Adipocytes control food intake and weight regain via Vacuolar-type H⁺ ATPase

The study addresses why individuals with obesity commonly regain weight after weight loss despite behavioral efforts. Prior work suggests a persistent ‘metabolic memory’ of obesity in adipose tissue that may influence energy balance and appetite post-weight loss. The authors hypothesize that obesity-induced transcriptional changes in white adipose tissue (WAT) that persist after weight loss contribute to altered feeding behavior and weight regain. They aim to identify adipocyte genes underlying this peripheral metabolic memory and test their roles in food intake and weight regulation.

Multiple studies show obesity induces durable changes across the transcriptome, metabolome, proteome and microbiome that can persist after weight loss. Adipose tissue, particularly WAT, appears central to sustained metabolic alterations, including a refractory response to dietary restriction in previously ad libitum–fed mice. Prior work by the authors demonstrated obesity-driven metabolite changes persisting in adipose tissue but largely resolving in liver, muscle, and hypothalamus after weight loss. Concepts of nutritional memory and adipose-driven weight regain have been proposed, yet molecular effectors linking adipose memory to feeding behavior remain unclear.

• Diet-switch mouse model: Male C57BL/6J mice were fed low-fat diet (LFD, 10%) or high-fat diet (HFD, 60%) for 9 weeks; a subset of HFD mice were switched to LFD for 3 weeks to induce ~10% weight loss (formerly obese, SW). Epididymal WAT was fractionated into adipocytes and stromal vascular cells (SVCs) for RNA-seq.
• RNA-seq: Prepared from purified adipocytes and SVCs; differential expression analyzed with limma, FDR control; identification of genes altered by HFD vs LFD and those persisting after weight loss (‘metabolic memory’ genes).
• Cross-species orthology and worm feeding screen: Mouse metabolic-memory genes mapped to C. elegans orthologs using DIOPT and ranked by conservation. Available mutant strains (28) were screened in a high-throughput feeding assay (bacterial consumption OD600 depletion per worm). Serotonin challenge distinguished hypophagia due to sickness vs regulatory effects.
• Expression analyses across models: Assessed Atp6v0a1/V-ATPase subunits in mouse (diet-induced obesity and ob/ob) and human adipocytes (GEO datasets; adipose biopsies from lean vs obese) at gene and protein levels. Cellular fractionation and immunoblotting quantified V1 and V0 subunits in membrane and cytosol.
• Adipocyte-specific Atp6v0a1 knockout (KO): Generated by crossing floxed Atp6v0a1 mice with adiponectin-Cre. KO efficiency measured by qPCR and Western in adipocytes; SVCs and BAT served as controls. Phenotyping included chow and HFD cohorts.
• Metabolic phenotyping: Food intake (daily), body weight, DEXA body composition, histology (adipocyte size), indirect calorimetry (energy expenditure via ANCOVA, respiratory quotient, spontaneous activity), hypothalamic and WAT qPCR (Agrp, Npy, Pomc, Cart; Adipoq, Lep, lipogenic/oxidative and ER stress genes), plasma hormones (leptin, insulin, ghrelin) and Gdf15.
• Glucose homeostasis: Glucose tolerance tests (GTT), insulin tolerance tests (ITT), glucose-stimulated insulin secretion; acute insulin signaling assessed by pSer473-AKT immunoblotting in WAT, liver, skeletal muscle (basal vs insulin-stimulated).
• Pharmacological inhibition: Bafilomycin B1 (BFM, 100 nmol/kg, IP daily) administered to WT and KO mice during HFD initiation for 14 days; food intake, weight gain, GTT/ITT, insulin secretion measured; hypothalamic and WAT gene expression post-treatment.
• Weight rebound model: Mice rendered obese (9 weeks HFD), weight loss on LFD (3 weeks), then re-fed HFD with Veh or BFM; outcomes included food intake, weight regain, fat mass, hypothalamic Pomc, WAT Gdf15, V-ATPase subunit localization.
• In vitro adipocytes: 3T3-L1 adipocytes transfected with Atp6v0a1 siRNA vs control; outcomes included Atp6v0a1 and Gdf15 mRNA/protein, Adipoq and Lep expression, ER stress markers (Atf4, Ddit3).
• Statistics: Normality testing (Shapiro–Wilk); t-tests, one- or two-way ANOVA with multiple-comparison correction (Benjamini–Krieger–Yekutieli), repeated-measures ANOVA where appropriate; energy expenditure by ANCOVA with body weight covariate; α = 0.05.

