Histone lactylation mediated by Fam172a in POMC neurons regulates energy balance

Abstract

Glycolysis-derived lactate was identified as substrate for histone lactylation, which has been regarded as a significant role in transcriptional regulation in many tissues. However, the role of histone lactylation in the metabolic center, the hypothalamus, is still unknown. Here, we show that hypothalamic pro-opiomelanocortin (POMC) neuron-specific deletion of family with sequence similarity 172, member A (Fam172a) can increase histone lactylation and protect mice against diet-induced obesity (DIO) and related metabolic disorders. Conversely, overexpression of Fam172a in POMC neurons led to an obesity-like phenotype. Using RNA-seq and CUT&Tag chromatin profiling analyzes, we find that knockdown of Fam172a activates the glycolytic process and increases peptidylglycine α-amidating monooxygenase (PAM), which affects the synthesis of α-MSH, via H4K12la (histone lactylation). In addition, pharmacological inhibition of lactate production clearly abrogates the anti-obesity effect of PFKO (POMC-Cre, Fam172a^loxP/loxP, POMC neurons Fam172a knockout). These findings highlight the importance of Fam172a and lactate in the development of obesity and our results mainly concern male mice.

Fig. 1. Knockout of Fam172a in the neurons of the hypothalamic Arc protects mice against HFD induced metabolic dysregulation.

A Adult C57 BL/6 mice were fed a chow diet or HFD (4 or 8 weeks), and western blot analysis of Fam172a in Arc of hypothalamus was performed. n = 3 mice per group. B Expression of Fam172a in Arc of hypothalamus. Brain section of adult C57 BL/6 mice under a chow diet or HFD 8 weeks were immunostained for Fam172a (red). n = 5 mice per group. Cell nuclei were counterstained with DAPI (blue). Arc, arcuate nucleus; 3 V, third ventricle. Scale bars, 100 μm. C Expression of Fam172a in Arc of hypothalamus. Brain section of adult C57 BL/6 mice was coimmunostained for Fam172a (red) and Neu N (green). n = 4 mice. Cell nuclei were counterstained with DAPI (blue). 3 V, third ventricle. Scale bars, 100 μm. D Schematic diagram of virus injection in mice. Created in BioRender. Chen, Z. (2024) https://BioRender.com/j97w406. E AAV-Syn-GFP and AAC-Syn-Cre were injected into the Arc nucleus of adult Fam172a^loxP/loxP mice. Immunofluorescence staining for Fam172a (red) and GFP (green) was then performed on brain sections at 4 weeks post-surgery. n = 4 mice per group. Cell nuclei were counterstained with DAPI (blue). 3 V, third ventricle. Scale bars, 100 μm. F AAV viruses were injected into the Arc of adult male Fam172a^loxP/loxP mice fed a HFD. Body weight was then assessed. n = 8 mice per group. G–I AAV viruses were injected into the Arc of adult male Fam172a^loxP/loxP mice fed a HFD. Representative DEXA images (G), fat mass (H) and lean mass (I) were then assessed. n = 8 (AAV-Syn-GFP) or 7 (AAV-Syn-Cre) mice per group. J, K Glucose tolerance test (GTT, J) and the area under the curve (AUC) of the GTT (K) of mice. n = 10 mice per group. L, M Oxygen consumption (VO₂, L) and energy expenditure (EE, M) of the mice. lbm, lean body mass; Dark, dark cycle; Light, light cycle. n = 6 (AAV-Syn-GFP) or 5 (AAV-Syn-Cre) mice per group. Data are presented as mean ± SEM. two-tailed Student’s t-test (H, I, K), two-way ANOVA with Bonferroni’s post hoc test (F, J, L, M). Source data are provided as a Source Data file.

Fig. 2. Deletion of Fam172a in POMC neurons protects mice against DIO and its associated comorbidities.

