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. 2024 Feb 21;82:101904. doi: 10.1016/j.molmet.2024.101904

Effects of chronic high fat diet on mediobasal hypothalamic satiety neuron function in POMC-Cre mice

Özge Başer 1,4, Yavuz Yavuz 1,4, Deniz Öykü Özen 1, Hüseyin Buğra Özgün 1, Sami Ağuş 1, Cihan Civan Civaş 1, Deniz Atasoy 2, Bayram Yılmaz 1,3,∗
PMCID: PMC10910127  PMID: 38395148

Abstract

Objective

The prevalence of obesity has increased over the past three decades. Proopiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus (ARC) play a vital role in induction of satiety. Chronic consumption of high-fat diet is known to reduce hypothalamic neuronal sensitivity to hormones like leptin, thus contributing to the development and persistence of obesity. The functional and morphological effects of a high-calorie diet on POMC neurons and how these effects contribute to the development and maintenance of the obese phenotype are not fully understood. For this purpose, POMC-Cre transgenic mice model was exposed to high-fat diet (HFD) and at the end of a 3- and 6-month period, electrophysiological and morphological changes, and the role of POMC neurons in homeostatic nutrition and their response to leptin were thoroughly investigated.

Methods

Effects of HFD on POMC-satiety neurons in transgenic mice models exposed to chronic high-fat diet were investigated using electrophysiological (patch-clamp), chemogenetic and Cre recombinase advanced technological methods. Leptin, glucose and lipid profiles were determined and analyzed.

Results

In mice exposed to a high-fat diet for 6 months, no significant changes in POMC dendritic spine number or projection density from POMC neurons to the paraventricular hypothalamus (PVN), lateral hypothalamus (LH), and bed nucleus stria terminalis (BNST) were observed. It was revealed that leptin hormone did not change the electrophysiological activities of POMC neurons in mice fed with HFD for 6 months. In addition, chemogenetic stimulation of POMC neurons increased HFD consumption. In the 3-month HFD-fed group, POMC activation induced an orexigenic response in mice, whereas switching to a standard diet was found to abolish orexigenic behavior in POMC mice.

Conclusions

Chronic high fat consumption disrupts the regulation of POMC neuron activation by leptin. Altered POMC neuron activation abolished the neuron's characteristic behavioral anorexigenic response. Change in nutritional content contributes to the reorganization of developing maladaptations.

Keywords: Obesity, POMC neurons, Electrophysiology, Chemogenetic, High fat diet, Behavioral

Highlights

  • •

    HFD did not alter POMC neuron projections.

  • •

    HFD reversed the anorexigenic behavior of POMC neurons.

  • •

    HFD impaired the response of POMC neurons to leptin.

  • •

    HFD altered the firing frequency and post-synaptic currents of POMC neurons.

  • •

    HFD altered POMC neuron excitability.

1. Introduction

Obesity is a chronic and multifactorial disease that is defined as the accumulation of fat in the body as a result of an imbalance between calorie intake and energy expenditure [1,2]. This complex condition not only creates health risks of its own, but also increases the likelihood of other diseases affecting various body systems. It affects multiple body systems, including cardiovascular and metabolic systems [3]. Understanding obesity and its associated risks is crucial for promoting overall health and preventing development of complications [4].

Today, sedentary lifestyle along with easy access to high-calorie diets is considered to be one of the causes of obesity [5,6]. The effects of continuous consumption of certain diets on metabolic regulation in the peripheral and central nervous systems have been extensively investigated. Maladaptations in the neural networks and endocrine system in pathophysiology of obesity has recently gained more attention [1,[7], [8], [9]]. It has been suggested that excessive consumption of a high-fat diet may affect energy metabolism in the long term, disrupt hypothalamic neural networks and lead to obesity [[10], [11], [12]].

Neural networks located in the hypothalamic arcuate nucleus (ARC) receive and integrate peripheral signals and projections from other regions of the brain to regulate energy homeostasis [7,13,14]. These neurons in the ARC act as an interface, receiving and processing information on neurotransmitters, neuromodulators and signals related to hormones and nutrients. Agouti-related peptide (AgRP)/Neuropeptide Y (NPY) neural circuit located in this hypothalamic region conveys orexigenic signals related to food intake [15,16], while neurons expressing proopiomelanocortin (POMC) play a role in satiety (anorexigenic) neurotransmission [17]. Dysfunction in these neural networks can lead to metabolic disorders such as obesity.

