J.Q. Yan

Ghrelin signaling in the ventral tegmental area mediates both reward-based feeding and fasting-induced hyperphagia on high-fat diet

Ghrelin is a potent orexigenic hormone that acts in the central nervous system to stimulate food intake via the growth hormone secretagogue receptor (GHSR) that is abundantly expressed in the ventral tegmental area (VTA). Not only does ghrelin modulate feeding behavior via a homeostatic mechanism, but numerous studies have identified ghrelin as a key regulator of reward-based hedonic feeding behaviors. Nutritional states influence ghrelin and GHSR expression as well as the behavioral sensitivity to reward-inducing stimuli. In the current study, we examined the role of ghrelin at the VTA level in food intake in two different nutritional states, satiety and hunger, by using a restricted feeding model. In this model, rats were conditioned to a daily 3-h (h) feeding session on standard chow for 10days and a high-fat diet (HFD) was supplied either in the third hour after 2h of chow diet intake, or at the beginning of a daily meal on the test day. We found that intra-VTA microinjection of 1, 2, and 4μg of ghrelin, induced a dose-related increase of 1h of reward-based feeding on HFD in sated rats, as well as a 24-h body weight gain. The overconsumption stimulated by ghrelin could be attenuated by 10μg of direct infusion of the ghrelin receptor antagonist D-Lys3-GHRP-6 into the VTA. Moreover, our data showed that the injection of 1, 2, and 4μg of ghrelin in the VTA, enhanced fasting-induced hyperphagia on HFD in a dose-related manner following a 21-h food restriction as well as a 24-h body weight gain. Conversely, hyperphagia on HFD that is potentiated by ghrelin could be blocked by pretreatment with a 10-μg D-Lys3-GHRP-6 intra-VTA microinjection. Collectively, these data demonstrate that ghrelin signaling at the VTA level mediates both reward-based eating and fasting-induced hyperphagia and provides a primary target for the control of the intake of rewarding food.

Dietary sodium deprivation evokes activation of brain regional neurons and down-regulation of angiotensin II type 1 receptor and angiotensin-convertion enzyme mRNA expression.

Previous studies have indicated that the renin-angiotensin-aldosterone system (RAAS) is implicated in the induction of sodium appetite in rats and that different dietary sodium intakes influence the mRNA expression of central and peripheral RAAS components. To determine whether dietary sodium deprivation activates regional brain neurons related to sodium appetite, and changes their gene expression of RAAS components of rats, the present study examined the c-Fos expression after chronic exposure to low sodium diet, and determined the relationship between plasma and brain angiotensin I (ANG I), angiotensin II (ANG II) and aldosterone (ALD) levels and the sodium ingestive behavior variations, as well as the effects of prolonged dietary sodium deprivation on ANG II type 1 (AT1) and ANG II type 2 (AT2) receptors and angiotensin-convertion enzyme (ACE) mRNA levels in the involved brain regions using the method of real-time polymerase chain reaction (PCR). Results showed that the Fos immunoreactivity (Fos-ir) expression in forebrain areas such as subfornical organ (SFO), paraventricular hypothalamic nuclei (PVN), supraoptic nucleus (SON) and organum vasculosum laminae terminalis (OVLT) all increased significantly and that the levels of ANG I, ANG II and ALD also increased in plasma and forebrain in rats fed with low sodium diet. In contrast, AT1, ACE mRNA in PVN, SON and OVLT decreased significantly in dietary sodium depleted rats, while AT2 mRNA expression did not change in the examined areas. These results suggest that many brain areas are activated by increased levels of plasma and/or brain ANG II and ALD, which underlies the elevated preference for hypertonic salt solution after prolonged exposure to low sodium diet, and that the regional AT1 and ACE mRNA are down-regulated after dietary sodium deprivation, which may be mediated by increased ANG II in plasma and/or brain tissue.

Characterization of the expression pattern of adrenergic receptors in rat taste buds.

