High-fat diet disrupts a septal control on feeding to promote obesity in male mice methods
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Results
To investigate how HFD-induced obesity modulates the transcriptional profiles of cells within the septal region, adult mice were assigned to either a HFD or control chow diet. Both groups had ad libitum access to food. Mice on the HFD exhibited significantly higher energy intake compared to those on the control diet, resulting in a significant increase in bodyweight that surpassed that of control mice as early as the first week (Fig. ). The caloric intake continued to increase, leading to the development of an obesity phenotype (Fig. ). After a 4-week dietary regimen, septal tissues were harvested for single-nucleus RNA sequencing (snRNA-seq) (Fig. ). Fig. 1 The HFD alters the transcriptional profiles of septal area neurons. a Left: quantification of daily energy intake for the normal food and high-fat diet groups (diet: F 1,60 = 157.2; p <...
Overexpression of Hcn1 restores LS neuronal excitability and prevents HFD-induced obesity
Next, we examine the effect of Hcn1 overexpression on the neuronal excitability of LS GABA neurons and HFD-induced obesity by utilizing adeno-associated virus vectors (Fig. ). Immunofluorescence confirmed successful Hcn1 overexpression following this viral strategy (Fig. ). Overexpression of Hcn1 led to a significantly higher voltage sag amplitude and enhanced excitability of LS GABA neurons (Fig. ). In the liquid food intake assays, overexpression of Hcn1 did not affect the standard liquid food intake, but significantly decreased the lick numbers and total intake of palatable sucrose solution and Ensure (Fig. ). A separate cohort of naïve Gad2-Cre mice were used to test whether Hcn1 overexpression in LS GABA neurons could prevent HFD-induced overeating and obesity. We found mice with Hcn1 overexpression resisted excessive energy intake on an HFD, maintain...
Downregulation of Gad2 in the LS contributes to HFD-induced obesity
To explore whether restoring GABA levels in the LS could rescue HFD-induced obesity, AAV-DIO-Gad2-EGFP was bilaterally injected into LS of Gad2-Cre mice for LS GABA neurons Gad2 overexpression (Fig. ). Western blot and LC-MS analyses showed a significant increase in Gad2 and GABA protein in LS (Fig. ). In the liquid food intake assays, similar to overexpression of Hcn1, overexpression of Gad2 in LS GABA neurons did not affect the standard liquid food intake, but significantly decreased the lick numbers and total intake of sucrose solution and Ensure (Fig. ). In a distinct cohort of mice, 3 weeks after virus injection, the mice were switched to a 4-week HFD or chow diet. The results demonstrated that Gad2 overexpression in LS significantly reduced HFD intake and prevented obesity (Fig. and Supplementary Fig. ). Overexpression of Gad2 did not affect chow in...
Methods
In this study, adult male C57BL/6 J mice were obtained from the Guangdong Medical Laboratory Animal Center, Guangzhou, China. Additionally, the experimental cohorts included Gad2-Cre (male, Jax No. 010802) mice. All mice were housed in a temperature (22-25 °C) and humidity (55-65%) controlled environment, with ad libitum access to food and water, except during experimental sessions. A 12-hour light-dark cycle was maintained (lights on from 7:00 am to 7:00 pm). All efforts were made to minimize animal suffering and the number of animals used. A total of 338 mice were used in the different experiments. All experiments were conducted in accordance with relevant guidelines and regulations, and approved by the IACUC (Institutional Animal Care and Use Committee) of SIAT, Chinese Academy of Sciences (CAS, SIAT-IACUC-230927).
