Article Figures & Data
Figures
- Figure 1.
CCK-dependent expression of CART in vagal afferent neurons. Nodose ganglia from rats are shown. A, B, Rats fed ad libitum exhibiting CART-immunoreactive neurons. C, Reduced CART immunoreactivity after fasting for 24 h. D, Stimulation of CART immunoreactivity after fasting for 24 h and 1 h after an intraperitoneal injection of 10 nmol of CCK. E, F, Stimulation of CART after fasting for 24 h and refeeding for 1 h (E), which was inhibited by administration of the CCK-1 receptor antagonist lorglumide (F; 10 mg · kg−1, i.p.) 15 min before refeeding. Representative images from 10 independent experiments are shown. Magnification: A, 20×; B–F, 40×.
- Figure 2.
CART expression in cultured vagal afferent neurons. Vagal afferent neurons cultured for >48 h showing CART immunoreactivity (A, C, E, G, I, K) or nuclei stained with DAPI in the respective fields (B, D, F, H, J, L). A, B, CART is undetectable after transfer of neurons to serum-free medium for 2 h. C, D, CART immunoreactivity is readily detected in cells in serum-free medium 2 h after the addition of 10 nm CCK. E, F, CART is also induced in cells in serum-free medium 2 h after the addition of 100 nm PMA. G, H, The effect of CCK is reduced by the PKC inhibitor Ro-32-0432 (1 μm). I–L, In serum-free medium, ghrelin (10 nm) has no effect on CART abundance (I, J) but inhibits the effect of CCK (K, L). Arrows identify a representative neuron in each pair of images; filled arrows show CART-positive neurons, and open arrows show CART-negative neurons. Representative images from six independent experiments are shown. Scale bar, 50 μm.
- Figure 3.
CCK induces CREB phosphorylation through activation of PKC in vagal afferent neurons. Vagal afferent neurons cultured for >48 h and stained with antibody to phosphoCREB (A, C, E, G, I, K) or CREB (M, O) and DAPI (B, D, F, H, J, L, N, P), in the respective fields, are shown. A, B, Nuclear phosphoCREB staining was undetectable after transfer to serum-free medium for 2 h. C–F, Nuclear phosphoCREB was readily detectable after treatment with 10 nm CCK (C, D) and 100 nm PMA (E, F) for 30 min. G, H, The effect of CCK was inhibited by pretreatment with 1 μm Ro-32-0432. I, J, Nuclear phosphoCREB was undetectable after treatment with ghrelin (10 nm). K, L, Importantly, ghrelin (10 nm) in the presence of 10 nm CCK led to exclusion of phosphoCREB from the nucleus. M–P, Total CREB (M, N) was unchanged by CCK and showed a cytoplasmic localization in the presence of ghrelin plus CCK (O, P). A–L, Arrows identify a representative neuron in each pair of images; filled arrows show phosphoCREB-positive neurons, and open arrows show phosphoCREB-negative neurons. M–O, Arrows indicate a representative neuron exhibiting nuclear localization of total CREB (M, N) and a representative neuron exhibiting cytosolic localization of total CREB (M, O). Representative images from six independent experiments are shown. Scale bar, 50 μm.
- Figure 4.
CCK regulates CART transcription in luciferase promoter–reporter assays. A, Cultured vagal afferent neurons were transfected with empty vector (empty) and deletional mutants of the CART promoter of 3451, 3100, 620, and 120 bp upstream of the transcriptional start site in luciferase reporter vectors (empty, empty vector). Studies were performed 16 h after transfection when cells were transferred to serum-free medium and stimulated (filled bars) or not (open bars) with 10 nm CCK for 6 h. B, In vagal afferent neurons transfected with 3451CART-Luc, the response to 10 nm CCK was inhibited by 1 μm Ro-32-0432. C, In vagal afferent neurons transfected with 3451 CART-Luc, the effect of 10 nm CCK was inhibited by preincubation for 30 min with 10 nm ghrelin (Gh). Responses to CCK are expressed relative to the appropriate control (1.00). *p < 0.05, **p < 0.01,*** p < 0.001 (t test; n = 6–15). Error bars indicate SEM.
- Figure 5.
CCK-stimulated CART transcription is mediated by Cre, CREB, and CBP in vagal afferent neurons. A, In cultured vagal afferent neurons cotransfected with 3451CART-Luc and ACREB, stimulation of luciferase activity by CCK was inhibited compared with a control vector (EMPTY). B, An inhibitor of the CREB transcriptional coactivator CBP, E1A, inhibited the CART luciferase vector response to CCK compared with an appropriate E1A control vector (E1AΔ2-36). WT, Wild type. C, Mutation of the Cre site in the 3451CART-Luc vector (ΔCRE) abolished the response to CCK. ***p < 0.001 (n = 6). Error bars indicate SEM.
- Figure 6.
CCK switches between expression of CART and MCH in vagal afferent neurons. A–D, Immunocytochemical localization of CART and MCH in vagal afferent neurons exposed to serum-free medium for 8 h (A, DAPI; B, MCH; C, CART; D, overlay of B and C). Note the expression of MCH but not CART. E–H, In neurons incubated in serum-free medium with the addition of CCK (10 nm) for 85 min, coexpression of CART and MCH in the same neuron can be demonstrated (E, DAPI; F, MCH in neurite terminals; G, CART in the cell soma and neurite terminals; H, overlay of F and G). I–L, In neurons incubated in serum-free medium (6 h) with the addition of CCK (10 nm) for 2 h, there is loss of MCH and stimulation of CART (I, DAPI; J, MCH; K, CART; L, overlay of J and K). M–P, In neurons transfected with ACREB, incubated in serum-free medium (6 h) with the addition of CCK (10 nm) for 2 h, the loss of MCH is inhibited (M, DAPI; N, ACREB revealed by immunostaining for FLAG epitope; O, MCH; P, overlay of N and O). Filled arrows indicate the position of nuclei for reference, and open arrows indicate the position of a terminal neurite containing both CART- and MCH-immunoreactive vesicles. Representative images from six independent experiments are shown. Scale bar, 50 μm.
