Our experiments addressed the hypothesis that CART peptide’s effects on energy balance cannot be characterized as simply catabolic in nature. CART peptide was applied to the 4th ventricle (hindbrain-directed), the 3rd ventricle (hypothalamus-directed), and directly to the NTS parenchyma and its effects on energetic (TC and activity), cardiovascular (HR), food intake, body weight, and plasma glucose parameters were measured. Our data show that CNS delivery of CART results in a hypothermia. We discuss the hypothermic effect first, the GLP-1R mediation of this response next, and then review the other effects observed.
These are the first data to show that hindbrain CART55–102
delivery induced a pronounced and long-lasting hypothermic
response (a 1.5°C and 1.6°C decrease in TC
for the 1.0 and 2.0 µg doses relative to vehicle treatment; >6h duration). Injections of CART into the forebrain ventricle (providing access of drug to forebrain and hindbrain sites due to the caudal flow of CSF), hindbrain ventricle, and hindbrain parenchyma (intra-NTS) were each effective in decreasing TC
. The potent and long duration of hypothermia triggered by injections selective to the hindbrain ventricle or NTS parenchyma, that bypass forebrain, indicated that CART responsive neurons located within the hindbrain and at least partially within the NTS mediated the hypothermic response. Additional experiments evaluating hypothalamic or forebrain parenchymal sites are needed to directly exclude the possibility of forebrain contribution to the hypothermic response. However, the few published studies that examine the energy expenditure effect of hypothalamic (ARC; PVN) CART injection show responses (increases in brown adipose tissue (BAT) UCP-1) that are typically associated with a hyperthermic
action (Wang et al., 2000
; Kong et al., 2003
). That the thermic response to hypothalamic CART injection is opposite in direction to the current findings strengthens the hypothesis that the hypothermia was mediated by hindbrain CART responsive neurons.
We recently showed (Hayes et al., 2008a
) that stimulation of hindbrain GLP-1R induces hypothermia similar in duration and size to that produced by CART in the present study. Current data establish that GLP-1 neurons and hindbrain GLP-1Rs are downstream mediators of the effect of CART55–102
on Tc as the hypothermic effect of CART was eliminated by blockade of hindbrain GLP-1Rs with Exendin 9–39. It is interesting to note that the hypothermic effect of hindbrain GLP-1R stimulation does not require intact forebrain-hindbrain communication as the effect was unaltered by supracollicular decerebration (Hayes et al., 2008a
). By contrast, we showed here that the hypothermic effect of CART requires neural connections between the forebrain and hindbrain and forebrain processing is required, as the hypothermia was not present in rats with chronic supracollicular transsection. Therefore the circuitry mediating the hypothermic effect of CART might involve hindbrain neurons responsive to CART that project to the forebrain. The identity of those forebrain-projecting neurons is not known, however our data showing hypothermia in response to NTS CART stimulation suggests that at least part of CART’s effects on temperature is mediated by potential CART receptors located in or near the NTS. NTS neurons project heavily in a rostral fashion (e.g. PBN) to nuclei that relay peripheral thermal information to thermoregulatory neurons in the hypothalamic preoptic area (Nakamura and Morrison, 2008
). The forebrain-relayed information would be subsequently transmitted back to the hindbrain resulting in putative GLP-1 release from the NTS proglucagon expressing neurons and subsequent hindbrain GLP-1R stimulation. Once activated, the output pathway following hindbrain GLP-1R activation inducing hypothermia is contained within the hindbrain (Hayes et al., 2008a
). Several nuclei expressing GLP-1Rs within the hindbrain contain sympathetic premotor neurons (Yamamoto et al., 2002
). It is possible that inhibition of those neurons results in the observed hypothermia. Experiments evaluating hindbrain CART and GLP-1 effects on SNS neurons and BAT activity are needed to evaluate this possibility. Another potential mechanism contributing to hypothermia is SNS-mediated vasodilatation. A role of other neurotransmitters (e.g. serotonin and dopamine) that are associated with hypothermic responses (Cryan et al., 2000
; Catalina et al., 2002
) in this pathway is possible and remains to be investigated.
