; Figure 4 C). For functional evaluation, Npymice injected with the AAV-FLEX-NPY vector were allowed to recover for 2 weeks before entering the HFDS paradigm. Importantly, NPY overproduction in the CeM led to a significant increase in body weight gain ( Figure 4 D) as a result of increased calorie intake ( Figure 4 E). Body fat mass was also significantly increased after 2 weeks of HFDS ( Figure 4 F) while there was no significant change in lean mass, BMD, and BMC ( Figures 4 G–4J), indicating that the increased feeding because of the overexpression of NPY specifically impacted fat metabolism. NPY overproduction in the CeM did not further enhance the serum corticosterone level response to stress ( Figure 4 K).

To determine the role of CeA NPY in the development of obesity under HFD and stress conditions in more detail, we utilized our Npymice and selectively ablated Npy from the CeA via bilateral injection of an AAV-Cre vector using an AAV-GFP vector as control. Injection coordinates were confirmed by injecting an AAV-FLEX-mCherry vector into Npy;TRAPmice showing specific expression only in the medial nuclei of the central amygdala (CeM), but not in the lateral nuclei of the central amygdala (CeL), nor in the dorsal striatal region ( Figures 3 A and S4 A). The successful deletion of Npy in the CeM was verified by RNAscope ( Figure 3 B). In addition, in situ hybridization ( Figure S4 C) was also performed showing a significant knockdown of Npy mRNA ( Figure 3 C). For in vivo evaluation, male Npymice injected the same way as described above were allowed to recover for 2 weeks before entering the HFDS paradigm. These mice also showed the typical increase in corticosterone levels ( Figure 3 D), suggesting that Npy in CeA neurons is not essential for the upregulation of corticosterone. Interestingly, however, CeA Npy ablated mice showed a significant reduction in body weight gain compared to control injected mice ( Figure 3 E), which also manifested itself as a significantly lower absolute body weight ( Figures 3 F and 3G). Consistent with that, DXA analysis performed 2 weeks after the start of the HFDS paradigm revealed a significantly reduced body fat mass under the HFDS paradigm ( Figure 3 H) because of a smaller gain of fat mass during the treatment period ( Figure 3 I), while no significant difference was found in lean mass, BMD, or BMC ( Figures 3 J–3L). The leaner phenotype in the Npy;AAV-Cre mice was further confirmed in individual fat deposits that showed a significant reduction in weight, both in absolute values and when normalized to body weight ( Figures 3 M and S4 D). Consistent with the observed increase in fat and liver weight in the control group, the selective deletion of Npy from the CeA also caused a reduction in liver weight but had no influence on any other tissues ( Figures S4 E and S4F). Npy;AAV-Cre mice showed a significant reduction in caloric intake ( Figure 3 N) accompanied by reduced EE ( Figure 3 O), while RER was not significantly influenced (data not shown). Physical activity was significantly increased in the Npy;AAV-Cre mice during the onset of the dark phase but otherwise not different ( Figure 3 P).

To investigate the expression of Npy in neurons of the amygdala more precisely in our four models, translating ribosome affinity purification (TRAP) technology was used (). For this we crossed Npymice with conditional TRAP mice (TRAP) to eventually generate Npy;TRAPmice in which the GFP-L10a ribosomal fusion protein is only produced in NPY neurons ( Figures S3 G and S6 A). These mice were then exposed to the same treatment regimens as above, and the amygdala region and the Arc of these mice were isolated and GFP+ ribosomal complexes were purified. Using qPCR analysis, Npy mRNA was shown to be significantly increased in the amygdala of the ChowS mice ( Figure S3 H), while under HFD conditions Npy was significantly downregulated (HFD; Figure S3 H). Interestingly, when HFD feeding was combined with the stressor, Npy was significantly increased compared to the HFD mice ( Figure S3 H). Similarly, in the Arc, Npy was significantly higher under chronic stress in both chow (ChowS) and HFD (HFDS) condition ( Figure S3 H). Together, this demonstrates that chronic stress activates the NPY system in both the Arc and the CeA, thereby reinforcing food consummatory behavior and also inducing a stress-dependent energy conservation state leading to an exacerbated development of obesity.