Electrophysiological recordings
Procedures for preparing acute brain slices were similar to those described previously. Mice were anesthetized with isoflurane. Under sterile conditions, we perfused the mice with 4 °C slicing solution containing (in mM) 110 choline chloride, 2.5 KCl, 0.5 CaCl 2, 7 MgCl 2, 1.3 NaH 2 PO 4, 1.3 Na-ascorbate, 0.6 Na-pyruvate, 25 glucose, and 25 NaHCO 3 ), then placed the tissue in 4 °C slicing solution saturated with 95% O 2 and 5% CO 2. Coronal slices (250-300 µm) containing the LS, LH or TN were prepared using a vibratome (Leica, VT-1000S). Slices were incubated in 37 °C oxygenated artificial cerebrospinal fluid (in mM: 125 NaCl, 2.5 KCl, 2 CaCl 2, 1.3 MgCl 2, 1.3NaH 2 PO 4, 1.3 Na-ascorbate, 0.6 Na-pyruvate, 25 glucose, and 25 NaHCO 3 ) for at least 30 min for recovery. Then the slices were transferred to a recording cham...
Virus-mediated neural tracing
To trace the downstream projections of LS GABA neurons, AAV2/9-hSyn-FLEX-tdTomato-T2A-synaptophysin-EGFP-WPRE-pA was injected into the LS. Brain sections from these mice were obtained following the procedures described above. For immunostaining, brain sections were rinsed with PBS (3 × 10 min) and then blocked with 10% normal goat serum and 0.3% TritonX-100 in PBS for 2 hr at room temperature. Next, sections were incubated with primary antibody (Rabbit anti-GFP, 1:1000, ThermoFisher, Cat# A-11122) diluted in 10% normal goat serum and 0.3% TritonX-100 in PBS for 24-48 hr at 4 °C. After washing with PBS (3 × 10 min), sections were incubated with secondary antibody (Goat anti-Rabbit 488, 1:1000, ThermoFisher, Cat# A-11008) diluted in 10% normal goat serum and 0.3% Triton X-100 in PBS at room temperature for 2 hr and the...
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To investigate how HFD-induced obesity modulates the transcriptional profiles of cells within the septal region, adult mice were assigned to either a HFD or control chow diet. Both groups had ad libitum access to food. Mice on the HFD exhibited significantly higher energy intake compared to those on the control diet, resulting in a significant increase in bodyweight that surpassed that of control mice as early as the first week (Fig. ). The caloric intake continued to increase, leading to the development of an obesity phenotype (Fig. ). After a 4-week dietary regimen, septal tissues were harvested for single-nucleus RNA sequencing (snRNA-seq) (Fig. ). Fig. 1 The HFD alters the transcriptional profiles of septal area neurons. a Left: quantification of daily energy intake for the normal food and high-fat diet groups (diet: F 1,60 = 157.2; p < 0.001; time: F 4,60 = 132.6, p < 0.001; interaction: F 4,60 = 9.5, p < 0.001). Right: quantification of bodyweight between the Chow and HFD groups (diet: F 1,60 = 49.3; p < 0.001; time: F 4,60 &...
Next, we examine the effect of Hcn1 overexpression on the neuronal excitability of LS GABA neurons and HFD-induced obesity by utilizing adeno-associated virus vectors (Fig. ). Immunofluorescence confirmed successful Hcn1 overexpression following this viral strategy (Fig. ). Overexpression of Hcn1 led to a significantly higher voltage sag amplitude and enhanced excitability of LS GABA neurons (Fig. ). In the liquid food intake assays, overexpression of Hcn1 did not affect the standard liquid food intake, but significantly decreased the lick numbers and total intake of palatable sucrose solution and Ensure (Fig. ). A separate cohort of naïve Gad2-Cre mice were used to test whether Hcn1 overexpression in LS GABA neurons could prevent HFD-induced overeating and obesity. We found mice with Hcn1 overexpression resisted excessive energy intake on an HFD, maintaining bodyweights similar to chow-fed groups and significantly lower than HFD-fed EGFP groups (Fig. ). Crucially, this metabolic protection was diet-dependent. Overexpression of Hcn1 did not alter food intake or body weight in mice maintained on a standard chow diet (Fig, and Supplementary Fig.&...