While the hypothermic
response to hindbrain CART55–102
peptide stimulation contrasts with the hyperthermic
effect of hindbrain melanocortin receptor stimulation, the anorexia and acute hyperglycemic effects of CART injection are similar to those observed with melanocortin delivery (Fan et al., 2000
). The anorexic effect of hindbrain CART stimulation observed here is consistent with several studies (Kristensen et al., 1998
; Rogge et al., 2008
). Furthermore, Aja et al. (Aja et al., 2006
) show that GLP-1Rs are downstream of the anorexia triggered by hindbrain CART as measured by alterations in palatable diet (Ensure®) intake. Our studies confirm this GLP-1R mediation of anorexic effects of CART and extend the conclusion to effects on consumption of standard chow (food of lesser palatability). We show, for the first time, that acute CART55–102
stimulation increased blood glucose levels. This is consistent with the direction of acute effect of CNS melanocortin stimulation on blood glucose (Fan et al., 2000
). Unlike CART’s hypothermic and anorexic effect, the hyperglycemic effect was not mediated by hindbrain GLP-1Rs as blockade of hindbrain GLP-1Rs did not alter CART-induced hyperglycemia. Other effects of CART (e.g. gastric emptying) are mediated by CRF-Rs (Smedh and Moran, 2003
), but not GLP-1Rs. Here we show that CART-induced hyperglycemia does not require hindbrain CRF-R mediation, indicating that a different mechanism might govern the hyperglycemic effect of CART.
CART peptides are not only widely present in the CNS but also detected in some areas relevant to energy balance outside of the CNS including the adrenal medulla, gut, and pancreas (Thim et al., 1998
; Jensen et al., 1999
; Thim et al., 1999
). In order to evaluate the potential contribution of CART stimulation outside of the CNS to energetic and anorexic effects of CART rats were injected ip with the highest dose of CART applied in the ventricle. Peripheral CART55–102
application did not have any effects on any of the measured parameters, suggesting that effects seen after central CART application do not result from stimulation of potential receptors accessed by ip injection.
Our current data, along with those of others [see (Kuhar et al., 2000
) for review], suggest that CNS CART peptide plays a role in several anatomically separable circuits. CART stimulation within the cortical and mesolimbic regions elicits reward and motivation responses (Jaworski and Jones, 2006
). CART stimulation in the mesolimbic nucleus accumbens produces anorexia (Yang et al., 2005
). Hypothalamic CART injection has varied effects on food intake, but appears to increase energy expenditure (UCP1 activity) (Wang et al., 2000
; Kong et al., 2003
). In hypothalamic explants CART suppresses release of MSH and induces agouti-related protein release(Stanley et al., 2001
; Dhillo et al., 2002
). In addition, while CART and POMC are co-expressed in arcuate hypothalamus, CART is also co-expressed with the orexigenic peptide melanin-concentrating hormone in lateral hypothalamic neurons (Broberger, 1999
). Hindbrain CART application decreases food intake, gastric emptying (Smedh and Moran, 2003
), and as shown here core body temperature. Further evaluation of the complexity of CART’s functional effects and the mediating neurocircuitry awaits the discovery of CART receptors.
The observed hypothermia was not associated with other anabolic indicators. Our measurements included several parameters that are associated with energy expenditure and sympathetic control (TC
, activity, HR). Melanocortin treatment for example alters all three parameters in the catabolic direction consistent with increased energy expenditure and sympathetic activation (Fan et al., 2000
; Gutierrez-Juarez et al., 2004
; Skibicka and Grill, 2008
) . CART injection triggered a different pattern of response – hypothermia without alteration in activity and HR. If CART hypothermia is not a part of a coordinated pattern of energy balance effects, then what is the physiological utility of potent and sustained hypothermic response? Several processes have been associated with a marked hypothermia. Conditioned taste aversion (CTA) is often accompanied by a hypothermic response. Some studies (Aja et al., 2002
) suggest that CART plays a role in CTA response. LiCl, a common inducer of CTAs, produces hypothermia via α1-adrenergic receptor (Amaro et al., 1996
). It has been proposed that hypothermia might enhance CTA formation by extending the associative interval between presentation of taste conditioned stimuli and the unconditioned aversive effects of LiCl or other agents (Hinderliter et al., 2002
; Hinderliter et al., 2004
). Therefore the elicited CART hypothermia might be adaptive in facilitating CTA formation. Another process enhanced by hypothermia is neuroprotection (Sahuquillo and Vilalta, 2007
). Neuronal survival after conditions of decreased oxygen supply (e.g. ischemia) is often increased if core temperature is decreased after the ischemic episode; this phenomenon is observed in rat models of ischemia and is utilized as a treatment strategy in ischemic patients (see (Hoesch and Geocadin, 2007
) and (Nagel et al., 2008
) for review). CART is associated with a neuroprotective effect in ischemia (Xu et al., 2006
; Jia et al., 2008
). Induction of hypothermia could be a part of the mechanism by which CART exerts a neuroprotective effect. Other experiments are needed to evaluate this hypothesis and whether the CART-induced hypothermia is physiological in nature. Our results indicate that the hypothermia induced by CART is long lasting and shows little tolerance upon repeated exposure (data not shown), two properties highly sought after in potential hypothermia-inducing neuroprotective treatments.