Therapeutic knockdown of MLKL reduces diet-induced obesity and improves insulin signalling in mature adipocytes

Abstract

Obesity affects one in three adults and is complicated by adipose inflammation, lipotoxicity and cell death. We previously identified RIPK1 as a genetic determinant of human obesity risk and adipose inflammation. Because RIPK1 is the apical kinase in the necroptosis pathway upstream of RIPK3 and the executioner protein MLKL, and emerging evidence links MLKL to lipid metabolism, MLKL has surfaced as a potential metabolic regulator. However, conflicting findings in Mlkl knockout mice fed a high fat diet have left its therapeutic relevance unresolved. MLKL has not been previously targeted through therapeutic knockdown in vivo in the context of diet-induced obesity. Here, we evaluated two independent MLKL antisense oligonucleotides (ASOs) in high fat diet (HFD)-fed C57BL/6J mice. In a 24-week progression model, MLKL ASO markedly reduced body weight, fat mass and hepatic steatosis compared with controls, while preserving lean mass. MLKL knockdown also lowered the respiratory exchange ratio, indicating a shift toward increased fat oxidation. In the intervention model, once obesity was established after 12 weeks of HFD feeding, both MLKL ASOs, and similarly, two independent RIPK1 ASOs, reversed weight gain and improved systemic glucose control. In vitro, MLKL-CRISPR/Cas9 knockout blocked 3T3-L1 adipogenesis, indicating a requirement for MLKL during adipocyte differentiation. However, in mature adipocytes, MLKL siRNA reduced palmitic acid-induced lipid accumulation, increased isoprenaline-stimulated lipolysis, and prevented TNF-mediated suppression of insulin-mediated AKT signalling and glucose uptake. Collectively, these findings demonstrate that partial MLKL suppression reprograms whole-body energy metabolism, enhances insulin sensitivity and limits diet-induced adiposity. MLKL, therefore, represents a promising and mechanistically novel therapeutic target for obesity and insulin resistance.