To explore whether restoring GABA levels in the LS could rescue HFD-induced obesity, AAV-DIO-Gad2-EGFP was bilaterally injected into LS of Gad2-Cre mice for LS GABA neurons Gad2 overexpression (Fig. ). Western blot and LC-MS analyses showed a significant increase in Gad2 and GABA protein in LS (Fig. ). In the liquid food intake assays, similar to overexpression of Hcn1, overexpression of Gad2 in LS GABA neurons did not affect the standard liquid food intake, but significantly decreased the lick numbers and total intake of sucrose solution and Ensure (Fig. ). In a distinct cohort of mice, 3 weeks after virus injection, the mice were switched to a 4-week HFD or chow diet. The results demonstrated that Gad2 overexpression in LS significantly reduced HFD intake and prevented obesity (Fig. and Supplementary Fig. ). Overexpression of Gad2 did not affect chow intake or weight gain (Fig. ). qPCR analysis confirmed that Gad2 expression was significantly higher in the overexpression group after 4 weeks of HFD feeding compared to the Chow+GFP control group (Fig. ). Importantly, neither silencing of LS GABA neurons, Gad2 overexpression, nor 4-week h...
In this study, adult male C57BL/6 J mice were obtained from the Guangdong Medical Laboratory Animal Center, Guangzhou, China. Additionally, the experimental cohorts included Gad2-Cre (male, Jax No. 010802) mice. All mice were housed in a temperature (22-25 °C) and humidity (55-65%) controlled environment, with ad libitum access to food and water, except during experimental sessions. A 12-hour light-dark cycle was maintained (lights on from 7:00 am to 7:00 pm). All efforts were made to minimize animal suffering and the number of animals used. A total of 338 mice were used in the different experiments. All experiments were conducted in accordance with relevant guidelines and regulations, and approved by the IACUC (Institutional Animal Care and Use Committee) of SIAT, Chinese Academy of Sciences (CAS, SIAT-IACUC-230927).
Procedures for preparing acute brain slices were similar to those described previously. Mice were anesthetized with isoflurane. Under sterile conditions, we perfused the mice with 4 °C slicing solution containing (in mM) 110 choline chloride, 2.5 KCl, 0.5 CaCl 2, 7 MgCl 2, 1.3 NaH 2 PO 4, 1.3 Na-ascorbate, 0.6 Na-pyruvate, 25 glucose, and 25 NaHCO 3 ), then placed the tissue in 4 °C slicing solution saturated with 95% O 2 and 5% CO 2. Coronal slices (250-300 µm) containing the LS, LH or TN were prepared using a vibratome (Leica, VT-1000S). Slices were incubated in 37 °C oxygenated artificial cerebrospinal fluid (in mM: 125 NaCl, 2.5 KCl, 2 CaCl 2, 1.3 MgCl 2, 1.3NaH 2 PO 4, 1.3 Na-ascorbate, 0.6 Na-pyruvate, 25 glucose, and 25 NaHCO 3 ) for at least 30 min for recovery. Then the slices were transferred to a recording chamber and superfused with 2 ml min -1 artificial cerebrospinal fluid. Recording was performed at room temperature (23 °C) with a Multiclamp 700B amplifier and a Digidata 1550B acquisition system (Molecular Devices). Data were sampled at 10 kHz and analyzed with Clamp...
To trace the downstream projections of LS GABA neurons, AAV2/9-hSyn-FLEX-tdTomato-T2A-synaptophysin-EGFP-WPRE-pA was injected into the LS. Brain sections from these mice were obtained following the procedures described above. For immunostaining, brain sections were rinsed with PBS (3 × 10 min) and then blocked with 10% normal goat serum and 0.3% TritonX-100 in PBS for 2 hr at room temperature. Next, sections were incubated with primary antibody (Rabbit anti-GFP, 1:1000, ThermoFisher, Cat# A-11122) diluted in 10% normal goat serum and 0.3% TritonX-100 in PBS for 24-48 hr at 4 °C. After washing with PBS (3 × 10 min), sections were incubated with secondary antibody (Goat anti-Rabbit 488, 1:1000, ThermoFisher, Cat# A-11008) diluted in 10% normal goat serum and 0.3% Triton X-100 in PBS at room temperature for 2 hr and then counterstained with DAPI (1:3000).
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