• Adipocyte-centric metabolic memory: HFD induced substantial gene expression changes in adipocytes (925 DE genes) and SVCs (354). After weight loss, 752/925 adipocyte genes and 247/354 SVC genes remained persistently dysregulated, localizing most metabolic memory to adipocytes.
• C. elegans feeding screen: Of 28 mutants tested for conserved orthologs, 19 were significantly hypophagic (3–74% of normal intake), 2 hyperphagic (150–180%), and 8 unchanged vs N2 controls; most hypophagic strains (15/19) responded to serotonin. The unc-32/Atp6v0a1 mutant was hypophagic; prior IMPC data showed heterozygous Atp6v0a1 knockout males with reduced fat mass (p = 1.7 × 10^−6).
• Atp6v0a1/V-ATPase upregulation in obesity and persistence after weight loss: Atp6v0a1 expression increased in adipocytes from obese mice (diet-induced and ob/ob) and remained elevated after weight loss; multiple V-ATPase subunits showed similar patterns. In humans, adipocyte ATP6V0A1 mRNA and protein were higher in obesity and did not decrease after 5–15% weight loss.
• V-ATPase assembly state: Obesity increased both membrane and cytoplasmic abundance of V0 and V1 subunits in WAT; elevated levels persisted after weight loss. In KO mice, membranal V0a1 was significantly depleted.
• Adipocyte-specific Atp6v0a1 KO reduces intake and weight: KO mice had ~73% Atp6v0a1 mRNA reduction and ~92% protein reduction in adipocytes, with lower chow food intake and body weight; on HFD, KO mice ate less, gained less weight, had lower fat mass and smaller adipocytes. RQ and activity were unchanged; energy expenditure was slightly lower (mainly light period). Pair-feeding WT to KO intake recapitulated KO body weight, supporting reduced intake as the driver.
• Central and adipose signals: KO mice showed increased hypothalamic Pomc (~4-fold) and reduced Npy; in WAT, increased Adipoq and reduced Lep, proportional to weight. Gdf15 increased in WAT and plasma; ER stress markers Atf4 and Ddit3 were upregulated, suggesting a link to Gdf15 induction.
• Improved glucose homeostasis with KO: KO mice displayed better GTT and ITT performance (partly weight-related) and greater insulin-stimulated pSer473-AKT specifically in adipose tissue; basal insulin and glucose-stimulated insulin were not significantly different.
• Pharmacological validation: In WT mice, bafilomycin decreased average daily food intake by ~9%, reduced HFD-induced weight gain, and improved GTT/ITT without altering insulin secretion; these effects were not observed in KO mice, indicating dependence on adipocyte Atp6v0a1. BFM increased hypothalamic Pomc and WAT Gdf15 in WT.
• Weight regain model: During HFD refeeding after weight loss, BFM reduced food intake, blunted weight regain, decreased fat mass, increased hypothalamic Pomc, elevated WAT and plasma Gdf15, and reduced membranal V1/V0 in WAT.
• In vitro: Atp6v0a1 knockdown in 3T3-L1 adipocytes increased Gdf15 mRNA/protein and ER stress markers (Atf4, Ddit3) without altering Adipoq or Lep, supporting a cell-autonomous link between V-ATPase inhibition, ER stress, and Gdf15.

Findings demonstrate that a substantial portion of obesity-induced transcriptional changes persist after weight loss predominantly in adipocytes, constituting a peripheral ‘metabolic memory.’ Cross-species functional screening identified Atp6v0a1, a V-ATPase subunit, as a regulator of food intake. Genetic ablation of Atp6v0a1 in adipocytes or pharmacologic inhibition of V-ATPase reduced food intake by approximately 10–15%, lowered body weight and fat mass, and improved glucose handling. Mechanistically, obesity increased V-ATPase expression and assembly in adipocytes, and this upregulated state persisted after weight loss. Disrupting V-ATPase reduced membranal V0 levels and was associated with elevated adipose and circulating Gdf15 and increased hypothalamic Pomc, consistent with an adipose-to-brain signaling axis that suppresses feeding. Enhanced insulin-stimulated AKT phosphorylation in adipose tissue of KO mice suggests improved adipose insulin signaling accompanies reduced intake and weight. Overall, adipocyte V-ATPase activity appears to be a key effector of metabolic memory that promotes increased intake and weight regain; its inhibition modulates neuroendocrine pathways to counter these effects.

The study identifies adipocyte ATP6v0a1/V-ATPase as a central regulator of peripheral metabolic memory that influences food intake and susceptibility to weight regain. Adipocytes retain most obesity-induced transcriptional changes after weight loss. Genetic and pharmacologic inhibition of V-ATPase in adipocytes consistently reduces food intake, attenuates weight gain and regain, and improves glucose homeostasis, potentially via increased adipose Gdf15 and hypothalamic Pomc. These results suggest adipocyte V-ATPase as a therapeutic target for obesity management and weight-loss maintenance. Future work should define the precise mechanisms linking V-ATPase activity to Gdf15/ER stress, evaluate long-term efficacy and safety of targeted V-ATPase modulation, and explore translatability across sexes and diverse dietary contexts.

• The obesity model used a very high-fat diet (60% kcal), which may not fully reflect typical human dietary patterns, potentially affecting generalizability.
• Experiments were performed in male mice only; sex-specific effects were not assessed.
• Pharmacological inhibition used systemic bafilomycin, a broad V-ATPase inhibitor, over relatively short durations; long-term safety, specificity, and translational feasibility were not evaluated.
• Sample sizes for some assays were modest, and some physiological measures (e.g., energy expenditure) showed small or unexpected differences that warrant further investigation.
• While associations with Gdf15 and Pomc were demonstrated, causality and the full signaling pathway from adipose V-ATPase to central appetite circuits require further mechanistic dissection.