Caveolin-1 and Mitochondrial Alterations in Regenerating Rat Liver MARIA MASTRODONATO, 1 * PIERO PORTINCASA, 2 DONATELLA MENTINO, 1 ROBERTA ROSSI, 3 LEONARDO RESTA, 3 DOMENICO FERRI, 1 AND GIUSEPPA ESTERINA LIQUORI 1 1 Department of Biology, ‘‘Aldo Moro’’ University, Bari, Italy 2 Department of Internal Medicine, ‘‘Aldo Moro’’ University, Bari, Italy 3 Department of Pathological Anatomy, Laboratory of Ultrastructural Pathology, ‘‘Aldo Moro’’ University, Bari, Italy KEY WORDS liver steatosis; metabolic syndrome; oxydative stress; caveolin-1; mitochondria ABSTRACT The liver has a remarkable ability to regenerate after partial hepatectomy (PH), although the factors governing such ability are still poorly understood. During the prereplicative phase of the regeneration, ultrastructural alterations of periportal hepatocytes were seen, includ- ing mitochondrial swelling, abnormal accumulation of lipids, and myelin figures which could lead to the formation of lipid droplets. As it has been hypothesized that caveolin-1 is involved in lipido- genesis and in mitochondrial homeostasis, we aimed to study the subcellular distribution of caveo- lin-1 in hepatocytes at an early stage following PH. Liver samples were processed for light and elec- tron microscopy at 0 h, 24 h, and 96 h after PH. The expression and subcellular distribution of cav- eolin-1 was assessed by immunohistochemical and immunocytochemical techniques. Following PH, at 24 h, membranes of altered mitochondria of periportal hepatocytes exhibited significant decrease of caveolin-1 expression compared with control. Myelin figures showing high expression of caveolin-1 were also seen. At 96 h, hepatocytes became ultrastructurally similar to the control liver, and the expression of caveolin-1 on mitochondria showed a moderate increase compared with 24 h after PH. Decrease of expression of caveolin-1 in the altered liver mitochondrial membranes at 24 h fol- lowing PH, and the high expression of caveolin-1 observed on myelin figures, suggests involvement of caveolin-1 is in both mitochondrial homeostasis and lipidogenesis. Addressing the role played by caveolin-1 during liver regeneration might disclose additional features of mitochondrial homeosta- sis and lipidogenesis during frequent metabolic liver diseases. Microsc. Res. Tech. 00:000–000, 2012. V V C 2012 Wiley Periodicals, Inc. INTRODUCTION The liver exhibits the peculiar ability to regenerate after an insult, as the partial hepatectomy (PH) or the use of toxic agents with necrotizing effect. The subtle mechanisms governing liver regeneration, however, are still largely unclear. This complex process involves the effect of growth factors, cytokines, hormones lead- ing signals to kinases, and transcription factors. The proliferative response of regenerating hepatocytes fol- lowing PH is characterized by a prereplicative phase, with a drastic change in the metabolism of the remnant liver parenchyma; a replicative phase characterized by DNA synthesis, which activated within 15–18 h, is also involved (Chamuleau and Bosman, 1988). During the prereplicative phase, mitochondria exhibit morphologi- cal and functional changes which are associated with increased production of reactive oxygen species (ROS). It has been reported that mitochondria are the most important producers of ROS and that ROS production causes oxidative stress, with an increase in the ratio of oxidized/reduced mitochondrial glutathione (GSSG/ GSH) (Ferri et al., 2005; Guerrieri et al., 2002; Martı ´- nez-Outschoorn et al., 2010; Schafer and Buettner, 2001), protein thiol oxidation and lipid peroxidation, which promote the permeability transition causing mi- tochondrial swelling and calcium release, accompanied by change in organelle ultrastructure (Ferri et al., 2005). The release of calcium ions may play a key role in transduction of signals of cell proliferation and thus can be considered a comitogen factor (Bernardi and Petronelli, 1996). The mitochondria recover their own functions during the replicative phase. In the early phases of regeneration, hepatocytes accumulate lipid droplets (triglycerides, phospholipids, and cholesterol), which represent the main energy source of ATP for remaining hepatocytes, in view of liver reconstruction. Part of lipids is then secreted in bile as cholesterol or as bile acids (which represent end-products of choles- terol metabolism) or used in the production of lipopro- teins or new membranes. Of note, both lipids and mito- chondria play a fundamental role in the processes of liver regeneration. In mice with impaired lipogenesis, e.g., that induced by leptin, impairment liver regenera- tion is observed (Frank and Lisanti, 2007). Caveolin-1, the main structural protein of caveolae, is involved in M.M. and P.P. contributed equally to this work. *Correspondence to: Maria Mastrodonato, Department of Biology, ‘‘Aldo Moro’’ University, Via Orabona, 4, I-70125 Bari, Italy. E-mail: m.mastrodonato@ biologia.uniba.it Abbreviations used: GSH, reduced glutathione; GSSG, oxidized glutathione; PH, partial hepatectomy; RER, rough endoplasmic reticulum; ROS, reactive oxygen species; TBS, Tris-buffered saline Received 22 December 2011; accepted in revised form 6 February 2012 DOI 10.1002/jemt.22027 Published online in Wiley Online Library (wileyonlinelibrary.com). V V C 2012 WILEY PERIODICALS, INC. MICROSCOPY RESEARCH AND TECHNIQUE 00:000–000 (2012)