To determine whether the growth factor activity in LIRKO serum is a product of hepatocytes or nonhepatic cells, we used conditioned media from cultures of primary hepatocytes (HCM), isolated from control or LIRKO mice in an in vitro cell proliferation assay. is a major economic burden in the world. A promising but still unrealized goal of efforts to improve diabetes therapy is the identification of novel factors that promote cell regeneration, with the long-term N-Carbamoyl-DL-aspartic acid goal of increasing functional cell mass in patients with either type 1 or type 2 diabetes. Reduced functional cell mass is a central feature in both forms of the disease and in diabetes associated with obesity (Muoio and SPRY1 Newgard, 2008). While autoimmune destruction of cells is the major cause of cell loss in type 1 diabetes, a failure of cells to compensate for ambient insulin resistance leads to uncontrolled hyperglycemia in type 2 diabetes. Lending encouragement to therapeutic strategies aimed at enhancing cell mass, decades of research indicate that cells possess the capacity to compensate for both N-Carbamoyl-DL-aspartic acid physiological (pregnancy) and pathological (obesity) insulin resistance (Ogilvie, 1933; Van Assche et al., 1978). Although cell growth in both humans and rodents has been documented to occur through N-Carbamoyl-DL-aspartic acid self-duplication of preexisting cells (Dor et al., 2004; Meier et al., 2008; Teta et al., 2007), albeit at low levels, the source of putative growth factor(s) mediating this process, especially in the context of insulin resistance, remains unknown. Among possible systemic regulators of cell mass, gut-derived incretins such as glucagon-like peptide-1 (GLP-1), glucose-dependent insulin-tropic polypeptide (GIP) (Renner et al., 2010; Saxena et al., 2010), adipocyte-derived adipokines including leptin (Morioka et al., 2007) and adiponectin (Holland et al., 2011), muscle-derived myokines such as IL-6 (Ellingsgaard et al., 2008; Suzuki et al., 2011), macrophage-derived cytokines including IL-1, IFN, and TNF- (Wang et al., 2010), bone-derived osteocalcin (Ferron et al., 2008), thyroid-derived T3/T4 hormones (J?rns et al., 2010; Verga Falzacappa et al., 2010), platelet-derived growth factor (PDGF) (Chen et al., 2011), serotonin (Kim et al., 2010), and FGF21 (Wente et al., 2006) have each been implicated. However, the lack of significant and consistent alterations in these known factors in the peripheral blood that can fully account for the cell proliferation in the insulin-resistant LIRKO mouse model (Table S1) prompted us to explore the presence of an as yet unidentified factor that is derived from an insulin-resistant liver. To test the hypothesis that crosstalk between the liver and pancreatic islets, communicated via a systemic humoral factor, mediates compensatory cell regeneration in the LIRKO mouse, we used in vivo (parabiosis, transplantation) and in vitro (primary islet cell proliferation assay) models to identify blood-borne and hepatocyte-produced soluble factors on cell proliferation. RESULTS AND DISCUSSION Concerted efforts in diabetes research are aimed at identifying molecules that specifically promote cell regeneration without adverse proliferation of cells in other tissues. To determine whether LIRKO mice, which manifest a dramatic hyperplasia of the endocrine pancreas, exhibit increased proliferation in extrapancreatic tissues, we injected bromodeoxyuridine (BrdU; 100 mg/kg body weight) intraperitoneally in 3-month-old LIRKO mice and assessed proliferation of cells, cells, and cells in metabolic organs such as the liver, adipose and skeletal muscle, and in nonmetabolic tissues such as the lung, kidney, and spleen. We observed a 2-fold increase N-Carbamoyl-DL-aspartic acid in cell mass (LIRKO 1.32 0.2 versus control 0.68 0.08 mg; p < 0.05; n = 6) in LIRKO mice compared to littermate controls that was due to enhanced cell proliferation evidenced by a 2.5-fold increase in BrdU incorporation (LIRKO 1% 0.08% versus control 0.4% 0.07% BrdU+ cells; p < 0.001; n = 6) and Ki67 staining (LIRKO 1.34% 0.1% versus control 0.51% 0.08% Ki67+ cells; p < 0.001; n = 6) in the LIRKOs. TUNEL staining did not reveal significant differences in the number of apoptotic cells between groups. We also observed no difference.
To determine whether the growth factor activity in LIRKO serum is a product of hepatocytes or nonhepatic cells, we used conditioned media from cultures of primary hepatocytes (HCM), isolated from control or LIRKO mice in an in vitro cell proliferation assay
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