To identify the critical regions in the brain and the neuronal pathway that may be responsible for the enhancement of the obese phenotype in the HFDS mice, we used a candidate gene approach. One major anxiolytic neurotransmitter known to be strongly upregulated during prolonged stress is NPY, and the amygdala is a key site for this action (). Importantly, NPY is also one of the strongest inducers of feeding, and these two properties combined make NPY a strong potential candidate. In the arcuate nucleus (Arc) of the hypothalamus, chronic activation of the NPY system can trigger an energy conservation state by reducing EE (). Therefore, the dynamic changes of Npy expression in these two brain regions by stress might alter both behavior and energy homeostasis. To test this, we initially employed NPY-GFP reporter mice in which GFP expression is controlled by the Npy promoter () and exposed them to the same stress paradigm. Body weight and fat mass in these NPY-GFP mice were affected the same way by the treatment interventions as in the WT (wild-type) mice (data not shown). Brains from these mice were processed and sectioned and NPY levels were estimated by counting the number of GFP-positive (GFP+) cells in the amygdala and Arc. Under stress treatment, with and without HFD, both amygdala and Arc showed altered GFP expression compared to their corresponding diet controls, reflecting a change in the Npy promoter activity regulated by both diet and stress ( Figure S3 A). In response to stress alone in ChowS mice, more GFP+ cells were found in the lateral (LA) and BLA region, but not in the CeA ( Figure S3 A and quantified in Figures S3 B and S3C). Under HFD alone, there was no significant difference found in the number of GFP+ cells ( Figures S3 A–S3C). Strikingly, when mice were stressed in combination with HFD feeding (HFDS), the GFP+ cells significantly increased in the CeA. Interestingly, more GFP+ cells were found in the Arc of ChowS and HFDS mice when compared to their corresponding diet group ( Figures S3 E and S3F), data consistent with the observed reduction in EE in the ChowS and HFDS WT mice (). Taken together, increased Npy expression in both the CeA and Arc could be the underlying mechanism that facilitates excessive feeding with reduced EE under HFDS condition.

The increased caloric intake because of an HFD was also associated with a strong reduction in physical activity ( Figures 2 J and S2 D). Importantly, while there was also a trend toward reduced activity in response to stress in the ChowS group ( Figure 2 K), this was not further exacerbated in the HFDS group ( Figures 2 L and S2 D). Investigating the influence of HFD and stress on behavioral aspects of these mice revealed some interesting alterations in their movement patterns. Plotting the preferred location of the mice in the different groups identified that ChowS mice were much less explorative than Chow or HFD mice, and they restricted themselves to a much smaller area close to the water and food hoppers ( Figure 2 M). Interestingly, this mobility restraining effect was less severe in the HFDS cohort ( Figure 2 M), supporting the concept that a highly palatable food acts as a “comforting agent” to attenuate stress-induced anxiety behavior (). Activity categorization of these mice further supported that HFDS mice showed a trend toward increased interaction with the hopper to take food more frequently than HFD mice, while ChowS mice showed a significantly reduced interaction with the food hopper compared to Chow mice ( Figure S2 E). This may also indicate that stressed mice were attracted to consume a more palatable diet, which acted to dampen their stress-induced anxiety.

