Protection against overfeeding-induced weight gain is preserved in obesity but does not require FGF21 or MC4R

Body weight and fat mass are under homeostatic regulation, with feedback mechanisms influencing energy intake and expenditure to counter energy balance disruptions. This aligns with the 'dual intervention point' model, suggesting weight maintenance within upper and lower boundaries rather than a single set point. This homeostatic system likely evolved to balance predation risk (excess fat) and starvation (insufficient fat). Remarkably, the body effectively counteracts weight loss attempts by increasing appetite and reducing energy expenditure, hindering sustained weight loss in many. In chronic negative energy balance, reduced leptin levels are a key driver of weight regain. However, homeostatic mechanisms also protect against excessive weight gain. Overfeeding studies in animals and humans show weight recovery after cessation of overfeeding, but the underlying mediators remain unclear. It's also unknown if obesity affects this homeostatic recovery. Rodent parabiosis studies suggest endocrine regulation, but leptin's limited role in countering chronic overfeeding points to other signaling molecules and pathways. This study uses an intragastric overfeeding mouse model to investigate obesity's influence on homeostatic weight recovery, the roles of FGF21 and MC4R, hypothalamic transcriptional and vascular changes, and potential endocrine regulators.

The literature extensively documents the homeostatic regulation of body weight and fat mass, highlighting feedback mechanisms that counteract perturbations in energy balance. The 'dual intervention point' model proposes that body weight is maintained within a range defined by upper and lower boundaries rather than a precise set point. This model is supported by the body's robust compensatory mechanisms that resist both weight loss and weight gain attempts. Several studies have investigated the role of various hormones and neuropeptides in these regulatory processes, particularly leptin, a key hormone signaling nutritional status. However, the specific molecular mediators of the homeostatic response to overfeeding remain largely unidentified, particularly those operating independently of leptin. While parabiosis studies have suggested a role for circulating factors, the precise nature of these factors and their mechanisms of action require further elucidation.

This study employed an intragastric overfeeding (ExpOF) mouse model. Lean and diet-induced obese (DIO) male mice underwent 14 days of intragastric infusion of a hypercaloric liquid diet (50% energy surplus). Control groups received water infusions. Body weight, food intake, and various metabolic parameters (plasma glucose, insulin, leptin, ghrelin, triglycerides, NEFA, cholesterol) were monitored. Tissue samples (eWAT, IWAT, IBAT, liver, muscle, hypothalamus) were collected for histological, transcriptomic (RNA-sequencing), and proteomic analyses. Targeted plasma proteomics investigated circulating factors linked to overfeeding. The roles of FGF21 and MC4R were investigated using knockout mice subjected to ExpOF. Recombinant DLL1 and LGMN proteins were administered to DIO mice to assess their effects on body weight and food intake. Hypothalamic vascularization was assessed using iDISCO 3D imaging. Statistical analyses included one-way and two-way ANOVA, followed by post-hoc tests where appropriate. RNA sequencing data was processed using STAR aligner, featureCounts, and DESeq2. Gene Ontology enrichment analysis was performed using clusterProfiler.

Both lean and DIO mice exhibited a robust and prolonged suppression of voluntary food intake following ExpOF, leading to a rapid return to baseline body weight. FGF21, although significantly elevated in response to overfeeding, was not essential for this weight recovery. Targeted proteomics identified legumain (LGMN) as a potential modulator of the overfeeding response. Recombinant LGMN administration reduced body weight and food intake in DIO mice. Overfeeding was associated with reduced hypothalamic vascularization (in DMH and VMH, but not ARC) and sustained reductions in *Npy* and *Agrp* gene expression. MC4R KO mice showed a slower weight recovery compared to WT mice, suggesting a role for MC4R in the speed but not the overall efficacy of weight regulation after overfeeding. Overfeeding induced subtle changes in markers of adaptive thermogenesis, predominantly in brown adipose tissue, but these were less prominent than the hypophagic response. RNA sequencing of the hypothalamus revealed distinct transcriptional changes during overfeeding and recovery, with persistent suppression of *Npy* expression.

The study's findings highlight the potent homeostatic defense against overfeeding-induced weight gain, which persists even in obesity. The primary mechanism appears to be a profound and prolonged suppression of voluntary food intake, surpassing the impact of alterations in energy expenditure or adaptive thermogenesis. The dispensability of FGF21 and MC4R in this response suggests that other yet-to-be-identified factors play critical roles. The identification of LGMN as a potential modulator opens up new avenues for research. The hypothalamic changes, including reduced vascularization and altered gene expression, support the involvement of central nervous system mechanisms in this homeostatic regulation. Future research should focus on the precise role of LGMN, further characterization of other potential circulating factors, and a deeper understanding of the central nervous system pathways involved in this compensatory response.

This study demonstrates the robustness of the homeostatic defense against overfeeding-induced weight gain in both lean and obese mice, independent of FGF21 and MC4R. The identification of LGMN as a circulating factor that impacts body weight and food intake in DIO mice provides a novel target for future research. The study underscores the importance of hypothalamic signaling in regulating weight homeostasis and offers potential new avenues for obesity treatment.

The study primarily used male mice, limiting the generalizability to females. The intragastric overfeeding model, while effective, might not fully replicate natural eating behaviors. Further studies are needed to validate the physiological role of LGMN and explore its potential therapeutic implications. The relatively short-term nature of the study may not capture long-term metabolic consequences of overfeeding.