Cocaine- and amphetamine-regulated transcript is the neurotransmitter regulating the action of cholecystokinin and leptin on short-term satiety in rats

Abstract

Vagal CCK-A receptors (CCKARs) and leptin receptors (LRbs) interact synergistically to mediate short-term satiety. Cocaine- and amphetamine-regulated transcript (CART) peptide is expressed by vagal afferent neurons. We sought to demonstrate that this neurotransmitter regulates CCK and leptin actions on short-term satiety. We also examined the signal transduction pathways responsible for mediating the CART release from the nodose ganglia (NG). ELISA studies coupled with gene silencing of NG neurons by RNA interference elucidated intracellular signaling pathways responsible for CCK/leptin-stimulated CART release. Feeding studies followed by gene silencing of CART in NG established the role of CART in mediating short-term satiety. Immunohistochemistry was performed on rat NG neurons to confirm colocalization of CCKARs and LRbs; 63% of these neurons contained CART. Coadministration of CCK-8 and leptin caused a 2.2-fold increase in CART release that was inhibited by CCK-OPE, a low-affinity CCKAR antagonist. Transfection of cultured NG neurons with steroid receptor coactivator (SRC) or phosphatidylinositol 3-kinase (PI3K) small-interfering RNA (siRNA) or STAT3 lentiviral short hairpin RNA inhibited CCK/leptin-stimulated CART release. Silencing the expression of the EGR-1 gene inhibited the CCK/leptin-stimulated CART release but had no effect on CCK/leptin-stimulated neuronal firing. Electroporation of NG with CART siRNA inhibited CCK/leptin stimulated c-Fos expression in rat hypothalamus. Feeding studies following electroporation of the NG with CART or STAT3 siRNA abolished the effects of CCK/leptin on short-term satiety. We conclude that the synergistic interaction of low-affinity vagal CCKARs and LRbs mediates CART release from the NG, and CART is the principal neurotransmitter mediating short-term satiety. CART release from the NG involves interaction between CCK/SRC/PI3K cascades and leptin/JAK2/PI3K/STAT3 signaling pathways.

Fig. 1.

Immunostaining of rat nodose ganglia (NG) neurons shows colocalization of leptin receptors (LRb), CCK-A receptors (CCKAR), and cocaine- and amphetamine-regulated transcript (CART). A: representative photomicrograph of a rat NG shows that 36.7 ± 4.4% of the NG neurons stained positive for LRb. B: 55.9 ± 5.1% of the NG neurons stained positive for the CCKAR. C: 24.5 ± 6.3% of the NG neurons stained positive for CART. D: 18.7 ± 2.8% of the NG neurons contained both CCKARs and LRbs. E: 11.5 ± 5.1% contained CART immunoreactivity as well as CCKARs and LRbs. F: histogram shows the percentage of the total number of NG neurons that exhibited immunoreactivities for LRb, CCKAR, CART, or for both receptors and CART, n = 4, scale bar = 50 μm. Data are representative of 4 independent experiments (n = 4 rats).

Fig. 2.

CCK-8 and leptin stimulate CART release in a dose-dependent manner. A: NG neurons stimulated with CCK-8 (0.1–100 nM) for 2 h exhibited a significant increase in CART release at 1, 10, and 100 nM. B: leptin significantly stimulated CART release at 1, 10, and 100 nM. Data are representative of 5 independent experiments. *P < 0.05 indicates statistical significance from unstimulated control.

Fig. 3.

CCK-8 and leptin potentiate CART release via low-affinity CCKARs. NG neurons were stimulated for 2 h with leptin (0.1 nM) and CCK-8 (0.1 nM) alone and in combination. Neither leptin nor CCK-8 increased CART release; however, when combined, leptin (L) and CCK-8 (C) caused a significant 2.27 ± 0.21-fold increase in CART release. The synergistic CCK/leptin-stimulated CART release was inhibited >70% by the CCK-OPE analog (100 nM), which acts as a low-affinity CCKAR antagonist. Data are representative of 5 independent experiments, n = 5 rats. *P < 0.05, significantly different from unstimulated control; **P < 0.05, significantly different from CCK/leptin-stimulated CART release.

Fig. 4.

Silencing the phosphatidylinositol 3-kinase (PI3K) and SRC genes inhibits CCK-8 and leptin synergistically stimulated CART release. A: 70% of the synergistic CCK/leptin-stimulated CART release was significantly inhibited by silencing the PI3K gene in NG neurons transfected with PI3K small interfering RNA (siRNA). B: representative Western blot confirms inhibition of PI3K expression in NG neurons transfected with PI3K siRNA. C: 79% of the CCK/leptin synergistic CART release was significantly inhibited by silencing the SRC gene. D: representative Western blot confirms inhibition of SRC expression in NG neurons transfected with SRC siRNA. Bars represent means ± SE from 5 independent experiments; *P < 0.05 compared with unstimulated controls (con); **P < 0.05 compared with CART stimulated by CCK-8 (1 nM) and leptin (1 nM).