Leptin has a stimulatory effect on the activity of POMC neurons [18,19]. This is caused by the activation of a generic cation channel and the simultaneous inhibition of inhibitory synaptic connections in POMC cells [18,20,21]. Progression and prevention of obesity involve disruption of POMC neuronal function and loss of hormone sensitivity of POMC neurons (such as leptin) due to hypothalamic inflammation caused by chronic high-fat consumption [21]. Leptin resistance has been demonstrated in both short-term (6 days) [22] and long-term (12 weeks) HFD-fed mice [23]. The loss of leptin sensitivity has been linked to disrupted calcium balance of POMC neurons following short-term HFD consumption [24]. In addition to disrupted calcium balance, the frequency and amplitude of inhibitory postsynaptic currents (IPSC) to POMCARC neurons increased after short-term (3 days) use of HFD [25]. In addition, induction of satiety is regulated through a different neural pathway via NPY2R of POMC neurons during HFD consumption [26]. However, the question of how chronic high-calorie diet affects the functional and morphological characteristics of the ARC satiety neurons and how these effects could causally contribute to the formation and maintenance of the obese phenotype has not yet been fully elucidated. For this purpose, in the present study, POMC-Cre transgenic mice were exposed to HFD, and at the end of a 3- and 6-month periods, electrophysiological and morphological changes in these neurons were thoroughly investigated. Feeding behavior was also monitored, and relationship with potential changes in the electrophysiological and morphological findings was elucidated.

2. Methods

2.1. Animals

Sixty POMC-Cre mice (#010714, Jackson Laboratoires, Bar Harbor, ME, USA) were used in our study. All experiments were performed on adult mice (8–32 weeks old, both male and female). Animals were housed at 21 ± 2 °C on a 12:12 h light:dark cycle with ad libitum access to water and standard chow [27]. Cre recombinase-expressing POMC-Cre mouse line was backcrossed with C57BL/6J wild-type mice (Noncarrier x Hemizygote) for maintenance. The breeding of the mice was carried out at Yeditepe University Faculty of Medicine Experimental Research Center (YUDETAM), Istanbul, Turkey. Experimental procedures were approved by the Yeditepe University experimental animal research ethics committee.

2.2. Stereotaxic surgery

Intracranial injection was performed as described previously [[28], [29], [30]]. Anterograde labeling using AAV injections was performed using a stereotaxic device (David Kopf, USA) under isoflurane gas anesthesia (1.5–2%). Following a scalp incision, the skull was exposed and drilled to create a tiny injection hole. Utilizing a pulled glass pipette (Drummond Scientific, Wiretrol, Broomall, PA, USA) with a 50 μm tip diameter, 300 nL pAAV-FLEX-GFP/pAAV-hSyn-DIO-hM3D(Gq)-mCherry viruses were intracranially injected into each side according to groups. Virus injections were performed on the ARC bilaterally (AP: 1.35 mm; ML: 0.35 mm; DV: 5.75 mm) [31] for 40 nL/min by a micromanipulator allowing 15 min for each injection. After taking out the pipette, the scalp was sutured. Mice were allowed to recover for at least two weeks before performing any procedure.

2.3. Overall experimental design

Following stereotaxic surgery, all mice were randomly divided into three groups as imaging (n = 12), electrophysiology (n = 12) and behavior groups (n = 36). Mice were fed with a high-fat (EF D12492-I; Ssniff Diet) and standard chow diet, considering the experimental design. Mice had access to standard chow diet or HFD and water ad libitum. The chow diet was given control groups (6 months) and Post-HFD mice (last 3 months). HFD was given to HFD groups (6 months) and Post-HFD groups (first 3 months). Experimental time schedule and feeding types are shown in Figure 1B.

Figure 1.

