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

Obesity is a significant global health concern, increasing the risk of various comorbidities and reducing life expectancy. While weight loss can be achieved through dietary changes and increased physical activity, maintaining this weight loss proves challenging, with a substantial portion of individuals regaining the lost weight within a year. This weight regain is attributed to physiological adaptations, including alterations in appetite-regulating hormones, changes in energy expenditure, and increased appetite. Although these physiological changes are well-documented, the underlying molecular mechanisms driving weight regain remain poorly understood. Previous research introduced the concept of "metabolic memory," where obesity induces lasting metabolic changes that persist even after weight loss. These persistent changes manifest in the transcriptome, metabolome, proteome, and microbiome. Adipose tissue, crucial for energy balance regulation, is central to theories explaining the persistence of a biological drive to regain weight. Studies have shown that obesity-induced changes in metabolite levels in adipose tissue endure after weight loss, while other tissues revert to pre-obese levels. The concept that adipose-derived metabolic memory influences food intake and metabolism is further supported by studies demonstrating a refractory transcriptional and metabolic response in white adipose tissue (WAT) to dietary restriction in mice previously fed ad libitum. This study hypothesized that obesity-induced changes persisting after weight loss might significantly impact feeding behavior and contribute to weight regain. The researchers aimed to identify the regulators of this peripheral metabolic memory by functionally analyzing transcriptional changes in obese WAT that persist after weight loss and to investigate the potential role of these genes in feeding behavior and weight regain.

The literature review section of the paper extensively covers the challenges associated with long-term weight loss maintenance and the concept of "metabolic memory." It cites numerous studies demonstrating persistent changes in various biological systems (transcriptome, metabolome, proteome, and microbiome) following weight loss in individuals with obesity. A key focus is the role of adipose tissue in these persistent metabolic changes and its contribution to weight regain. The review highlights previous findings indicating that the "metabolic memory of obesity" predominantly resides in adipose tissue, where changes in metabolite levels persist even after successful weight loss. It also mentions studies showing that switching mice from dietary restriction to ad libitum feeding acutely increases mortality, while the reverse switch causes only a gradual increase in survival, suggesting a memory of prior nutritional states. Overall, the review lays a strong foundation for the study's hypothesis by emphasizing the lack of understanding of the molecular mechanisms underlying the drive for weight regain and the importance of adipose tissue in this process.

The study employed a multi-pronged approach, utilizing various techniques and model organisms to investigate the role of adipocytes in weight regain: 1. **Diet Switch Mouse Model:** Male C57BL/6J mice were fed either a low-fat diet (LFD) or a high-fat diet (HFD) for 9 weeks. Half of the HFD-fed mice were then switched to LFD for 3 weeks to induce weight loss. Epididymal white adipose tissue (WAT) was collected and fractionated into adipocytes and stromal vascular cells (SVCs) for RNA sequencing analysis. 2. **RNA Sequencing:** RNA sequencing was performed on adipocytes and SVCs from lean, obese, and formerly obese mice to identify persistently dysregulated genes after weight loss. The data were analyzed using the limma statistical algorithm. 3. **C. elegans Food Intake Assay:** A high-throughput food intake assay in *C. elegans* was used to screen for metabolic memory genes identified in the mouse model. *C. elegans* mutants lacking orthologous mouse genes were screened to determine their impact on food intake. A serotonin challenge was used to differentiate between hypophagia due to metabolic changes and developmental defects. 4. **Adipocyte-Specific Atp6v0a1 Knockout Mice:** Adipocyte-specific *Atp6v0a1* knockout mice were generated using the adiponectin-cre system. These mice were fed normal chow or HFD, and food intake, body weight, body composition, and energy expenditure were measured. Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed. 5. **Bafilomycin Treatment:** Wild-type and knockout mice were treated with bafilomycin B1, a V-ATPase inhibitor, to assess the effect of pharmacological V-ATPase inhibition on food intake, weight gain, and glucose homeostasis. GTT and ITT were also conducted in these mice. 6. **Weight Rebound Model:** Formerly obese mice were re-fed HFD and treated with bafilomycin to determine the effect of V-ATPase inhibition on weight regain. 7. **3T3-L1 In Vitro Studies:** 3T3-L1 adipocytes were used to investigate the effects of *Atp6v0a1* knockdown on gene and protein expression levels of GDF15, ATF4 and DDIT3. 8. **Statistical Analysis:** Various statistical methods were employed, including Student's t-tests, one-way and two-way ANOVAs, and ANCOVA, to analyze the data. Appropriate post-hoc tests and corrections for multiple comparisons were used where necessary.

