Human Milk Oligosaccharide L‐N‐Tetraose Activates GPR35 on Vagal Afferent Neurons In Vitroand Reduces High Fat Diet‐Induced Obesity in Male Mice

Obesity is associated with altered gut microbiota; dietary supplementation with prebiotics improves metabolic outcomes in obesity, an effect ascribed to improved microbial dysbiosis. However, alternative mechanisms have not been fully explored. We have shown that prebiotic milk oligosaccharides (MOs) decrease weight gain and food intake in mice fed a high-fat diet (HFD), and improve cholecystokinin (CCK)-induced satiety signaling. It has recently been shown that the human MO lacto-N-tetraose (LNT) is a ligand for the G-protein coupled receptor, GPR35. GPR35 is highly expressed by vagal afferent neurons (VAN) where it is co-expressed with the CCKA R. HFD reduces endogenous GPR35 ligands and sensitivity to CCK, but the role of GPR35 and its ligands eg LNT, in satiety signaling via the vagal afferent pathway have not been investigated. Here we test the hypothesis that LNT reduces HFD-induced weight gain and hyperphagia in mice via a direct effect on GPR35 expressed by VAN.Mice were maintained on low-fat diet (10% kcal fat), HFD (45% fat kcal), or HFD supplemented with 10% LNT for 8 weeks (male, n=5-9/group). To determine if VAN are directly activated by GPR35 ligands, VAN from adult male Wistar rats were cultured and treated with the GPR35 agonist zaprinast (100 µM), LNT (10 mM), or D-lactose (constituent of LNT; 10 mM) for 15 minutes after 1 hour pre-incubation with the GPR35 competitive antagonist CID-2745687 (100 µM) or vehicle (n=4/treatment). Phosphorylation of extracellular regulated kinase (ERK) relative to total ERK (pERK/ERK) was determined by western blot and used as an indicator of VAN activation.LNT administration significantly reduced body weight, fat mass, and improved HFD-induced hyperphagia (p<0.05). Treatment of VAN with the GPR35 agonist zaprinast increased pERK/ERK (p<0.05) relative to vehicle; zaprinast-induced activation was prevented by the CID-2745687. LNT, but not D-lactose, also activated VAN, an effect inhibited by the GPR35 antagonist.LNT may decrease food intake via a direct effect on VAN, possibly by increasing sensitivity to peripheral satiety signals. This work reveals a potential peripheral therapeutic target for ameliorating diet-induced weight gain.