In order to determine the underlying cause of the altered body weight gain seen in the different treatment groups, we next analyzed food intake. As expected, caloric intake in the HFD mice was significantly higher than in the Chow mice ( Figure 2 A). Interestingly, however, while ChowS mice displayed a significantly reduced ad libitum basal cumulative caloric intake ( Figures 2 B and S2 A), under HFDS conditions this effect was reversed ( Figures 2 C and S2 A), demonstrating that the causative effect of stress on body weight gain under high-fat-diet conditions is increased intake of the highly palatable food. EE was significantly higher in the HFD group compared to that of the Chow group ( Figure 2 D), consistent with their increased energy intake and increased thermogenesis. In contrast, EE was significantly lower in the ChowS mice compared to Chow mice, again consistent with their observed lower energy intake ( Figures 2 E and S2 B). Interestingly, this stress-induced reduction in EE was also recorded in the HFDS compared to the HFD mice despite these mice consuming an increased amount of calories ( Figures 2 F and S2 B), indicating that stress can override the otherwise typical increase in EE caused by an HFD ( Figures 2 D and S2 B). The fuel source preference determined by the respiratory exchange ratio (RER) shifted toward a preferential use of fat as an energy source in the HFD compared to the Chow group ( Figure 2 G). Importantly, this shift was also induced by the stressor in the ChowS group ( Figure 2 H) and further enhanced when combined with the high-calorie diet in the HFDS group ( Figures 2 I and S2 C).

(M) Visualization of 24 h footprint records of Chow, ChowS, HFD, and HFDS mice. Each dot represents the XY coordinate of the mice within the chamber. H 2 O, water hopper; F, food hopper; Home, resting area. Data are represented as means ± SEM, 4 mice per group. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Shaded area from (A) to (L) indicates dark phase and unshaded area indicates light phase. Bar graphs on the top left corner show the average 24 h measurement of each group of mice. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

In order to capture the early responses to these treatments and avoid the potential complications from compensatory effects that may occur during prolonged stress, the initial experiments were performed over a 2-week period. Animals were maintained on an HFD in which 43% of the calories came from fat. Importantly, after only 5 days of treatment, HFD compared to Chow mice began to show significant increases in body weight, both in absolute values ( Figure 1 F) and when expressed as body weight gain ( Figure 1 G). Mice on chow exposed to the stressor (ChowS) showed no difference in body weight compared to unstressed mice (Chow) throughout the 2-week period ( Figures 1 F and 1G). Importantly, when combining the highly palatable diet with the stressor, the HFDS group showed significantly accelerated body weight gain and increased body weight compared to all other groups ( Figures 1 F and 1G). This significantly increased body weight gain in the HFDS group was almost entirely due to an increase in fat mass ( Figure 1 H). Although lean mass was not affected ( Figure 1 I), whole-body mineral density (BMD) and bone mineral content (BMC) were significantly reduced in the ChowS group ( Figures 1 J and 1K), consistent with the known negative impact of chronic stress on bone mass (). Analysis of the weight of individual fat depots revealed that combined HFD and stress treatment promoted white adipose tissue (WAT) gain uniformly in all major depots, including inguinal (i), epididymal (e), mesenteric (m), and perirenal fat (r), both in absolute values ( Figure 1 L) and when normalized to body weight ( Figure S1 A), without affecting other tissue weights ( Figures S1 B and S1C). While BAT weight was also increased by HFD feeding, it was not further changed in HFDS mice ( Figure 1 L). To exclude any impact of the 10°C water-altering thermogenesis and affecting the phenotype, we repeated our experiment using room-temperature (RT) water ( Figure S1 D). This paradigm also induced significant increases in serum corticosterone in both ChowS and HFDS mice ( Figure S1 E); the HFDS mice developed the same degree of obesity ( Figure S1 F) and metabolic phenotype ( Figures S1 I–S1K) as the cold-water-induced mice ( Figure 1 L) without affecting overall body temperature ( Figures S1 G and S1H), demonstrating that the development of obesity driven by our stress protocol is unlikely to have been caused by the 10°C water.