Fig. 5.

Silencing the STAT gene, but not the Erk1/2 genes inhibits CCK-8 and leptin synergistically stimulated CART release. A: 90% of the CCK/leptin synergistic CART release was significantly inhibited by silencing the STAT3 gene. B: representative Western blot confirms inhibition of STAT3 expression in NG neurons transfected with retroviral STAT3 short hairpin RNA (shRNA). C: CCK/leptin-stimulated CART release was not inhibited by silencing the Erk1/2 gene. D: representative Western blot confirms inhibition of Erk1/2 expression by Erk1/2 siRNA. Bars represent means ± SE from 5 independent experiments; *P < 0.05 compared with unstimulated controls; **P < 0.05 compared with CART stimulated by CCK-8 (1 nM) and leptin (1 nM).

Fig. 6.

Silencing the EGR-1 gene inhibits leptin plus CCK-8 synergistic stimulation of CART release. A: 80% of the synergistic CCK/leptin-stimulated CART release was significantly inhibited by silencing the EGR-1 gene in NG neurons transfected with EGR-1 siRNA. Data are representative of 5 independent experiments; *P < 0.05, significantly different from unstimulated control; **P < 0.05, significantly different from CCK/leptin-stimulated CART release. B: representative CART RT-PCR confirms that silencing the EGR-1 gene results in decreased CART mRNA expression 5 days after transfection of cultured NG neurons. C: representative EGR-1 RT-PCR confirms that EGR-1 siRNA inhibits both control and CCK/leptin-stimulated EGR-1 expression. D: representative EGR-1 immunoblot confirms >80% inhibition of EGR-1 expression 5 days after transfection with EGR-1 siRNA, n = 5.

Fig. 7.

Silencing the EGR-1 gene did not affect neuronal firing evoked by leptin/CCK-8. A: representative nodose ganglia neuron transfected with EGR1 siRNA and identified by the presence of Cy3 marker (red). B: continuous membrane potential recording demonstrates that silencing EGR-1 did not abolish the synergistic leptin (1 nM) and CCK-8 (1 nM) excitatory action in the recorded neuron. The neuronal input resistance was tested every 40 s by injecting 0.5-s, 100-pA negative-amplitude current pulses (negative membrane potential deflections). C: in contrast, continuous membrane potential recording (performed in the same neuron as in shown in B) demonstrates that superfusion of leptin (1 nM) or CCK-8 (1 nM) separately did not produce significant changes in neuronal excitability. These findings are similar to those observed in nontransfected nodose ganglia neurons.

Fig. 8.

Silencing the expression of CART in rat NG inhibited c-Fos expression in the hypothalamus. Representative photomicrographs showing immunostaining of rat hypothalamic c-Fos-positive nuclei stained red. c-Fos expression in rat lateral hypothalamus (LH; A), dorsomedial hypothalamus (DMH; C), paraventricular nucleus (PVN; E), and arcuate nucleus (ARC; G) in response to CCK-8 (3.5 μg/kg) plus leptin (Lep; 120 μg/kg) ip, 5 days after electroporation with control siRNA. Elimination of c-Fos expression in rat LH (B), DMH (D), PVN (F), and ARC (H), 5 days after electroporation of the nodose ganglia with CART siRNA, followed by CCK-8 (3.5 μg/kg) plus leptin (120 μg/kg ip). I: histogram shows the percentage of neurons that exhibited c-Fos immunoreactivities over the total number of neurons in the hypothalamus. Note that silencing of CART in the NG markedly reduced the CCK/leptin-stimulated c-Fos expression in the LH, DMH, PVN, and ARC; *P < 0.05 compared with electroporation with control siRNA. Data are representative of 5 independent experiments, n = 5 rats in each study group.

Fig. 9.

Silencing the STAT3 and CART genes abolishes the effects of CCK-8/leptin on satiety. Feeding studies in rats 5 days after electroporation of NG with control siRNA or CART siRNA (A) and with control siRNA or STAT3 siRNA (B). Rats were fasted overnight and injected with CCK-8 (3.5 μg/kg ip) and leptin (120 μg/kg ip), and, after a 1-h recovery, cumulative food intake was measured for 3 h. Data are representative of 5 independent experiments; *P < 0.05 compared with food intake by control rats that received no treatment to nodose ganglia. C: representative RT-PCR confirms silencing of CART gene expression, 5 days after electroporation of NG. D: representative RT-PCR confirms silencing of STAT3 gene expression, 5 days after electroporation of NG (n = 5).