Effects of leptin receptor mutation on Agrp gene expression in fed and fasted lean and obese (LA/N-faf) rats.

Agouti-related protein provides an orexigenic signal, probably through interaction with central melanocortin receptors. Expression of Agrp is markedly increased in the hypothalamus of mice deficient in leptin (Lep(ob)/Lep(ob)) or its receptor (Lepr(db)/Lepr(db)), suggesting that leptin mediates signals suppressing Agouti-related protein production. The regulation of Agrp expression in the rat hypothalamus has not been reported. We, therefore, analyzed the expression of Agrp in the medial basal hypothalamus of lean (+/+, +/fa(f)) and obese leptin receptor-deficient (fa(f)/fa(f)) LA/N rats. Using a sensitive solution hybridization/S1 nuclease protection assay, we found no significant difference in Agrp messenger RNA (mRNA) levels (pg/microg total RNA +/- SEM) in obese rats (n = 5), compared with lean controls (n = 5): 0.46 +/- 0.06 vs. 0.47 +/- 0.06 (P = 0.9). Similarly, no difference in Agrp expression was found using in situ hybridization or semiquantitative RT-PCR. In contrast to Agrp, Pomc mRNA levels were significantly suppressed in the obese, compared with the lean, rats (P = 0.001). Thus, the ratio of Pomc to Agrp mRNA is decreased in the obese rats and may be an important modulator of food intake. To assess the physiological regulation of Agrp in rats, we examined the effect of food deprivation in lean Sprague Dawley (SD) rats. There was a 273% increase in medial basal hypothalamus Agrp mRNA in SD rats fasted for 48 h (n = 8), compared with rats fed ad libitum (n = 8): 0.82 +/- 0.23 vs. 0.30 +/- 0.08 (P = 0.0001). Lean LA/N rats (n = 7) fasted for 48 h also showed a 231% increase in Agrp expression, compared with fed lean controls (n = 8): 0.74 +/- 0.11 vs. 0.32 +/- 0.03 (P = 0.002), whereas Pomc expression was decreased by 32% in fasted animals from the same experiment (0.34 +/- 0.05 vs. 0.50 +/- 0.07; P = 0.03). There were no significant differences in Agrp or Pomc mRNA levels between fasted and fed obese LA/N-fa(f) rats. These results suggest that, in the rat, the Agrp response to fasting may involve leptin-mediated phenomena, but factors in addition to leptin must also be involved in the regulation of Agrp gene expression.