A, B Immunofluorescence staining for Fam172a (green) indicated that Fam172a protein has been depleted from POMC neurons of the POMC-Cre, Fam172a^loxP/loxP (PFKO) mice (A) and colocalization quantification of immunofluorescence (B). Both PFKO and POMC-Cre mouse lines were crossed with the tdTomato reporter/Ai14 mice, so that the POMC neurons could be identified as tdTomato (red)-positive cells in the Arc. Cell nuclei were counterstained with DAPI (blue). 3 V, third ventricle. n = 3 mice per group. Scale bars, 100 μm. C Body weight of male mice under HFD feeding. Fam172a^L/L, Fam172a^loxP/loxP mice; PFKO, POMC-Cre, Fam172a^loxP/loxP mice. n = 8 (Fam172a^L/L) or 7 (PFKO) mice per group. D–F Representative DEXA images (D), fat mass (E) and lean mass (F) of male mice under HFD feeding. n = 8 mice per group. G The weights of three adipose tissues, such as BAT, sWAT and eWAT. n = 8 mice per group. (H) Representative H&E staining images of BAT (upper panel), sWAT (middle panel) and eWAT (lower panel) tissues of the mice fed a HFD. Scale bars, 100 μm. (I, J) GTT (I) and AUC of GTT (J) of the mice fed a HFD. n = 7 mice per group. K, L ITT (K) and AUC of ITT (L) of the mice fed a HFD. n = 8 mice per group. (M) Average food intake of the mice during HFD feeding. n = 9 (Fam172a^L/L) or 7 (PFKO) mice per group. (N, O) Oxygen consumption (VO₂, N) and energy expenditure (EE, O) of the mice after HFD feeding. lbm, lean body mass; Dark, dark cycle; Light, light cycle. n = 6 mice per group. Data are presented as mean ± SEM. two-tailed Student’s t-test (B, E, F, G, J, L, M), two-way ANOVA with Bonferroni’s (C, I, K, N, O). Source data are provided as a Source Data file.

Fig. 3. Activation of Fam172a in POMC neurons leads to an obesity-like phenotype.

A AAV-GFP or AAV-Fam172a viruses were injected into the Arc of male adult POMC-Cre mice. Immunofluorescence staining for Fam172a (red) was then performed on brain sections. When Cre recombinase is present, infection with the AAV-GFP or AAV-Fam172a virus can lead to GFP expression. n = 4 mice per group. Cell nuclei were counterstained with DAPI (blue). 3 V, third ventricle. Scale bars, 100 μm. B AAV viruses were injected into the Arc of male adult POMC-Cre mice fed a normal chow. Body weight was then assessed. n = 7 (AAV-GFP) or 6 (AAV-Fam12a) mice per group. C–E AAV viruses were injected into the Arc of male adult POMC-Cre mice fed a normal chow. Representative DEXA images (C), fat mass (D) and lean mass (E) were then assessed. n = 8 (AAV-GFP) or 7 (AAV-Fam12a) mice per group. F The weight of three adipose tissues, such as BAT, sWAT and eWAT. n = 8 (AAV-GFP) or 7 (AAV-Fam12a) mice per group. G Representative H&E staining images of mouse adipose tissues. Scale bars, 100 μm. H, I GTT (H) and the AUC of GTT (I) of the mice. n = 7 mice per group. (J, K) ITT (J) and the AUC of ITT (K) of the mice. n = 7 mice per group. (L) Average food intake of the mice was assessed. n = 7 mice per group. (M, N) Oxygen consumption (VO₂, M) and energy expenditure (EE, N) of the mice. lbm, lean body mass; Dark, dark cycle; Light, light cycle. n = 6 mice per group. Data are presented as mean ± SEM. two-tailed Student’s t-test (D, E, F, I, K, L), two-way ANOVA with Bonferroni’s post hoc test (B, H, J, M, N). Source data are provided as a Source Data file.

Fig. 4. Intracellular lactate levels were increased by glycolytic process after Fam172a knockdown.

