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Sexually Dimorphic Ano2 Expression in Nodose Neurons Determines CCK‐mediated Satiation and Obesity in Heterozygote Male Mice

The gut peptide cholecystokinin (CCK) contributes to neurally mediated post prandial satiety. Our previous results indicate that high fat diet downregulates the CCK‐sensitive anoctamins (Ano1 and Ano2) that function as calcium‐activated chloride channels (CaCCs) in intestinal vagal afferents. Using Cre‐loxP system, we generated heterozygote (HET) Nav1.8CreAno2fl/WT mice. The endogenous Scn10a promoter drives Cre expression (Nav1.8Cre) selectively in sensory neurons of the nodose, dorsal root and trigeminal ganglia where loxP sites flanked Ano2 exon 12. The male HET mice exhibited an obese phenotype and decreased sensitivity to CCK. To assess the Ano2 expression in HET vs. control (Nav1.8Cre+/−) mice, we first did qRT‐PCR on whole nodose ganglia obtained under anesthesia at 50 weeks of age and found no significant difference in Ano2 mRNA expression (n=4, p>0.05). Ano2 gene may be expressed in non‐neuronal cells of nodose ganglia while Cre expression is selective to neurons. We measured Ano2 mRNAs in nodose neurons cultured for 16–18 hours using single cell qRT‐PCR. Cre positive neurons were 57% (12 of 21) in HET and 74% (29 of 39) in control, indicating a lower efficiency of Cre expression in HET. Correspondingly, Ano2 positive neurons were 48% (10 of 21) and 62% (24 of 39), respectively, indicating the Ano2 allelic deletion in HET. The combined Cre and Ano2 positivity in HET was 29% (6 of 21) vs. 54% (21 of 39) in control. Most importantly the Ano2 mRNA levels in HET neurons (n=10) were 20% of the levels measured in control neurons (n=24, p<0.05) (0.20±0.16 vs. 1.00±0.57). The Ano2 expression in individual nodose neurons of female mice did not show a reduction or even higher in HET (n=27) vs. control (n=28) (2.58±1.48 vs. 1.42±1.27, p>0.05). The female HET mice remained CCK sensitive indicating a sexual dimorphism. We conclude that differences in expression of Ano2 mRNA support our findings of CCK insensitivity and selective obesity in male but not female HET mice (HL14388).