Figure 1

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The effects of chronic HFD on POMCARC neurons A) Schematic images illustrating intracranial targeting to ARC and a representative image of AAV-FLEX-GFP-infected ARC POMC neurons Scale bar: 200 μm B) Schematic illustration of experimental groups T0, T1, and T2 are time points (3-month intervals) for collecting data. Brown pellets represent the standard diet. Blue pellets represent 60% fat containing HFD. C) Representative images showing projections from GFP-infected ARC neurons to the paraventricular nucleus (PVN) in all experimental groups Scale bar: 100 μm. Effects of chronic HFD on POMC neurons D) dendritic spines and E) projection numbers. (Control n = 11, HFD n = 7, Post- HFD n = 4 mice; Data are mean ± standard deviation. Statistical analysis: One-way ANOVA followed by Tukey's multiple comparison test). (For interpretation of the references to color/colour in this figure legend, the reader is referred to the Web version of this article.)

2.4. Electrophysiology

Slice preparation was performed as described previously [32,33]. Briefly, mice were sacrificed, and brains were immersed in N-Methyl-d-glutamine (NMDG)-HEPES containing artificial cerebrospinal fluid (aCSF) cutting solution (in mM): 92 NMDG, 2.5 KCl, 1.25 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 2 thiourea, 5 Na-ascorbate, 3 Na-pyruvate, 0.5 CaCl2·2H2O, and 10 MgSO4·7H2O. Brain tissue was kept in 95% O2/5% CO2 aerated ice-cold cutting solution and 250 μm fresh slices containing the hypothalamus were obtained with vibratome and transferred to 95% O2/5% CO2 aerated and HEPES containing aCSF incubation solution containing (in mM): 92 NaCl, 2.5 KCl, 1.25 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 2 thiourea, 5 Na-ascorbate, 3 Na-pyruvate, 2 CaCl2·2H2O, and 2 MgSO4·7H2O. The sections were incubated in this solution for at least 1h and placed in the recording chamber containing the recording aCSF (in mM): 124 NaCl, 2.5 KCl, 1.25 NaH2PO4, 24 NaHCO3, 12.5 glucose, 5 HEPES, 2 CaCl2·2H2O, and 2 MgSO4·7H2O. Cell-attached and whole-cell patch clamp recordings were performed using electrodes with 7–10MΩ tip resistances. Leptin (100 nM) was perfused into the bath, and population recordings were obtained from POMC neurons. Whole cell voltage-clamp recordings were performed for spontaneous inhibitory postsynaptic currents (sIPSCs) and spontaneous excitatory postsynaptic currents (sEPSCs) in the presence of glutamate receptor blockers CNQX (10 μM) + AP5 (50 μM) and GABAA (PTX 10 μM), respectively. For whole-cell voltage clamp recordings the internal solution was composed of (in mM): 125 CsCl, 5 NaCl, 10 HEPES, 0.6 EGTA, 4 Mg-ATP, 0.3 Na2GTP, 10 lidocaine N-ethyl bromide (QX-314) at pH 7.35 and 290 mOsm. MultiClamp 700B Amplifier (Molecular Devices, San Jose, CA) and Axon™ pCLAMP™ 11.3 software (Molecular Devices, San Jose, CA) were used to record and analyze data [8,32].

2.5. Food intake analysis

Mice were individually housed to monitor feeding behavior during T0, T1, and T2 time points (Figure 5A). For chemogenetic activation experiments, 3 mg/kg CNO or saline was intraperitoneally (i.p.) administered to POMC-Cre mice at the dark onset [34]. Food measurements were taken every hour between 7:00 and 11:00 pm.

Figure 5.

Figure 5

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HFD reverses anorexigenic pattern of POMC neurons by chemogenetic activation A) Schematic illustration of the food intake experiment schedule. The average of food intake in B) control group (n = 7 mice) C) HFD group (n = 13 mice) D) Post-HFD group (n = 12 mice). (Data are mean ± standard deviation. Statistical analysis: Student's paired t-test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001).

2.6. Measurement of plasma leptin and blood glucose levels

Thirty minutes after CNO administration, 200 μl blood samples were collected. The blood glucose level was measured using the Vivacheck Eco glucometer. Blood was centrifuged at 3000 rpm for 20 min at 4 °C, and plasma was separated. Plasma leptin concentration was measured using an enzyme immunoassay (ELISA) kit (Elabscience, E-EL-M3008, USA) according to manufacturer's instructions.