The study yielded several key findings: 1. **Metabolic Memory in Adipocytes:** RNA sequencing revealed that a substantial portion of obesity-induced gene expression changes persisted in WAT after weight loss, predominantly in adipocytes. This "metabolic memory" was far more pronounced in adipocytes than in stromal vascular cells (SVCs). 2. **ATP6v0a1 as a Regulator of Food Intake:** A *C. elegans* food intake screen identified *Atp6v0a1*, encoding a V-ATPase subunit, as a gene whose loss significantly reduced food intake. This finding highlighted its potential role in regulating energy balance. 3. **Elevated ATP6v0a1 in Obesity:** The expression of *Atp6v0a1* was significantly elevated in adipocytes of obese mice and humans, and this elevation persisted after weight loss. Other V-ATPase subunits showed similar patterns. 4. **Adipocyte-Specific Atp6v0a1 Knockout Reduces Food Intake and Weight Gain:** Adipocyte-specific *Atp6v0a1* knockout mice exhibited significantly lower food intake and body weight, both on normal chow and HFD. This effect was primarily attributed to reduced food intake, not changes in energy expenditure or intestinal absorption. 5. **Improved Glucose Metabolism in Knockout Mice:** The knockout mice displayed improved glucose tolerance and insulin sensitivity, mainly driven by enhanced insulin signaling in adipose tissue. 6. **Bafilomycin Inhibits Weight Gain and Improves Glucose Tolerance:** Pharmacological inhibition of V-ATPase using bafilomycin resulted in reduced food intake, weight gain, and improved glucose tolerance in wild-type mice. This effect was dependent on adipocyte *Atp6v0a1* expression, as bafilomycin had no effect on knockout mice. 7. **V-ATPase Inhibition and Appetite Regulators:** Both genetic and pharmacological V-ATPase inhibition were associated with increased hypothalamic *Pomc* (satiety) expression and increased expression of *Gdf15* in adipose tissue, a hormone linked to appetite suppression and potentially ER stress. Inhibition of V-ATPase also led to a decrease in hypothalamic *Npy* (orexigenic) expression. 8. **Effect of V-ATPase Inhibition on Weight Regain:** In a weight rebound model, V-ATPase inhibition decreased food intake and blunted weight regain in formerly obese mice. 9. **In vitro 3T3-L1 study:** In vitro studies using 3T3-L1 adipocytes, showed that siRNA knockdown of Atp6v0a1 significantly reduced Atp6v0a1 gene and protein expression, and increased Gdf15 expression, which is suggestive of ER stress.

This study successfully identified *ATP6v0a1*, a component of the vacuolar H+-ATPase, as a key regulator of peripheral metabolic memory in obesity. The findings strongly suggest that increased V-ATPase activity in adipocytes contributes to the persistent drive for increased food intake and weight regain after weight loss. The observed effects of both genetic knockout and pharmacological inhibition of *Atp6v0a1* on food intake, weight gain, and glucose metabolism underscore its crucial role in regulating energy balance. The increased *Pomc* expression in the hypothalamus and elevated *Gdf15* levels in adipose tissue and plasma suggest a mechanism through which adipocyte V-ATPase activity influences the brain-adipose axis, modulating appetite and energy expenditure. The multi-species approach strengthens the study's conclusions, highlighting the conserved role of V-ATPase in regulating food intake across diverse organisms. The findings have implications for the development of novel anti-obesity therapies targeting peripheral metabolic pathways, potentially minimizing the risks associated with centrally acting agents.

This comprehensive study demonstrates a crucial role for adipocyte V-ATPase, particularly the *ATP6v0a1* subunit, in regulating food intake and weight regain in obesity. Both genetic and pharmacological inhibition of V-ATPase consistently led to reduced food intake, weight loss, and improved glucose metabolism. The observed increase in *Gdf15* and altered hypothalamic neuropeptide expression suggests a mechanism involving the adipose-hypothalamic axis. These findings position *ATP6v0a1* as a promising therapeutic target for obesity management, offering a peripheral approach that may mitigate the potential mental health side effects of centrally acting treatments. Future research should focus on further elucidating the precise mechanisms by which V-ATPase influences the brain-adipose axis and investigating the long-term effects of V-ATPase inhibition on weight maintenance and metabolic health.

While the study provides strong evidence for the role of *ATP6v0a1* in regulating energy balance, certain limitations should be noted. The mouse models, while useful, may not perfectly replicate the complexity of human obesity. The pharmacological approach using bafilomycin, a broad V-ATPase inhibitor, lacks specificity. Future studies should utilize more targeted V-ATPase inhibitors or genetic modifications to confirm these findings. Furthermore, the study primarily focused on white adipose tissue; investigating other adipose tissue depots and their contribution to metabolic memory would be beneficial. Finally, the long-term effects of V-ATPase inhibition on metabolic health and potential side effects require further exploration.