Hypothalamic responses to reduced food intake in healthy animals and rats bearing small tumors

Anorexia is frequently observed during cancer and associated with increased morbidity and mortality. Our previous work has shown the presence of transcription factors in the hypothalamus of anorectic tumor-bearing rats. The aim of the present study was, therefore, to assess expression of neuropeptides, neurotransmitter-synthesizing enzymes and receptors in this structure. Morris hepatoma 7777 cells injected subcutaneously in Buffalo rats provoked a 15% reduction in food intake and 10% lower body weight when the tumor represented 1-2% of body mass. Real-time PCR showed that tumor-bearing rats did not display the increase in hypothalamic agouti-related peptide mRNA observed in food-restricted weight-matched animals. These findings indicate that blunted hypothalamic AgRp mRNA expression underlies cancer-associated anorexia. A pool of cDNA for each experimental group containing an equal amount of cDNA of each individual (n5-8) was used for in vitro transcription to yield multiple copies of amino-allyl-modified antisense RNA (aRNA) using amino-allyl-labeled UTP (Ambion, Austin, TX, USA). Subsequently, aimine groups of the fluorescent Cy3 or Cy5 dyes (Amersham Biosciences, GE Healthcare, Munich, Germany ) were coupled to the a RNA amino allyl groups following manufacturer’s instructions. Gene expression profiles were examined using Agilent whole rat genome aRNA microarrays (G4131a, Agilent Technologies, Santa Clara, CA, USA) using 0.75 μg of Cy3-labelled RNA and 0.75 μg of Cy5-labelled RNA. A dye-swap chip was included for each comparison to control for differences in dye incorporation.