7 research outputs found
β-Adrenergic Agonist and Antagonist Regulation of Autophagy in HepG2 Cells, Primary Mouse Hepatocytes, and Mouse Liver
<div><p>Autophagy recently has been shown to be involved in normal hepatic function and in pathological conditions such as non-alcoholic fatty liver disease. Adrenergic signalling also is an important regulator of hepatic metabolism and function. However, currently little is known about the potential role of adrenergic signaling on hepatic autophagy, and whether the β-adrenergic receptor itself may be a key regulator of autophagy. To address these issues, we investigated the actions of the β<sub>2</sub>-adrenergic receptor agonist, clenbuterol on hepatic autophagy. Surprisingly, we found that clenbuterol stimulated autophagy and autophagic flux in hepatoma cells, primary hepatocytes and <i>in vivo</i>. Similar effects also were observed with epinephrine treatment. Interestingly, propranolol caused a late block in autophagy in the absence and presence of clenbuterol, both in cell culture and <i>in vivo</i>. Thus, our results demonstrate that the β<sub>2</sub>- adrenergic receptor is a key regulator of hepatic autophagy, and that the β-blocker propranolol can independently induce a late block in autophagy.</p></div
Clenbuterol increases autophagic flux <i>in vivo</i>.
<p><b>A–B.</b>) Clenbuterol increases autophagosomal marker LC3-II in the liver of mice treated for 3 days. A further increase is seen with co-administration of chloroquine, indicating an increase in autophagic flux. Asterisk represents significance vs. ctrl, hash represents significance vs. CQ, and ampersand represents significance vs. Clen as per Tukey's post-hoc test following one-way ANOVA. <b>C–D.</b>) SQSTM1/P62 levels in the livers of the same mice. Asterisk indicates p<0.05. <b>E–F.</b>) Electron micrographs of the same mice, showing isolation membranes, autophagosomes, and autolysosomes. A significant increase in the number of autophagic vesicles per cell was observed. Asterisk represents p<0.05. Bar = 2 mm.</p
Long acting β<sub>2</sub>-agonist clenbuterol increases autophagosome number in HepG2 cells and in mouse primary hepatocytes.
<p><b>A–B.</b>) Clenbuterol increases LC3-II 24 hours after addition in HepG2 cells, at concentrations as low as 300 nM. Asterisk represents significance vs. ctrl, and hash represents significance vs. 30 nM as per Tukey's post-hoc test following one-way ANOVA. <b>C–D.</b>) Clenbuterol increases puncta formation in HepG2 cells transiently transfected with eGFP-LC3 plasmid. Image taken at 20× magnification. <b>E.</b>) Clenbuterol increases endogenous LC3 puncta in mouse primary hepatocytes. Image taken at 40× magnification, <b>F–G.</b>) Clenbuterol increases LC3-II 24 hours after addition in mouse primary hepatocytes. Error bars represent SEM. Unless otherwise noted, asterisk represents p<0.05 relative to control using Student's T-test.</p
Propranolol inhibits autophagic flux in HepG2 cells.
<p><b>A–B.</b>) Propranolol increases LC3-II levels in HepG2 cells, even in the absence of adrenergic agonist. Asterisk represents significance vs. ctrl, hash represents significance vs. Clen, and ampersand represents significance vs. Prop as per Tukey's post-hoc test following one-way ANOVA. <b>C–D.</b>) Propranolol inhibits autophagic protein turnover in HepG2 cells. LC3-II and SQSTM1/p62 levels are increased with increasing doses of propranolol. Asterisk represents significance vs. ctrl, hash represents significance vs. 1 µM, and ampersand represents significance vs. 10 µM as per Tukey's post-hoc test following one-way ANOVA. <b>E–F.</b>) Propranolol increases autophagosome number, but decreases autolysosome number in HepG2 cells transiently transfected with GFP-RFP-LC3 plasmid. Image taken at 40× magnification. Asterisk represents p<0.05 as per Student's t-test with respect to control. <b>G–H.</b>) Co-treatment of HepG2 cells with chloroquine and propranolol shows no increased accumulation of LC3-II compared to control cells treated with chloroquine. Asterisk represents significance vs. ctrl as per Tukey's post-hoc test following one-way ANOVA. For all parts, error bars represent SEM.</p
Clenbuterol increases autophagic flux in HepG2 cells and mouse primary hepatocytes.
<p><b>A–B.</b>) Co-treatment of HepG2 with Clenbuterol and Chloroquine shows a greater accumulation of LC3-II compared to control cells. Asterisk represents significance vs. Clen/CQ, and hash represents significance vs. CQ as per Tukey's post-hoc test following one-way ANOVA. <b>C–D.</b>) Clenbuterol increases autolysosomes in HepG2 cells transiently transfected with GFP-RFP-LC3 plasmid. Image taken at 40× magnification. <b>E.</b>) Clenbuterol increases lysosomal acidification (orange/red structures) in HepG2 stained with Acridine Orange. Image taken at 20× magnification. <b>F–G.</b>) Clenbuterol induces SQSTM1/p62 degradation in HepG2 cells. <b>H–I.</b>) Clenbuterol induces SQSTM1/p62 degradation in mouse primary hepatocytes. Error bars represent SEM. Unless otherwise noted, asterisk represents p<0.05 relative to control using Student's T-test.</p
Propranolol inhibits autophagic flux in mouse primary hepatocytes and <i>in vivo</i>.
<p>Propranolol induces accumulation of both LC3-II and p62 in mouse primary hepatocytes (<b>A,B</b>), and in mouse liver (<b>C,D</b>). Asterisk represents p<0.05 as per Student's t-test with respect to control. For all parts, error bars represent SEM.</p
Loss of ULK1 increases RPS6KB1-NCOR1 repression of NR1H/LXR-mediated <i>Scd1</i> transcription and augments lipotoxicity in hepatic cells
<p>Lipotoxicity caused by saturated fatty acids (SFAs) induces tissue damage and inflammation in metabolic disorders. SCD1 (stearoyl-coenzyme A desaturase 1) converts SFAs to mono-unsaturated fatty acids (MUFAs) that are incorporated into triglycerides and stored in lipid droplets. SCD1 thus helps protect hepatocytes from lipotoxicity and its reduced expression is associated with increased lipotoxic injury in cultured hepatic cells and mouse models. To further understand the role of SCD1 in lipotoxicity, we examined the regulation of <i>Scd1</i> in hepatic cells treated with palmitate, and found that NR1H/LXR (nuclear receptor subfamily 1 group H) ligand, GW3965, induced <i>Scd1</i> expression and lipid droplet formation to improve cell survival. Surprisingly, ULK1/ATG1 (unc-51 like kinase) played a critical role in protecting hepatic cells from SFA-induced lipotoxicity via a novel mechanism that did not involve macroautophagy/autophagy. Specific loss of <i>Ulk1</i> blocked the induction of <i>Scd1</i> gene transcription by GW3965, decreased lipid droplet formation, and increased apoptosis in hepatic cells exposed to palmitate. Knockdown of ULK1 increased RPS6KB1 (ribosomal protein S6 kinase, polypeptide 1) signaling that, in turn, induced NCOR1 (nuclear receptor co-repressor 1) nuclear uptake, interaction with NR1H/LXR, and recruitment to the <i>Scd1</i> promoter. These events abrogated the stimulation of <i>Scd1</i> gene expression by GW3965, and increased lipotoxicity in hepatic cells. In summary, we have identified a novel autophagy-independent role of ULK1 that regulates NR1H/LXR signaling, <i>Scd1</i> expression, and intracellular lipid homeostasis in hepatic cells exposed to a lipotoxic environment.</p