Taste buds signal the presence of chemical stimuli in the oral cavity to the central nervous system using both early transduction mechanisms, which allow single cells to be depolarized via receptor-mediated signaling pathways, and late transduction mechanisms, which involve extensive cell-to-cell communication among the cells in the bud. The latter mechanisms, which involve a large number of neurotransmitters and neuropeptides, are less well understood. Among neurotransmitters, multiple lines of evidence suggest that norepinephrine plays a yet unknown role in the taste bud. This study investigated the expression pattern of adrenergic receptors in the rat posterior taste bud. Expression of alpha1A, alpha1B, alpha1D, alpha2A, alpha2B, alpha2C, beta1, and the beta2 adrenoceptor subtypes was observed in taste buds using RT-PCR and immunocytochemical techniques. Taste buds also expressed the biosynthetic enzyme for norepinephrine, dopamine beta-hydroxylase (DbetaH), as well as the norepinephrine transporter. Further, expression of the epinephrine synthetic enzyme, phenylethanolamine N-methyltransferase (PNMT), was observed suggesting a possible role for this transmitter in the bud. Phenotyping adrenoceptor expression patterns with double labeling experiments to gustducin, synaptosomal-associated protein 25 (SNAP-25), and neural cell adhesion molecule (NCAM) suggests they are prominently expressed in subsets of cells known to express taste receptor molecules but segregated from cells known to have synapses with the afferent nerve fiber. Alpha and beta adrenoceptors co-express with one another in unique patterns as observed with immunocytochemistry and single cell reverse transcription polymerase chain reaction (RT-PCR). These data suggest that single cells express multiple adrenergic receptors and that adrenergic signaling may be particularly important in bitter, sweet, and umami taste qualities. In summary, adrenergic signaling in the taste bud occurs through complex pathways that include presynaptic and postsynaptic receptors and likely play modulatory roles in processing of gustatory information similar to other peripheral sensory systems such as the retina, cochlea, and olfactory bulb.

Activation of μ-opioid receptors in the central nucleus of the amygdala induces hypertonic sodium intake.

Opioid mechanisms are involved in the control of water and NaCl intake and opioid receptors (ORs) are present in the central nucleus of the amygdala (CeA), a site of important facilitatory mechanisms related to the control of sodium appetite. Therefore, in the present study we investigated the effects of the activation of μ-ORs in the CeA on 0.3 M NaCl and water intake in rats. Male Sprague-Dawley rats with stainless steel cannulas implanted bilaterally in the CeA were used. In rats submitted to water deprivation-partial rehydration, bilateral injections of the selective μ-OR agonist [D-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin (DAMGO) in the doses of 1, 2, and 4 nmol into the CeA induced a dose-related increase of 0.3M NaCl intake and water intake, and bilateral injections of the selective μ-OR antagonist D-Phe-Cys-Trp-Arg-Thr-Pen-Thr-NH₂ (CTAP) in the doses of 0.5, 1, and 2 nmol into the CeA produced a dose-related decrease of 0.3 M NaCl and water intake induced by DAMGO 2 nmol into the same site. In rats treated with the diuretic furosemide (10 mg/kg b.w.) combined with the angiotensin-converting enzyme inhibitor captopril (5 mg/kg b.w.) injected subcutaneously, bilateral injections of DAMGO 2 nmol into the CeA increased 0.3 M NaCl intake and water intake and the blockade of μ-ORs with CTAP 1 nmol injected into the CeA reduced the increase in 0.3 M NaCl intake and water intake induced by DAMGO 2 nmol into the same site. Bilateral injections of DAMGO into the CeA did not change urinary volume, sodium urinary excretion and mean arterial pressure, but increased activity. Thus stimulating μ-ORs in the CeA increases hypertonic sodium intake, whereas antagonizing these sites inhibits hypertonic sodium intake. Together, our results implicate μ-ORs in the CeA in a positive regulation of sodium intake.

Natriorexigenic effect of DAMGO is decreased by blocking AT1 receptors in the central nucleus of the amygdala

μ-Opioid receptor (μ-OR) activation with agonist [D-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin (DAMGO) in the central nucleus of the amygdala (CeA) induces sodium (0.3M NaCl) intake in rats. The purpose of this study was to examine the effects of pre-injections of losartan (AT1 angiotensin receptor antagonist) into the CeA on 0.3 M NaCl and water intake induced by DAMGO injected bilaterally in the same area in rats submitted to water deprivation-partial rehydration (WD-PR) and in rats treated with the diuretic furosemide (FURO) combined with a low dose of the angiotensin-converting enzyme inhibitor captopril (CAP) injected subcutaneously (FURO/CAP). Male Sprague-Dawley rats with stainless steel cannulas implanted bilaterally into the CeA were used. In WD-PR rats, bilateral injections of DAMGO (2 nmol in 0.5 μL) into the CeA induced 0.3 M NaCl and water intake, and pre-treatment with losartan (108 nmol in 0.5 μL) injected into the CeA reduced 0.3 M NaCl and water intake induced by DAMGO. In FURO/CAP rats, pre-treatment with losartan (108 nmol in 0.5 μL) injected into the CeA attenuated the increase in 0.3M NaCl and water intake induced by DAMGO (2 nmol in 0.5 μL) injected into the same site. The results suggest that the natriorexigenic effect of DAMGO injected into the CeA is facilitated by endogenous angiotensin II acting on AT1 receptors in the CeA, which drives rats to ingest large amounts of hypertonic NaCl.

