Fig. 5: Inhibition of parasympathetic cholinergic neurons elevates blood glucose levels.

a Schematic diagram of the experimental configuration (left panel). RetroAAV2-FLEX-Jaws-GFP viruses were injected into the liver of ChAT-IRES-Cre mice. Right top panel: Images of confocal fluorescence microscopy showing co-expression of GFP (left) and ChAT (middle) in a subset of cholinergic neurons in the DMV (right panel). Scale bar: 10 μm. Right bottom panel: Images of confocal fluorescence microscopy showing the expression of GFP-positive fibers (left) around hepatocytes in the liver. Note that these are not cytoplasmic staining. Scale bar: 20 μm. b Pictures showing our noninvasive optogenetic stimulation. Small SMD-LED (2 mm × 2 mm) were directly placed on the skin of the abdomen following hair removal. c Pooled data from 12 (w/o Jaws) and 18 (w Jaws) mice showing changes in ACh content in the liver (two-tailed t test, **p = 0.002). All data are shown as mean ± SEM. Source data are provided as a Source Data file. d, e Pooled data showing the effect of inhibition of liver-projecting cholinergic nerves on hepatic glucose output in male mice. Optogenetic inhibition of cholinergic fibers in the liver of ChAT-IRES-Cre injected with retroAAV-FLEX-Jaws-GFP mice elevated blood glucose levels (control (ChAT-IRES-Cre), n = 8 mice, ChAT-IRES-Cre+Jaws, n = 17 mice; two-way RM ANOVA followed by Sidak multiple comparisons test, interaction F (5, 115) = 13.9, p < 0.001, time F (5, 115) = 24.6, p < 0.001, between the groups, F (1, 23) = 19.8, p < 0.001; 15 min, *p = 0.03; 30 min, ***p < 0.001; 45 min, ***p < 0.001; 60 min, ***p < 0.001; left panel). Right panel: Summary plots showing individual data before and after optogenetic stimulation (two-tailed t test, ***p < 0.001, vs. 0 min, n = 17 mice) (d). e Summary plot showing changes in blood glucose levels at the end of stimulation (mean blood glucose, 0 min, 120.8 ± 8.6 mg/dl, 60 min, 192.1 ± 11.5 mg/dl, two-tailed t test, ***p < 0.001, n = 17 mice). Mice were not fasted for these experiments. All data are shown as mean ± SEM. Source data are provided as a Source Data file. f, g Pooled data showing the effect of inhibition of liver-projecting cholinergic nerves on hepatic glucose output in female mice (control (ChAT-IRES-Cre), n = 7 mice, ChAT-IRES-Cre+Jaws, n = 8 mice); two-way RM ANOVA followed by Sidak multiple comparisons test, interaction F (5, 65) = 9.9, p < 0.001, time F (5, 65) = 12.8, p < 0.001, between the groups F (1, 13) = 14.8, p = 0.002; 30 min, **p = 0.007; 45 min, ***p < 0.001; 60 min, ***p < 0.001; ChAT-Cre, n = 7 mice, ChAT-IRES-Cre+Jaws, n = 8 mice (f). g Summary plot showing changes in blood glucose levels at the end of stimulation (mean blood glucose, 0 min, 87.6 ± 12.8 mg/dl, 60 min, 147.1 ± 8.4 mg/dl, two-tailed t test, **p = 0.002, n = 8 mice). Mice were not fasted for these experiments. All data are shown as mean ± SEM. Source data are provided as a Source Data file. h, i Bar graphs showing changes in G6pase and Pepck mRNA expression with and without Jaws following optogenetic stimulation in male (h) and female (i) mice (males, two-tailed t test, G6pase, ***p < 0.001; Pepck, *p = 0.01; ChAT-Cre, n = 8 mice, ChAT-IRES-Cre + Jaws, n = 14–15 mice; females, two-tailed t test, G6pase, ***p < 0.001; Pepck, ***p < 0.001; ChAT-Cre, n = 10 mice, ChAT-IRES-Cre+Jaws, n = 13 mice). All data are shown as mean ± SEM. Source data are provided as a Source Data file. j, k Bar graphs showing plasma glucagon (j) and insulin (k) levels following the inhibition of liver-projecting cholinergic nerves (two-tailed t test, glucagon, p = 0.3 ChAT-IRES-Cre, n = 13 mice, ChAT-IRES-Cre + Jaws, n = 13 mice; insulin, ChAT-IRES-Cre, n = 15 mice, ChAT-IRES-Cre + Jaws, n = 15 mice, two-tailed t test, *p = 0.03). All data are shown as mean ± SEM. Source data are provided as a Source Data file.