A, B Neuro2a cells were transfected with ADV-shCtrl or ADV-shFam172a at 37°Cfor 48 h, and then RNA-Seq was performed. Heatmaps of the top 15 genes with significant differences (A). Top ten KEGG pathways were also shown (B). n = 3 cell cultures per group. C Schematic of the glycolytic pathway, and highlighting (red) the participating enzymes. D, E Neuro2a cells were transfected with ADV-shCtrl or ADV-shFam172a at 37°C for 48 h, total RNAs or proteins were then extracted, and the relative mRNA (D) and protein (E) levels of the indicated genes were assessed. n = 5 cell cultures per group for qPCR or 3 cell cultures per group for western blot. F, G Seahorse metabolic analysis (ECAR) of neuro2a cells which were transfected with ADV-shCtrl or ADV-shFam172a at 37°C for 48 h. n = 5 cell cultures per group. H Intracellular lactate levels of neuro2a cells transfected with ADV-shCtrl or ADV-shFam172a at 37°C for 48 h. n = 4 cell cultures per group. I, J Infection with ADV-shFam172a increased mouse LDHA (I) and PDK1 (J) promoter activity in Neuro2a cells. n = 5 cell cultures per group for LDHA promoter activity analysis or 6 cell cultures per group for PDK1 promoter activity analysis. K, L Neuro2a cells were transfected with CMV-Ctrl or CMV-Fam172a at 37°C for 48 h, total RNAs or proteins were then extracted, and the relative mRNA (K) and protein (L) levels of the indicated genes were assessed. n = 5 cell cultures per group for qPCR or 3 cell cultures per group for western blot. M, N Seahorse metabolic analysis (ECAR) of neuro2a cells transfected with CMV-Ctrl or CMV-Fam172a at 37°C for 48 h. n = 5 cell cultures per group. O Intracellular lactate levels of neuro2a cells which were transfected with CMV-Ctrl or CMV-Fam172a at 37°C for 48 h. n = 5 (CMV-Ctrl) or 4 (CMV-Fam172a) cell cultures per group. Data are presented as mean ± SEM. Hypergeometric test with Bonferroni’s post hoc test (B), two-tailed Student’s t-test (D, G, H, I, J, K, N, O). Source data are provided as a Source Data file.

Fig. 5. Inhibition of LDHA activity abrogates the anti-obesity effect of PFKO.

A Schematic diagram of the Seahorse XF Assay test of the hypothalamus Arc. Created in BioRender. Chen, Z. (2024) https://BioRender.com/j97w406. B, C Seahorse metabolic analysis (ECAR) of hypothalamus Arc in Fam172A^L/L or PFKO mice. n = 4 mice per group. D Western blot analysis of LDHA and PDK1 in hypothalamus Arc of Fam172A^L/L or PFKO mice. n = 3 mice per group. E Intracellular lactate levels of hypothalamus Arc in Fam172A^L/L or PFKO mice. n = 8 mice per group. F, G Adult male Fam172a^L/L or PFKO mice were fed a chow diet, and were then placed cannula directed to lateral ventricle. After 2 wks of recovery, mice were switched from chow diet to HFD and were i.c.v. administered aCSF or oxamate every other day for 21 days (F), created in BioRender. Chen, Z. (2024) https://BioRender.com/j97w406. Body weight gain (G) was assessed. n = 8 (Fam12a^L/L, ICV aCSF) or 9 (PFKO, ICV OX) or 10 (PFKO, ICV aCSF) mice per group. H, I Fat mass (H) and lean mass (I) of treated mice were also assessed. n = 8 (Fam12a^L/L, ICV aCSF) or 9 (PFKO, ICV OX) or 9 (PFKO, ICV aCSF) mice per group. J Representative H&E staining images of mouse adipose tissues. Scale bars, 100 μm. K, L After oxamate treatment, the mice were subjected to the GTT (K). The AUC (L) of GTT is shown. n = 9 (Fam12a^L/L, ICV aCSF) or 8 (PFKO, ICV OX) or 9 (PFKO, ICV aCSF) mice per group. M Average food intake assessed during the treatment period. n = 8 mice per group. N, O Central administration of oxamate decreased oxygen consumption (VO2, N), and energy expenditure (EE, O) in PFKO mice. lbm, lean body mass. n = 6 mice per group. Data are presented as mean ± SEM. two-tailed Student’s t-test (C, E), one-way ANOVA with Turkey’s post hoc test (L, M, N, O), two-way ANOVA with Bonferroni’s post hoc test (G, H, I, K). Source data are provided as a Source Data file.

Fig. 6. Lactate regulates the transcriptional level of PAM through H4K12la.

