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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 18, 2003 as doi:10.1096/fj.02-0805fje. |
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Departments of
* Metabolic Medicine, Division of Investigative Sciences, Hammersmith Campus, Faculty of Medicine, and
Mathematics, Faculty of Physical Sciences, South Kensington Campus, Imperial College School of Science, Technology and Medicine, London, UK; and
Department of Physiology, St Georges Hospital Medical School, London, UK
3Correspondence: Endocrine Unit, 6th Floor Commonwealth Building, Hammersmith Hospital, DuCane Road, London W12 ONN, United Kingdom. E-mail: w.kong{at}imperial.ac.uk
SPECIFIC AIMS
The hypothalamic neuropeptide cocaine and amphetamine-regulated transcript (CART) was originally reported to act as an anorectic peptide; however, we have shown that direct injection of CART peptide into the arcuate nucleus (ARC) of the hypothalamus acutely stimulates food intake. Here, we characterize the role of ARC CART in the long-term regulation of food intake and brown adipose tissue (BAT) using repeated intra-arcuate nuclear injection of CART peptide and polyethylenimine (PEI)-mediated gene transfer of a CART-expressing plasmid (pcDNA-CART) delivered to the ARC.
PRINCIPAL FINDINGS
Adult, male Wistar rats (220–280 g), 8–10 per group, were used in all studies.
1. Repeated intra-arcuate administration of CART peptide for 7 days increases food intake and up-regulates BAT
Rats were injected twice daily for 7 days with 1 µl 0.2 nmol CART (55–102), the biologically active fragment of the peptide, or saline (control) via a permanent, indwelling, 26G stainless steel cannula. Food intake was 500% greater between 1 and 4 h in the CART (55–102)-injected animals [mean±SEM: 1.85±0.4 (CART) vs. 0.38±0.11 g (saline); P<0.005]. Cumulative daytime food intake was 60% greater in the CART (55–102)-injected group [day 6 cumulative daytime food intake: 32±2 (CART) vs. 20±3 g (saline); P<0.001] (Fig. 1
). There was no difference in total daily food intake or cumulative weight gain between the two groups. On day 7, the thermogenic response to the selective ß3 receptor agonist BRL 35135 was 85% greater in the CART (55–102) group [1.3±0.2 (CART) vs. 0.7±0.13°C (saline); P<0.05]. BAT uncoupling protein-1 (UCP-1) mRNA was over 60% greater in the CART (55–102) animals [day 8: 33.3±4.1 (CART) vs. 20.6±4.4 relative units (saline); P<0.05].
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2. Chronic ARC overexpression of CART cDNA increases hypothalamic CART peptide release
pcDNA-CART was produced by cloning full-length CART cDNA into the plasmid pcDNA1.1(amp). Rats were stereotactically injected into the ARC with 1 µg pcDNA-CART/PEI or pcDNA1.1/PEI (control) in a volume of 1 µl. On day 10, post-gene transfer brains were collected, and CART-immunoreactive (IR) release from 1.2 mm medio-basal hypothalamic slices was measured in a static incubation system. CART-IR release was 40% higher in the pcDNA-CART group [184±13 (pcDNA-CART) vs. 129±19 fmol/explant (control); P<0.01].
3. Chronic overexpression of CART cDNA increases food intake and up-regulates BAT UCP-1
Rats were injected with 1 µg pcDNA-CART/PEI or pcDNA1.1/PEI as described above. Cumulative food intake was significantly increased during ad libitum feeding [day 10, 4-day cumulative food intake: 135±3 (pcDNA-CART) vs. 125±3 g (control plasmid); P<0.02]. Re-feeding following a 24-h fast was also greater in the pcDNA-CART group [cumulative 4-day re-feed: 161±3 (pcDNA-CART) vs. 149±2 g (control plasmid); P<0.01] (Fig. 2
a). At day 25 post-gene transfer, cumulative weight gain was also increased in the CART-overexpressing animals [day 25: 79±3 (pcDNA-CART) vs. 73±3 g (control plasmid); P<0.05]. However, the 24-h fast resulted in significantly greater weight loss in the CART-overexpressing animals compared with the control plasmid group [–27±1 (pcDNA-CART) vs. –24±1 g (control plasmid); P<0.01]. Interscapular BAT UCP-1 mRNA was increased by 80% in the pcDNA-CART-treated animals [day 25: 47.3±4.7 (pcDNA-CART) vs. 26.4±6.1 relative units (control plasmid); P<0.02] (Fig. 2)
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4. Repeated CART (55–102) injection or chronic CART cDNA decreases thyrotrophin (TSH)
Chronic arcuate overexpression of CART cDNA for 10 days or 25 days significantly decreased plasma TSH compared with control animals [10 days: 2.32±0.3 ng/mL (pcDNA-CART) vs. 3.73±0.6 ng/mL (control plasmid); 25 days, TSH: 3.5±0.3 (pcDNA-CART) vs. 4.4±0.3 ng/mL (control plasmid); P<0.05]. Repeated administration of CART (55–102) also showed a trend toward reduced TSH [3.3±0.3 (CART) vs. 4.2±0.4 ng/mL (saline); P=0.1]. These changes were accompanied by a nonsignificant trend toward increased T4 in the CART (55–102)-treated animals (P
0.07).