To investigate the impact of chronic stress on the regulation of body composition and body weight under different diet conditions, four groups of mice (1) chow-fed, no stress (Chow); (2) chow-fed and stress (ChowS); (3) high-fat-diet fed, no stress (HFD); and (4) high-fat-diet fed and stress (HFDS), were tested in a comprehensive phenotyping paradigm. As an effective stressor, we used our established methodology () that involves placing individual mice alone in a home cage in which the bedding has been replaced by 1 cm deep, 10°C water where they can freely move about for 1 h, 3 times per week, at T21°C ( Figure 1 A). This protocol induced a consistent stress response as shown by significantly increased serum corticosterone levels ( Figure 1 B). Consistent with the known effect of high-fat diet on energy expenditure (EE), body temperature as measured by infrared monitoring of skin temperature over the brown adipose tissue (BAT) and lumbar spine region ( Figure 1 C) did increase in the HFD groups ( Figures 1 D and 1E). Importantly, however, the stress protocol itself did not influence body temperature with unaltered values observed in the stressed compared to unstressed animals on the same diet ( Figures 1 D and 1E).

(F and G) Body weight (F) and body weight gain (G) of Chow, ChowS, HFD, and HFDS mice starting after baseline recording at 12 weeks of age. Baseline refers to the body weight before the stress treatment while day 1 to day 12 refers to the day after the treatment has started. ∗∗∗ p < 0.001 Chow versus HFD; ###p < 0.001 HFDS versus ChowS; %%%p < 0.001 or %p < 0.05 HFDS versus HFD.

Discussion

Results from this study demonstrate the critical role of NPY neurons in the amygdala in controlling feeding and energy homeostasis, which is especially important under conditions of stress combined with the ingestion of highly palatable food. Specifically, we identified CeA NPY neurons as the principal contributor to the increased food consummatory behavior that promotes the development of obesity under high-stress conditions. Importantly, lack of NPY selectively in CeA neurons attenuated the obese phenotype, while overproduction of NPY in the CeA further enhanced it. Moreover, even when only a normal chow diet is provided, the acute specific activation of CeA NPY neurons by chemogenetic tools is sufficient to increase food intake and lower EE. Analysis of the transcriptomic nature of these CeA NPY neurons demonstrated that they belong to a specific subclass of neurons that have all the hallmarks for activation under stress conditions. Finally, through the use of TRAP-seq data and single-cell resolution mRNA in situ hybridization technology, we discovered that the insr, but not the lepr, is predominantly expressed in CeA NPY neurons and that the loss of insulin responsiveness in these NPY neurons under HFDS condition leads to excessive NPY levels that are the primary cause for the food over-consummatory behavior in these mice. Together, these data provide conclusive evidence that the coordinated activation of NPY neurons in the CeA and the Arc is a key driver for the exaggerated development of obesity under conditions of stress and high-calorie-diet consumption.

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et al. Arcuate NPY controls sympathetic output and BAT function via a relay of tyrosine hydroxylase neurons in the PVN. In our study, HFD combined with chronic stress facilitated both an increase in palatable food consumption and a decrease in EE as a result of the combined upregulation of NPY signaling in the CeA and Arc. In chronic stress models, stress normally reduces food intake, and in some cases body weight is also reduced correlating with the degree of stress severity (). Interestingly, in rats forced to swim repeatedly for 1 h a day, which induces hypophagia, body weight gain was not different from the control group, suggesting that compensatory mechanisms exist to restore energy balance (). Similarly, although caloric intake was reduced in our ChowS mice, we found that the overall body weight gain and body fat composition were not changed, suggesting that a new homeostatic set point was established in these mice. Our results also highlight that this restoration of energy balance is likely mediated via the activation of Arc-NPY signaling during chronic stress. We previously demonstrated that NPY derived from the Arc is critical for mediating sympathetic outflow and controlling BAT function, with chronic overexpression of NPY, specifically in the Arc, leading to significantly increased food intake and lower EE, establishing an energy conservation state in these mice (). In our ChowS mice, stress also increased NPY levels in the Arc, causing the mice to develop a condition with significantly reduced EE compared to the Chow mice. Interestingly, lowered EE was also found in our HFDS mice, contrasting to the HFD mice where over-consumption of calories led to an increase in EE in response to increased food intake, in an attempt to maintain the original homeostatic set point. Importantly, although the HFDS mice eat even more than the HFD group, they lost the ability to re-adjust their energy homeostatic system by raising EE, and instead HFDS mice develop an even lower EE set point than the Chow control mice.