2.7. Immunohistochemistry and imaging

All anesthetized mice were transcardially perfused with 4% paraformaldehyde in 0.1 M pH 7.4 phosphate buffer fixative and decapitated. Brains were collected, incubated in the same fixative for 4 h and transferred to 30% sucrose solution overnight. Brain sections (70 μm) were collected with vibratome, and the sections were permeabilized with permeabilization buffer (% 0.1 Triton-X in PBS) for 1 h at 4 °C. Then the sections were blocked with blocking buffer (%3 BSA with 0.3% Triton-X in PBS) for 90min at room temperature. Slices were then incubated with the primary antibody (anti-c-Fos, 1:2000, ab190289), which was dissolved in 1% BSA with 0.05% Triton-X in PBS. Following an overnight incubation at 4 °C, sections were rinsed with PBS three times for 10 min. Then the slices were incubated with the secondary antibody (anti-rabbit IgG Fab2 AlexaFlour 594, 1:2000, #8889 Cell signaling, Danvers, MA, USA) for 1h at room temperature. Sections were then rinsed, transferred to microscope slides and mounted with Fluoromount (Sigma, F4680, St. Louis, MO, USA). Imaging was performed on a confocal microscopy (Carl Zeiss, Thornwood, NY, USA). Image J software was used to quantify the density of dendrites and axonal extensions to the projection regions of infected neurons.

2.8. Statistical analysis

The statistical analysis was performed by using GraphPad Prism v.8.0 (GraphPad Software, CA, USA). All results were represented as mean ± standard deviation. Multiple group statistical comparisons were done by ANOVA followed by Tukey's multiple comparison test. Differences between two groups were tested with paired Student's t test. P < 0.05 was considered to be statistically significant.

3. Results

3.1. Chronic HFD does not affect ARC POMC neuron anatomy or projections

To examine the effects of HFD on ARC POMC neuron projections and dendritic density, POMC-Cre mice were intracranially injected with AAV-flex-GFP virus into the ARC to select POMC neurons (Figure 1A). An experimental food intake design was applied to the groups (Figure 1B). POMC projections to PVN, LH, and BNST, known as feeding regulatory centers [35,36] were observed (Figure 1C). Our data showed that there was no significant differences in the projection density of POMC neurons to the PVN, LH, and BNST regions and the number of dendritic spines of these neurons between all groups during the experimental period (Figure 1C–E, Supplementary Fig. 1).

3.2. Chronic HFD changes firing frequency and post-synaptic currents of POMCARC neurons

The consumption of HFD for 3 days increases the frequency and amplitude of postsynaptic inhibitory currents to POMCARC neurons [25]. We performed patch-clamp recordings of POMC-GFP neurons to elucidate leptin signaling in these neurons under chronic HFD conditions (Figure 2A). Leptin increased the firing frequency of POMCARC neurons in the control and 3-month HFD groups (Figure 2B,C). POMC neurons lost their leptin response in 6-month HFD and Post- HFD groups (Figure 2F,G).

Figure 2.

Figure 2

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The firing frequency of POMCARC neuron on chronic HFD exposed POMC-Cre mice A) GFP expression in POMC neurons containing brain slices of POMC-Cre mice. Inset: representative image depicting loose-seal recording from a POMC neuron; recording pipette highlighted with dashed lines, Scale bar: 15 μm. B) Representative loose-seal recording traces for 3m groups. C-D) Effect of leptin on firing rate of POMC neuron in control and HFD group. Each closed circle and squares represent individual neurons. Control (control n = 15 neurons, leptin n = 14 neurons; 3 mice) and HFD (control n = 17 neurons, leptin n = 23 neurons; 3 mice) E) Representative loose-seal recording traces for 6m groups. F-G) Effect of leptin on firing rate of POMC neuron in HFD and Post- HFD group. (HFD; control n = 44 neurons, leptin n = 47 neurons; 3 mice) and (Post- HFD; control n = 11 neurons, leptin n = 21 neurons; 3 mice). (Data are mean ± standard deviation. Statistical analysis: Student's paired t-test; ∗∗∗p < 0.001).

