Heterozygote Knockout of TMEM16B in Intestinal Vagal Afferents Causes Cholecystokinin Insensitivity and Obesity in Male but Not Female Mice

Cholecystokinin (CCK) is a well-known satiety peptide that inhibits food intake. The CCK induced satiety signal in intestinal vagal afferents is attenuated in mice on a High Fat Diet (HFD). We have shown that down regulation of a Ca 2+ -activated Cl - channel (CaCC) downstream of CCK receptors is responsible for the neuronal insensitivity to CCK. We also found that the CaCC subunit Ano2/TMEM16B is essential for the CCK-induced current in nodose neurons. In this study we tested the hypothesis that reduction of this subunit in vivo contributes to weight gain. One allele of the Ano2/TMEM16B was knocked out in sensory neurons by crossing the ano2 fl/ fl mice with Nav1.8Cre mice to generate the Nav1.8Cre;ano2 fl /wt mice. The Cre negative littermates were used as control. We found that CCK-induced suppression of food intake is eliminated in male Nav1.8Cre;ano2 fl /wt mice. Food intake measured over 4 hours was 1.21±0.11g (n=5) in male wild type (wt) mice injected with saline and was reduced to 0.77±0.18 g (n=7, p<0.05) in mice injected with CCK-8 (3μg/kg). Those values were 0.63±0.15 g (n=6) in saline injected and 0.91±0.13 g (n=7, p>0.05) in CCK injected male Nav1.8Cre;ano2 fl /wt mice. However, the CCK injection did not affect food intake in either female wt or Nav1.8Cre;ano2 fl /wt mice. The male Nav1.8/ano2 fl/wt mice were on the average 5.5g heavier than wt mice at 40 weeks of age (39.8±1.4 g, n=13 vs 34.3±1.4 g, n=14, p<0.01). Body weight of females was significantly lower than in males but was not different between wt and Nav1.8Cre;ano2 fl /wt mice (30.4±1.0 g, n=14 vs 28.5±1.0 g, n=11, p=0.20 respectively). Single cell mRNA level of Ano2 and CCK-induced TMEM16 currents in nodose neurons were reduced significantly in male Nav1.8Cre;ano2 fl/wt mice compared to Cre negative controls, but such changes were not seen in female mice. We conclude that heterozygote knockout of Ano2/TMEM16B specifically in sensory neurons causes neuronal insensitivity to CCK and excessive weight gain in male but not female mice. Reduction of this subunit may contribute to the HFD induced obesity. The reason for the phenotype and allele expression variability between sexes is unclear.