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Klein R. Central amygdala circuits modulate food consumption through a positive-valence mechanism. Leu31,Pro34NPY almost completely recapitulated the effect of NPY overexpression. Chronic overexpression of Leu31,Pro34NPY also triggered the establishment of a lower EE set point, indicating that activation of Y1R signaling favors the energy-conserving state. However, we did not see a further exacerbation of obesity in the Leu31,Pro34NPY-overexpressing mice, suggesting that chronic activation of Y1R signaling might also activate other potential compensatory mechanisms, which presumably lead to increased fatty acid usage as the primary fuel that is reflected by the lower RER compared to AAV-NPY-injected and control mice. The CeA is a complex forebrain structure composed of a highly interconnected network of neurons that control rewarding behavior and fear responses (). Our results now reveal that HFD combined with chronic stress significantly activate production of NPY in Npy-expressing neurons specifically in the CeM nuclei, but not the CeL subdivision (), to also promote food intake. This is consistent with recent findings demonstrating that the CeA contains neuronal populations that are linked to anorexigenic and orexigenic output (). Specifically, a group of molecularly defined neurons that express the Htr2a gene has been demonstrated to modulate feeding behavior (). Molecularly, this group of cells does not overlap with an anorexigenic neuronal cell population expressing PKCδ (), but partially overlaps with other CeA markers that play a role in positive reinforcement including somatostatin (Sst), corticotropin-releasing hormone (Crh), and neurokinin B (Tac2). Interestingly, our TRAP-seq data also revealed that Htr2a, Sst, Tac2, and Crh transcripts are all, to different degrees, significantly enriched in the NPY neurons, while Prkcd (encoding for PKCδ) was lacking, indicating that NPY neurons overlap predominantly with the orexigenic Hrt2a cell population. We also demonstrated functionally that acute chemogenetic activation of CeA NPY neurons leads to a robust increase in ad libitum food intake that is entirely dependent on the presence of NPY. The fact that these neurons only get activated when HFD is combined with a stressor confirms the critical role of these NPY CeA neurons in mediating excessive feeding behavior under these conditions. Our data also demonstrate that ablation of Npy in these CeA NPY neurons effectively attenuates the obese phenotype because of reducing feeding, while on the contrary the elevation of NPY further promotes an obese phenotype. The CeA NPY-dependent development of an obese phenotype is likely mediated via NPY-Y1R signaling since chronic overexpression of the Y1R-preferringNPY almost completely recapitulated the effect of NPY overexpression. Chronic overexpression ofNPY also triggered the establishment of a lower EE set point, indicating that activation of Y1R signaling favors the energy-conserving state. However, we did not see a further exacerbation of obesity in theNPY-overexpressing mice, suggesting that chronic activation of Y1R signaling might also activate other potential compensatory mechanisms, which presumably lead to increased fatty acid usage as the primary fuel that is reflected by the lower RER compared to AAV-NPY-injected and control mice.

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York D.A. High-fat diets induce a rapid loss of the insulin anorectic response in the amygdala. Our chemogenetic results also provide evidence that CeA NPY-derived signaling may critically influence neuronal populations in the Arc and the PVN, forming the basis for a coordinated and potentially synergistic activation and enhancement from these pathways. Importantly, the activation of these neurons causes not only increased food intake but also a subsequent reduction in EE, suggesting that the NPY neurons in the CeA critically contribute to these functions. In support of this notion, CeA insulin infusion has been shown to trigger neuronal cell responses (Akt phosphorylation) in the Arc and PVN (), indicating that the insulin-specific anorectic action is likely mediated via neurons residing in these regions (). Taken together, our results demonstrate a novel role of CeA NPY neurons, which most likely via Y1 receptor signaling control both feeding behavior and energy homeostasis through the coordinated activation of amygdala and hypothalamic pathways, which are particularly important under conditions of stress in combination with calorie-dense food.