Chronic central infusion of cocaine- and amphetamine-regulated transcript (CART 55-102): effects on body weight homeostasis in lean and high-fat-fed obese rats.

BACKGROUND: Cocaine- and amphetamine-regulated transcript (CART) is expressed within hypothalamic nuclei implicated in the regulation of feeding behaviour. It is up-regulated by leptin, and CART-derived peptides acutely inhibit food intake. OBJECTIVE: The present study was designed to assess the long-term effects of central CART administration on food intake, body weight, plasma levels of glucose, insulin, leptin, free fatty acids and triglycerides, and on fuel utilisation in normal and high-fat-fed obese rats. DESIGN: Normal and high-fat-fed obese rats were cannulated intracerebroventricularly (i.c.v.) and infused for 6 days with CART (55-102) or its vehicle. At day 4, animals were placed in an indirect calorimeter for a 24 h period during which the respiratory quotient and the energy expenditure were determined hourly. RESULTS: In both normal and obese animals, the chronic i.c.v. infusion of CART (55-102) had marked, sustained inhibitory effects on food intake and body weight gain that were accompanied by decreases in plasma insulin and leptin levels. Using indirect calorimetry, it was observed that CART infusion promoted an increase in lipid oxidation in normal and in obese animals, although this increase reached statistical significance only in the obese group. The hypothalamic CART mRNA expression was found to be higher in obese rats (displaying hyperleptinaemia) than in normal animals. CONCLUSION: The data together show that chronic i.c.v. CART infusion is effective in inhibiting food intake, favouring lipid oxidation and limiting fat storage, both in normal and high-fat-diet-induced obese rats. The CART pathway thus seems to be an important determinant of body weight homeostasis in normal animals as well as in a model of nutritionally induced obesity.

Leptin inhibition of the hypothalamic-pituitary-adrenal axis in response to stress.

Leptin is a newly identified protein hormone that is synthesized and secreted by adipose tissue. Absence of the mature hormone is responsible for the obese phenotype of ob/ob mice. The hypothalamic-pituitary-adrenal axis (HPAA) is activated in ob/ob mice, and chronic administration of leptin to ob/ob mice decreases plasma corticosterone levels, suggesting that the adipose hormone is capable of inhibiting the HPAA. The aim of this study was to determine whether leptin feeds back acutely to inhibit the HPAA of normal mice and rats. Male C57BL mice were injected ip with 100 microl saline and 2 or 4 microg/g BW mouse leptin in saline vehicle, and 4 h later they were subjected to 2 h of restraint stress by taping the hind limbs together or no stress. Hind leg restraint stimulated the HPAA as measured by significant (P < 0.05) elevation of both ACTH and corticosterone. Pretreatment with recombinant mouse leptin blocked the stress-mediated stimulation of both plasma hormones. To determine whether this inhibition was exerted at the hypothalamic level through inhibition of CRH, we studied leptin action on isolated rat hypothalami perifused with Krebs-Ringer buffer containing glucose (5.5 mM). CRH secretion was stimulated by decreasing the glucose concentration of the buffer to 1.1 mM. A surge of CRH was released over a 2-h period (basal integrated release was 14.4 +/- 1.6 pg/2 h, n = 5 and increased to 34.7 +/- 3.1 pg/2 h, n = 14). This response was blocked by mouse leptin in a dose-dependent manner (integrated stimulated CRH secretion was 30.6 +/- 2.5 pg/2 h, n = 5; 20.5 +/- 3.6 pg/2 h, n = 7; 15.3 +/- 4.3 pg/2 h, n = 3 for 1 nM, 3 nM and 30 nM, respectively). Leptin did not alter secretion of ACTH from rat primary cultured pituitary cells. These data demonstrate that leptin can inhibit hypothalamic CRH release, either directly or indirectly through another hypothalamic neuropeptide such as neuropeptide-Y. Dysfunctional leptin, insufficient leptin levels, or leptin resistance should each result in a partial open loop, thereby accounting for elevated glucocorticoid levels that accompany and contribute to many obese phenotypes. Leptin's ability to inhibit CRH release is the likely explanation for its ability to inhibit activation of the HPAA in response to stress.

Synthesis of PCR-derived, single-stranded DNA probes suitable for in situ hybridization.

We report the novel synthesis of polymerase chain reaction (PCR)-derived single-stranded DNA (ssDNA) probes and their subsequent application in in situ hybridizations. Serial transverse sections of an 11.5-day postcoitum mouse embryo were hybridized to a 33P-ssDNA, 33P-RNA, or 35S-RNA probe corresponding to the same 181-bp sequence in the myogenin cDNA. Signal obtained using 33P-ssDNA was more intense than that using 33P-RNA probe, while signal/noise ratios obtained with both 33P-probes were far superior to those obtained with 35S-probe. Digoxigenin-labeled chicken growth hormone (GH) ssDNA gave slightly more intense signal than did digoxigenin-labeled chicken GH RNA when hybridized to chicken pituitary sections. 32P-ssDNA probes were found to be suitable for Northern blot hybridization. Advantages of using ssDNA probes for in situ hybridization include: (1) The ssDNA technique is rapid and simple. There was no need to clone a DNA template into a special RNA vector or order special T7-containing PCR primers. ssDNA probes can be synthesized in less than 1 day using any primers which currently exist in a laboratory (optimal probe length for in situ hybridization is between 50 and 200 bp). (2) In three separate in situ experiments, ssDNA probes yielded more intense signal than RNA probes. (3) ssDNA probes are potentially more stable than RNA probes. (4) Since the RNAse rinse is eliminated, posthybridization rinses are shortened when hybridizing with ssDNA probes. The ssDNA probes produced by this protocol can be labeled with a variety of different isotopes (both radioactive and nonradioactive), and are excellent probes for use in in situ hybridizations.