Abstract Body: Introduction
Obesity affects ~66% of adults and features adipose inflammation, lipotoxicity and cell death. We previously linked RIPK1 to human obesity risk (OR 1.8) and adipose inflammation. RIPK1 signals via downstream effectors, RIPK3 and MLKL, to drive necroptosis. Emerging data implicate MLKL in lipid metabolism. Despite conflicting reports in Mlkl^-/- obese mice, MLKL’s therapeutic potential and role in adipocyte insulin signaling remain unclear.

Methods
C57Bl/6 mice (n = 8/grp) on a high-fat diet (HFD; 60% kCal) underwent 2 MLKL anti-sense oligonucleotides (ASO)-treatment arms: progression model with weekly injections of control ASO or MLKL ASO#2 (50mg/kg, 24 weeks) or intervention model where 12week-HFD-fed obese mice received control ASO, MLKL ASO#1 or #2 for 10 weeks. In vitro, differentiated 3T3-L1 adipocytes with MLKL siRNA or CRISPR KO were assessed for lipid loading, lipolysis and insulin signalling in inflammatory or lipotoxic stress.

Results
In the progression study, MLKL ASO reduced body weight from 52.2±6.6g to 37.3±6.2g (p<0.001), fat mass by 56.2% (p<0.001), and liver steatosis (p<0.05) relative to control, while preserving lean mass. Serum triglycerides fell 36.6% (p<0.001) with no ALT/AST change. Of note, the respiratory exchange ratio (RER) decreased (p<0.01) in MLKL ASO-mice, indicating enhanced fat oxidation; physical activity trended towards an increase. In the intervention arm, both MLKL ASOs reduced body weight (56.0±2.8g vs 47.7±5.0g or 47.2±3.6g; p<0.05) and fat mass, mirroring RIPK1 ASO effects. Insulin sensitivity improved in both arms ( GTT and ITT, p<0.05).

In vitro, MLKL-KO blocked 3T3-L1 differentiation by markedly reducing adipogenic genes (adiponectin, Cebpa, Pparg, Bmp4 and Ebf1). In mature adipocytes, MLKL siRNA reduced palmitic-acid-induced lipid accumulation by 85% (p<0.001), increased isoprenaline-stimulated lipolysis by 39% (p<0.0001), and prevented TNF-mediated suppression of insulin-stimulated phospho-AKT and glucose uptake.

Conclusion
Targeted MLKL knockdown markedly reduced diet-induced adiposity, steatosis and triglycerides and enhanced fat oxidation and insulin sensitivity. Mechanistically, MLKL loss in mature adipocytes limits lipid accumulation, promotes lipolysis and restores insulin signaling under inflammatory stress. Given that MLKL deletion impairs adipocyte differentiation, modulated suppression of MLKL in mature adipocytes offers a promising, nuanced metabolic therapy.