Inhibitory effect of activation of GABAA receptor in the central nucleus of amygdala on the sodium intake in the sodium-depleted rat

The present study investigated the effects of a microinjection of GABA(A) receptor agonist (muscimol) and antagonist (bicuculline) into the central nucleus of the amygdala (CeA) in sodium-depleted rats. We measured the sodium intake and identified the neuronal activation in the brainstem induced by activating the GABA(A) receptors in the CeA using Fos immunohistochemistry. Muscimol (0.20, 0.35 or 0.50 nmol, in 0.2μl) that was injected bilaterally into the CeA decreased the 0.3M NaCl and water intake in a dose-dependent manner. Microinjection of 0.02 nmol/0.2μl muscimol also decreased the NaCl intake, but had no effect on the water intake. The inhibitory effect of muscimol (0.20 nmol) on the sodium and water intake could be blocked by pretreatment with bicuculline intra-CeA microinjection (0.4 nmol). However, bilateral injections of bicuculline alone into the CeA did not affect the NaCl or water intake. Furthermore, microinjection of muscimol (0.20 nmol) into the CeA increased the number of Fos-like immunoreactive (FLI) neurons in the caudal and intermediate parts of the nucleus of the tractus solitarius (cNTS and iNTS) and the lateral parabrachial nucleus (LPBN). These results suggest that GABA(A) receptors within the CeA may be involved in mediating the sodium intake in the sodium-depleted rat, and the cNTS, iNTS and LPBN were probably involved in this mechanism.

AT1 receptor blockade in the central nucleus of the amygdala attenuates the effects of muscimol on sodium and water intake

The blockade of the central nucleus of the amygdala (CeA) with the GABAA receptor agonist muscimol significantly reduces hypertonic NaCl and water intake by sodium-depleted rats. In the present study we investigated the effects of previous injection of losartan, an angiotensin II type-1 (AT1) receptor antagonist, into the CeA on 0.3M NaCl and water intake reduced by muscimol bilaterally injected into the same areas in rats submitted to water deprivation-partial rehydration (WD-PR) and in rats treated with the diuretic furosemide (FURO). Male Sprague-Dawley rats with stainless steel cannulas bilaterally implanted into the CeA were used. Bilateral injections of muscimol (0.2 nmol/0.5 μl, n=8 rats/group) into the CeA in WD-PR-treated rats reduced 0.3M NaCl intake and water intake, and pre-treatment of the CeA with losartan (50 μg/0.5 μl) reversed the inhibitory effect of muscimol. The negative effect of muscimol on sodium and water intake could also be blocked by pretreatment with losartan microinjected into the CeA in rats given FURO (n=8 rats/group). However, bilateral injections of losartan (50 μg/0.5 μl) alone into the CeA did not affect the NaCl or water intake. These results suggest that the deactivation of CeA facilitatory mechanisms by muscimol injection into the CeA is promoted by endogenous angiotensin II acting on AT1 receptors in the CeA, which prevents rats from ingesting large amounts of hypertonic NaCl and water.

Taste sensitivity to sucrose is lower in outbred Sprague-Dawley phenotypic obesity prone rats than obesity resistant rats

The purpose of the present study was to better understand the role of sweet taste perception in dietary behavior and body weight in outbred Sprague-Dawley phenotypic obesity-prone and obesity-resistant rats by measuring sucrose taste sensitivity using a conditioned taste aversion paradigm. Rats were given a high fat diet for 2 weeks and were assigned as obesity-prone (P, upper tertile) or obesity-resistant (R, lower tertile) based on weight gain. Each group was then given either chow (C, 10% fat) or the high fat diet (F, 46% fat) for the remainder of the experiment (∼18 weeks) such that there were four groups - obesity-prone on chow (C-P), obesity-prone on high fat (H-P), obesity-resistant on chow (C-R), obesity-resistant on high fat (H-R). The sucrose sensitivity of phenotypic obesity-prone rats is lower than that of obesity-resistant rats in either H-fed or C-fed group, and all H-fed rats were more sensitivity than their C-fed counterparts (H-P vs. C-P; H-R vs. C-R). Body weight gain and total calories intake of phenotypic obesity-prone rats are more than that of obesity-resistant rats. The results suggest that lower sucrose taste sensitivity may contribute to body weight gain and total calories intake of phenotypic obesity-prone rats compared to obesity-resistant rats, and there is correlation between the change in the sweet taste threshold and diet treatment.