As POMCARC neurons lost their response to leptin, we characterized spontaneous excitatory (sEPSC) and inhibitory (sIPSC) postsynaptic currents that may contribute to responsiveness. It was found that leptin significantly increased excitatory current while significantly reducing inhibitory current in the standard group and 3-month HFD-fed mice group (Figure 3A–D).

Figure 3.

Figure 3

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The post-synaptic currents of POMCARCneurons on 3m HFD exposed POMC-Cre mice A-B) Effects of leptin on sEPSC frequency in control and HFD animals. Control (control n = 14 neurons, leptin n = 12 neurons) and HFD (control n = 15 neurons, leptin n = 18 neurons) n = 3 mice. C-D) Effects of leptin on sEPSC frequency in control and HFD animals. Control (control n = 14 neurons, leptin n = 13 neurons) and HFD (control and leptin n = 9 neurons) n = 3 mice. E-F) Representative whole-cell voltage-clamp recording traces for sIPSC and sEPSC. (Data are mean ± standard deviation. Statistical analysis: Student's paired t-test; ∗p < 0.05 and ∗∗p < 0.01).

The effect of leptin on excitatory and inhibitory postsynaptic currents on POMC neurons was disappeared in the 6-month-old HFD group. Although the excitatory and inhibitory postsynaptic properties of leptin on POMC neurons in the Post- HFD group were not statistically significant, they had a trend similar to the control group. (Figure 4A–D).

Figure 4.

Figure 4

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The post-synaptic currents of POMCARC neurons on 6m HFD exposed POMC-Cre mice A-B) Effects of leptin on sEPSC frequency in HFD and Post-HFD animals. HFD (control n = 21 neurons, leptin n = 16 neurons; 3 mice) and Post-HFD (control n = 4 neurons, leptin n = 9 neurons; 2 mice) C-D) Effects of leptin on sIPSC frequency in HFD and Post-HFD animals. HFD (control n = 25 neurons, leptin n = 13 neurons; n = 3 mice and Post-HFD (control n = 3 neurons, leptin n = 3 neurons; 2 mice; E-F) Representative whole-cell voltage-clamp recording traces for sIPSC and sEPSC. (Data are mean ± standard deviation. Statistical analysis: Student's paired t-test).

3.3. Chronic HFD reverses the anorexigenic pattern of POMCARC neurons

To investigate the effect of chronic HFD consumption on the homeostatic feeding pattern of POMC neurons, AAV-hM3D-mCherry viruses were injected bilaterally into POMC-Cre animals. The experimental design is shown in Figure 5A. We show that activation of POMC neurons before changing diet type reduces food intake in all groups (as shown in T0 - Figure 5B–D), as shown in the literature [37], but interestingly, stimulation of these neurons in mice exposed to 3 months of HFD increased food intake in the HFD and Post-HFD groups (T1, as shown in Figure 5C,D). The diet type in third months of Post-HFD group was HFD. This pattern disappeared when the HFD was cut off (as shown in T2 - Fig. 5D). The consumption of HFD was not change after CNO injection after 6-month on HFD group (as shown in T2 - Fig. 5C). Additionally, pattern changes in groups were analyzed for both genders (Supplementary Figs. 2A and B). The effects of this food type on POMC neurons were not evaluated in relation to gender. To demonstrate the neuronal activity of groups, we performed c-Fos staining on POMC-expressing neurons. The c-Fos-positive and POMC-hM3D-mCherry neurons were counted, and their ratio was used as an indicator of the distribution of active infected neurons (Figure 6C). HFD feeding reduced the ratio of active POMC neurons to all POMC neurons. Mice exposed to HFD for 6 days have been shown to develop glucose and leptin resistance in POMC neurons [22]. In contrast, leptin and glucose levels did not differ between the groups at 6 months (Supplementary Figs. 3A and D). However, comparing the groups individually, the blood glucose level of the Post-HFD group decreased during HFD, and then increased again after switching to a chow diet. (Supplementary Fig. 3B).

Figure 6.

