In vivo effect of an antilipolytic drug (3,5 0 -dimethylpyrazole) on autophagic proteolysis and autophagy-related gene expression in rat liver Alessio Donati a,1 , Annamaria Ventruti a,b,1 , Gabriella Cavallini a , Matilde Masini a , Simona Vittorini a , Isabelle Chantret b , Patrice Codogno b , Ettore Bergamini a, * a Centro di ricerca di Biologia e Patologia, dell’Invecchiamento, dell’Universita ` di Pisa, Via Roma 55, 56126 Pisa, Italy b INSERM U504, Glycobiologie et Signalisation cellulaire, Institut Andre ´ Lwoff, 16 avenue Paul-Vaillant-Couturier, 94807 Villejuif Cedex, France Received 3 December 2007 Available online 17 December 2007 Abstract Autophagy is an intracellular pathway induced by starvation, inhibited by nutrients, that is responsible for degradation of long-lived proteins and altered cell organelles. This process is involved in cell maintenance could be induced by antilipolytic drugs and may have anti-aging effects [A. Donati, The involvement of macroautophagy in aging and anti-aging interventions, Mol. Aspects Med. 27 (2006) 455–470]. We analyzed the effect of an intraperitoneal injection of an antilipolytic agent (3,5 0 -dimethylpyrazole, DMP, 12 mg/kg b.w.), that mimics nutrient shortage on autophagy and expression of autophagic genes in the liver of male 3-month-old Sprague–Dawley albino rats. Autophagy was evaluated by observing electron micrographs of the liver autophagosomal compartment and by monitoring protein degradation assessed by the release of valine into the bloodstream. LC3 gene expression, whose product is one of the best known markers of autophagy, was also monitored. As expected, DMP decreased the plasma levels of free fatty acids, glucose, and insulin and increased autophagic vacuoles and proteolysis. DMP treatment caused an increase in the expression of the LC3 gene although this occurred later than the induction of authophagic proteolysis caused by DMP. Glucose treatment rescued the effects caused by DMP on glucose and insulin plasma levels and negatively affected the rate of autophagic proteolysis, but did not suppress the positive regulatory effect on LC3 mRNA levels. In conclusion, antilipolytic drugs may induce both autophagic proteolysis and higher expression of an autoph- agy-related gene and the effect on autophagy gene expression might not be secondary to the stimulation of autophagic proteolysis. Ó 2007 Elsevier Inc. All rights reserved. Keywords: 3,5 0 -Dimethylpyrazole; Antilipolytic drugs; LC3 gene expression; Autophagy; Autophagic proteolysis; Amino acid; Insulin; Glucose Autophagy is the major catabolic pathway for long- lived proteins, cell organelles and is conserved from yeast to human [1]. Double membrane vesicles called autophago- somes enclose a portion of cytoplasm and deliver it to lyso- somes for degradation and for eventual recycling [2]. Under stress stimuli, such as lack of nutrients or decreased lipolysis, autophagic proteolysis is triggered while nutrients and insulin were shown to act like negative regulators [3]. Autophagy has a key role in cell reshaping, maintenance, and repair in physiological and pathological conditions, including atrophy and hypertrophy [4], disease [5], and aging [6]. Antilipolytic drugs can induce autophagy [7], removal of altered mitochondria [8], and anti-aging effects [9]. In this study, we explored the effect of the induction of autophagy by the administration of 3,5’-dimethylpyrazole (DMP) and the suppression of DMP-induced effects by the injection of glucose on rat liver autophagic proteolysis and on LC3 expression. LC3 protein is one of the two ubiq- 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.12.023 Abbreviations: DMP, 3,5 0 -dimethylpyrazole; FFA, free fatty acids. * Corresponding author. E-mail address: ebergami@med.unipi.it (E. Bergamini). 1 These authors contributed equally to this work. www.elsevier.com/locate/ybbrc Available online at www.sciencedirect.com Biochemical and Biophysical Research Communications 366 (2008) 786–792