Cholesterol-enriched membrane microdomains are needed for insulin signaling and proliferation in hepatic cells

Abstract

Hepatocyte proliferation during liver regeneration is a well-coordinated process regulated by the activation of several growth factor receptors, including the insulin receptor (IR). The IR can be localized in part to cholesterol-enriched membrane microdomains, but the role of such domains in insulin-mediated events in hepatocytes is not known. We investigated whether partitioning of IRs into cholesterol-enriched membrane rafts is important for the mitogenic effects of insulin in the hepatic cells. IR and lipid rafts were labeled in HepG2 cells and primary rat hepatocytes. Membrane cholesterol was depleted in vitro with metyl-β-cyclodextrin (MβCD) and in vivo with lovastatin. Insulin-induced calcium (Ca) signals studies were examined in HepG2 cells and in freshly isolated rat hepatocytes as well as in whole liver in vivo by intravital confocal imaging. Liver regeneration was studied by 70% partial hepatectomy (PH), and hepatocyte proliferation was assessed by PCNA staining. A subpopulation of IR was found in membrane microdomains enriched in cholesterol. Depletion of cholesterol from plasma membrane resulted in redistribution of the IR along the cells, which was associated with impaired insulin-induced nuclear Ca signals, a signaling event that regulates hepatocyte proliferation. Cholesterol depletion also led to ERK1/2 hyper-phosphorylation. Lovastatin administration to rats decreased hepatic cholesterol content, disrupted lipid rafts and decreased insulin-induced Ca signaling in hepatocytes, and delayed liver regeneration after PH. Therefore, membrane cholesterol content and lipid rafts integrity showed to be important for the proliferative effects of insulin in hepatic cells. NEW & NOTEWORTHY One of insulin's actions is to stimulate liver regeneration. Here we show that a subpopulation of insulin receptors is in a specialized cholesterol-enriched region of the cell membrane and this subfraction is important for insulin's proliferative effects.

Expression of the type 3 InsP3receptor is a final common event in the development of hepatocellular carcinoma

“…Mouse serum was obtained by centrifugation of total blood and used to assess liver function through the following panel of serological tests: alkaline phosphatase (Bioclin, Brazil), alanine aminotransferase (Labtest, Brazil), aspartate aminotransferase (Labtest, Brazil) and gamma-glutamyl transferase (Labtest, Brazil), as previously described. 15…”

“…The Novolink Polymer Detection System (Leica Biosystems, Germany) was used in the subsequent steps as described previously. 15 Primary antibodies against ITPR3 (1:100; Sigma), proliferating cell nuclear antigen (PCNA) (1:100; Abcam) and 5′-methylcytosine (5mC) (1:400; Zymo Research) were incubated overnight at room temperature followed by incubation with detection polymer for 40 min at room temperature. DAB was used for signal detection.…”

“…Livers were loaded with the Ca 2+ indicator dye Fluo-4/AM (Thermo Fisher Scientific) for 10 min at room temperature and placed onto the stage of a Nikon A1 confocal microscope, as described previously. 15 Arginine vasopressin (AVP), a known InsP3-dependent Ca 2+ release agonist, 22 23 was injected IV at a concentration of 100 ng/mL to trigger InsP3-dependent Ca 2+ release. Data are expressed as fluorescence/ baseline fluorescence×100%.…”

Emerging Role of HMGB1 in the Pathogenesis of Schistosomiasis Liver Fibrosis

“… Primary hepatocytes release HMGB1 in the presence of SEA. Mice were perfused through the portal vein with HANKS A and then HANKS B medium containing 0.05% collagenase (Roche Applied Science, Indianapolis, IN) and the perfused liver tissue was passed through a 40-μm nylon mesh filter ( 69 , 70 ). Primary hepatocytes were cultured at 37°C in 5% CO 2 /95% O 2 in Williams' medium E (Sigma-Aldrich, Missouri, USA) containing 10% fetal bovine serum (Gibco), 50 units/mL penicillin, 50 g/mL streptomycin (Sigma-Aldrich, Missouri, USA) were plated on collagen-coated coverslips (50 μg/mL) (BD Biosciences, San Jose, CA).…”

Akkermansia muciniphilasecretome promotes α-synuclein aggregation in enteroendocrine cells

“…Calcium (Ca 2+ ) is known to regulate several important cell functions, such as secretion, proliferation, apoptosis, protein biosynthesis and folding (Alvarenga et al, 2016; Carafoli & Krebs, 2016; Fonseca et al, 2018; Guimaraes et al, 2017). In order to study the effects of A. muciniphila conditioned media in the fluctuations of intracellular Ca 2+ signaling in STC-1 cells, we first stimulated Fluo-4/AM-loaded cells with 1 or 10% conditioned BHI medium (BHI CM) or unconditioned BHI medium (BHI).…”