Figure 6

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Effect of HFD on POMC neurons activity A) Representative illustration of intracranial injection into the hypothalamic ARC. B) Representative POMC neuron c-Fos staining. Scale bar: 50 μm. C) The ratio of the number of c-Fos-positive cells in the ARC to hM3D-infected POMC cells (control n = 8, HFD n = 15, Post-HFD n = 12 mice). (Data are mean ± standard deviation. Statistical analysis: One-way ANOVA followed by Tukey's multiple comparison test. ∗p < 0.05).

4. Discussion

Obesity is a major cause of the metabolic syndrome, which includes insulin resistance, type 2 diabetes, and dyslipidemia. Although the precise mechanisms by which obesity induces or exacerbates metabolic risk factors are still unknown, excessive fat accumulation, particularly increased visceral fat is associated with insulin resistance and abnormal glucose and lipid metabolism [2,4]. In addition, chronic low-grade inflammation in adipose tissue contributes to the pathogenesis of insulin resistance and metabolic syndrome [38]. The composition of the diet plays an important role in the development of obesity and related metabolic diseases [39,40]. HFDs are macronutrients that are high in energy density and cause less satiety than carbohydrates or proteins [41]. Long-term consumption of a HFD has been shown to induce hyperphagia, weight gain, fat accumulation and increased blood glucose and insulin levels in mice [[42], [43], [44]]. The adaptive changes of POMC neurons to HFD exposure morphologically and behaviorally were evaluated while looking at their electrophysiological properties. On the contrary, it has been reported that the total number of synaptic inputs in POMC cell bodies was reduced due to HFD-induced reactive astrogliosis [23,45], and no significant difference between groups was found in the number of dendritic endings in POMC-Cre mice exposed to HFD for 24 weeks.

Leptin plays a role in activating POMCARC neurons within the melanocortin system [18]. Chronic HFD consumption abolished the ability to respond to leptin stimuli in POMC neurons [24]. In the present study, we obtained a response to leptin from POMC neurons with HFD consumption for up to 12 weeks. However, at the end of 24 weeks of HFD consumption, POMC neurons appeared to lose their sensitivity to leptin. Leptin desensitization was permanent despite dietary changes of Post-HFD group. However, there was a trend toward improvement in postsynaptic currents to POMC neurons. As a result of ongoing desensitization, we showed that a 3-month diet change is not long enough to reverse the HFD effects.

In contrast to the classical leptin-melanocortin model, it has been established that POMCARC LEPR (+) neurons have a role in regulating glucose homeostasis [46]. There was no significant difference in blood glucose levels between the group exposed to the high-fat diet for 24 weeks and the control group. However, emerging evidence suggests that POMC neurons are highly heterogeneous, and other subtypes of neurons may take on their functions under metabolic stress [13]. The heterogeneity within POMC neurons also yields complex outcomes in the context of prolonged high-fat diet consumption [13,46]. Despite the limitations associated with the POMC-Cre mouse model in our study [[47], [48], [49]], we demonstrated that chronic activation of POMC neurons through a high-fat diet led to a shift in total balance in an orexigenic direction. The differing hormonal and metabolic regulations based on gender necessitate gender-specific testing of the identified findings [13]. In the present study, behavioral changes in food intake were found to be similar in male and female. HFD-induced orexigenic behavior and the later disappearance of this effect in the Post-HFD group were also similar in both sexes. It can be argued that maladaptations occurring in POMC neurons can be corrected with dietary changes. At the end of the 6th month period, the similar levels of leptin across all groups indicated that the recovery process in POMC neurons due to dietary changes was not leptin-dependent (Supplementary Fig. 3).

Cannabinoid receptor 1 (CB1R) activation on POMC neurons increases food intake [37]. The leptin resistance induced by a high-fat diet can be reversed through peripheral CB1R blockade [50]. Therefore, it may be suggested that CB1R (+) POMC neurons were activated in our model supported by DREADD technology and chronic HFD. It may be speculated that chronic exposure to a high-fat diet might have altered the balance in POMC neuron subpopulations towards increased β-endorphin release.

