Figure 2. High fat feeding impairs the EEC calcium response toward palmitate stimulation.

(A) Measurement of the EEC response to nutrient stimulation using Tg(neurod1:Gcamp6f). (B) Time lapse image of the EEC response to BSA conjugated palmitate stimulation in Tg(neurod1:Gcamp6f) using the EEC response assay. Note that palmitate responsive EECs are primarily in the proximal intestine. (C) Heat map image indicating the EEC calcium response at 0 and 3 min post palmitate stimulation from the highlighted area in B. (D) Change in Gcamp6f relative fluorescence intensity in 5 min with no stimulation or stimulation with egg water, BSA vehicle, palmitate, linoleate, dodecanoate or butyrate. Note that only palmitate, linoleate and dodecanoate induced EEC calcium responses. (E, F) Change in Gcamp6f relative fluorescence intensity in BSA stimulated (n = 4) and palmitate stimulated animals (n = 5). (G) Measurement of EEC calcium responses to palmitate stimulation following 4–8 hr of high fat (HF) meal feeding in 6 dpf Tg(neurod1:Gcamp6f) larvae. (H, I) Representative images of the EEC response to palmitate stimulation in control larvae (without HF meal feeding, (H) and 6 hr of HF feeding (I). (J) Measurement of EEC calcium responses to palmitate stimulation in Tg(neurod1:Gcamp6f) larvae following 4 and 8 hr HF feeding. Student t-test was used in F and one-way ANOVA with post-hoc Tukey test was used in J. **p<0.01, ***p<0.001.

Figure 2—figure supplement 1. EEC activity assay.

(A) Experimental design of EEC activity assay using Tg(neurod1:Gcamp6f) zebrafish. (B) Representative images of EEC calcium fluorescence analysis using FIJI template matching and background subtraction in Tg(neurod1:Gcamp6f) zebrafish stimulated with palmitate. (C) Relative fluorescence intensity in the proximal intestine in a series of video images from zebrafish in B. (D) Spatial-temporal resolution of the EEC response to palmitate, glucose and cysteine stimulation. (E) Representative images of the EEC nutrient response in a regional specific manner. Palmitate and glucose primarily activated EECs in the proximal intestine where most lipid and nutrient absorption occurs. In contrast, cysteine activated EECs in the mid-intestine (segment 2) where proteins and other macromolecules are digested andabsorbed by specialized intestinal epithelial cells in this region (Nakamura et al., 2004; Wang et al., 2010; Park et al., 2019).

Figure 2—figure supplement 2. Feeding a high fat meal did not impair subsequent fatty acid intake.

(A–C) Fatty acid was labeled with green fluorescence in BODIPY-C5 (Carten et al., 2011). BODIPY-C5 (in BSA complex) was delivered to zebrafish larvae that had been fed high fat (HF) meal for 6 hr, the same as the EEC activity assay. Within 5 min of delivery, green BODIPY-C5 was distributed throughout the entire zebrafish intestinal lumen. (D–F) Fatty acids were labeled with red fluorescence in BODIPY-C12 (Carten et al., 2011). BODIPY-C12 was delivered to zebrafish larvae that had been fed HF meal for 6 hr, the same as the EEC activity assay. Within 5 min of delivery, red BODIPY-C12 was distributed throughout the zebrafish intestinal lumen.