A Western blot analysis of H4K12la in neuro2a cells treated with NaCl or lactate at 37°C for 24 h. n = 3 cell cultures per group. B The binding density of H4K12la was visualized by deepTools: the heatmap presents the CUT&Tag tag counts on the different H4K12la binding peaks in neuro2a cells treated with NaCl or lactate at 37°C for 24 h, ordered by signal strength. C Genome-wide distribution of upregulated H4K12la-binding peaks in neuro2a cells treated with NaCl or lactate at 37°C for 24 h. D Genome browser tracks of CUT&Tag signals at the PAM target gene loci. E Neuro2a cells were treated with NaCl or latate at 37°C for 24 h, total RNA was then extracted, and the relative mRNA levels of the indicated genes were assessed. n = 6 (Ctrl) or 5 (Lactate) cell cultures per group. F Lactate treated neuro2a cells presented increased mouse PAM promoter activity. n = 5 (Ctrl) or 4 (Lactate) cell cultures per group. G A proposed working model for how lactate influenced the transcription of PAM. Created in BioRender. Chen, Z. (2024) https://BioRender.com/j97w406. H Neuro2a cells were transfected with ADV-shCtrl or ADV-shFam172a at 37°C for 48 h, after which RNA-Seq was performed. Heatmaps of POMC to α-MSH pathway target genes. n = 3 cell cultures per group. I Neuro2a cells were transfected with ADV-shCtrl or ADV-shFam172a at 37°C for 48 h, total RNA was then extracted, and the relative mRNA levels of the indicated genes were assessed. n = 6 (ADV-shCtrl) or 5 (ADV-shFam172a) cell cultures per group. J Knockdown of Fam172a in neuro2a cells increased mouse PAM promoter activity. n = 5 cell cultures per group. K Western blot analysis of PAM and H4k12la in neuro2a cells transfected with ADV-shCtrl or ADV-shFam172a at 37°C for 48 h. n = 3 cell cultures per group. Data are presented as mean ± SEM. two-tailed Student’s t-test (E, F, I, J). Source data are provided as a Source Data file.

Fig. 7. Disruption of Fam172a caused an increase of histone lactylation and α-MSH release in POMC neurons.

A–C Immunofluorescence staining for H4K12la (green, A) or H3K18la (green, B) and tdTomato (red) was then performed on POMC-Cre or PFKO mouse brain sections. Quantification was then performed (C). Both PFKO and POMC-Cre mouse lines had been crossed with the tdTomato reporter/Ai14 mice, so that the POMC neurons could be identified as tdTomato (red)-positive cells in the Arc nucleus. Cell nuclei were counterstained with DAPI (blue). n = 4 mice per group. 3 V, third ventricle. Scale bars, 100 μm. D Relative mRNA levels of α-MSH synthesis related genes in hypothalamus Arc of Fam172A^L/L or PFKO mice. n = 5 cell cultures per group. E Western blot analysis of PAM in hypothalamus Arc of Fam172A^L/L or PFKO mice. n = 3 mice per group. (F, G) Immunofluorescence staining for α-MSH (green) in brain sections from the PVN/DMH (F) and quantification of α-MSH fluorescence intensity (G). Cell nuclei were counterstained with DAPI (blue). n = 3 mice per group. Scale bars, 100 μm. (H) α-MSH levels in the hypothalamus of Fam172A^L/L or PFKO mice. n = 6 (Fam172a^L/L) or 5 (PFKO) mice per group. I Effects of SHU9119 on PFKO induced inhibition of food intake, and cumulative food intake measured at 0 h, 1 h, 2 h and 4 h after central administration of SHU9119. n = 10 (Fam172a^L/L, ICV aCSF), 7 (PFKO, ICV SHU9119) or 7 (PFKO, ICV aCSF) mice per group. (J, K) Central administration of SHU9119 decreased oxygen consumption (VO2, J), and energy expenditure (EE, K) in PFKO mice. lbm, lean body mass. n = 6 mice per group. Data are presented as mean ± SEM. two-tailed Student’s t-test (C, D, G, H), one-way ANOVA with Turkey’s post hoc test (J, K), two-way ANOVA with Bonferroni’s post hoc test (I). Source data are provided as a Source Data file.