It has been shown that exposed a chronic high-fat diet causes an increase in body weight [24]. However, our results show that despite the impairment of leptin sensitivity, hyperphagia does not develop and decreases in body weight (Supplementary Fig. 4). Several investigations have shown that HFD does not cause hyperphagia, hypertriglyceridemia, hyperglycemia, or hyperinsulinemia in mice [51,52]. In addition, mice with deletion of the Neuronal PAS domain protein 4 (Npas4) gene synthesized in POMC neurons could not gain weight when fed HFD [53]. It is thought that metabolic changes and/or adaptations in the POMC (Npas4) gene resulting from chronic fatty nutrition may have contributed to weight loss in HFD and Post-HFD groups.

5. Conclusion

Chronic consumption of high-fat diet leads to systemic changes by affecting the nervous system directly or triggering inflammation in the hypothalamus. POMC neurons, which are regulators of homeostatic food intake, respond to this process with leptin insensitivity and are also accompanied by a reorganization of hypothalamic system balances. When investigating these changes, the absence of changes in POMC neuron anatomy suggests systemic and molecular pathways. Activated POMC neurons affected by chronic HFD increased food intake, suggesting a disturbed balance in POMC neurons. Reverting the consumption of high-fat diet to a standard diet has led to a decrease in high-fat diet-induced overeating during POMC activation. Exploring plasticity and regulators of these systems could be a viable approach to combating metabolic diseases like obesity.

CRediT authorship contribution statement

Özge Başer: Writing – original draft, Investigation. Yavuz Yavuz: Writing – original draft, Resources, Investigation. Deniz Öykü Özen: Investigation. Hüseyin Buğra Özgün: Investigation. Sami Ağuş: Investigation. Cihan Civan Civaş: Investigation. Deniz Atasoy: Writing – review & editing, Writing – original draft, Investigation. Bayram Yılmaz: Writing – review & editing, Writing – original draft, Investigation.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This study was supported by the Scientific and Technological Research Council of Türkiye (TUBITAK project # 118S245).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.molmet.2024.101904.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

graphic file with name figs1.jpg

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Supplementary Fig. 1: The effect of chronic HFD on POMCARC Projections to LH and BNST. A) Representative images showing projections from GFP-infected POMCARC neurons to the lateral hypothalamus (LH) in all experimental groups. B) Bar graph representing the extent of POMCARC axonal projections received by LH neurons in 6 months of chronic feeding in all experimental groups (n = 16 in 4 mice per group) C) Representative images showing projections from GFP-infected POMCARC neurons to the bed nucleus stria terminalis (BNST) in all experimental groups. D) Bar graph representing the extent of POMCARC axonal projections received by BNST neurons in 6 months of chronic feeding in all experimental groups (n = 16 in 4 mice per group) (Data are mean ± standard deviation. Statistical analysis: One-way ANOVA followed by Tukey's multiple comparison test.).

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Supplementary Fig. 2: HFD reverses anorexigenic pattern of POMC neurons in both sexes. Average food intake at T2 for all groups A) female (Control n = 5, HFD n = 6, Post-HFD n = 5 mice) B) male (Control n = 6, HFD n = 7, Post-HFD n = 7 mice). (Data are mean ± standard deviation. Statistical analysis: Student's paired t-test ∗∗p < 0.01).

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Supplementary Fig. 3: Blood glucose, Serum hormone, and serum lipid levels in all groups. Measurement of blood glucose levels from mice at different time points A) HFD (n = 8 mice). B) Post-HFD (n = 7 mice). Chemogenetic activation of POMC neurons in all groups C) Blood glucose levels, D) Serum leptin levels (control n = 7, HFD n = 8, Post-HFD n = 7 mice). E) High density lipoprotein (HDL), F) Triglyceride (TG), G) Cholesterol, H) Low-density lipoprotein (LDL), I) Very low-density lipoprotein (VLDL) (control n = 6, HFD n = 8, Post-HFD n = 6 mice). (Data are mean ± standard deviation. Statistical analysis: One-way ANOVA followed by Tukey's multiple comparison test. ∗∗p < 0.01).

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Supplementary Fig. 4: Effects of diet type on mice body weight. (Control n = 8, HFD n = 8, Post-HFD n = 8 mice.) (Data are mean ± standard deviation. Statistical analysis: Two-way ANOVA followed by Tukey's multiple comparison test. ∗p < 0.05).

Data availability

Data will be made available on request.

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