5. CART mRNA in the ARC is up-regulated with cold acclimatization
In rats maintained at 4°C for 21 days, cumulative weight gain was 45% lower (P<0.005), and cumulative food intake was 40% greater (P<0.001) compared with rats maintained at 22°C.Using quantitative in situ hybridization, levels of CART mRNA in the ARC of cold, acclimatized animals were over double those of the warm, maintained animals [integrated density: 224±50 (4°C) vs. 100±21% (22°C); P<0.05]. There was no significant difference in CART mRNA expression in the paraventricular nucleus between the two groups (P=0.2).
CONCLUSIONS AND SIGNIFICANCE
We have shown that repeated intra-arcuate CART peptide injections resulted in a 60% increase in daytime food intake, but total daily food intake and weight gain were not significantly increased. We hypothesized that this might be a result of the relatively short half-life of the orexigenic effect of intra-arcuate CART (
4 h). To overcome this problem, we used targeted gene transfer to produce sustained, elevated CART in the ARC. This resulted in increased daily food intake and increased weight gain compared with controls.
These findings demonstrate that targeted gene transfer can be used to chronically manipulate hypothalamic neuropeptides in vivo. The technique is minimally invasive, making it particularly suitable for the investigation of behavioral changes such as feeding. We used in situ hybridization to confirm anatomical localization of the transgene. We confirmed increased transgene peptide expression using a static incubation system and demonstrated increased peptide release from hypothalamic explants taken from animals injected with the pcDNA-CART plasmid.
The anorectic effect of intracerebroventricular (ICV) CART (55–102) has been well documented. However, this effect is associated with pronounced abnormalities of movement and behavior, suggesting that the anorectic effects of ICV CART (55–102) may be indirect. Plugging the cerebral aqueduct greatly reduces these behavioral abnormalities and anorexigenic effects, suggesting that these are mediated at a hindbrain site. We recently reported acute orexigenic effects of intra-arcuate-injected CART peptide. The data here further support the hypothesis that the anorectic effect of ICV CART is a result of extra-hypothalamic effects.
We found that plasma TSH was significantly reduced in pcDNA-CART-treated animals compared with control animals. This was accompanied by a trend toward increased, circulating, free T3 levels. It is interesting that ICV CART increases TRH but with no effect on TSH. CART in the ARC may increase sympathetic outflow to the thyroid gland. Chronic sympathetic stimulation has been shown to increase serum T3, independent of TSH. Such changes could explain our observed reduction in plasma TSH.
A 24-h fast in animals overexpressing CART cDNA in the ARC was associated with greater weight loss than controls, suggesting increased energy expenditure. Consistent with this, we found increased BAT UCP-1 mRNA and increased thermogenic responses to BRL 35135 following CART gene transfer and repeated CART peptide injection. CART may activate thermogenesis by stimulation of sympathetic outflow to BAT. Increased BAT thermogenesis is likely to be undesirable in situations of food restriction, and this might explain why, despite its orexigenic action, arcuate CART is down-regulated with fasting.
In contrast to food restriction, the hyperphagia associated with cold acclimatization is associated with increased BAT thermogenesis. Little is known about the central neurogenic control of cold-induced hyperphagia. We have found that increased CART in the ARC increases food intake and stimulates BAT thermogenesis. Furthermore, animals maintained at 4°C for 21 days had almost double the levels of CART mRNA in the ARC compared with animals that had been maintained at 22°C. Our data provide further evidence that CART in the ARC is an important orexigenic peptide and suggest a possible role for ARC CART in cold adaptation and cold-induced hyperphagia in rodents (Fig. 3
).
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0805fje; doi: 10.1096/fj.02-0805fje 
Contributed equally to this study.
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). ***, P< 0.001, by three-way ANOVA.
