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• W2078570369 abstract "Nonalcoholic fatty liver disease is an increasingly prevalent spectrum of conditions characterized by excess fat deposition within hepatocytes. Affected hepatocytes are known to be highly susceptible to ischemic insults, responding to injury with increased cell death, and commensurate liver dysfunction. Numerous clinical circumstances lead to hepatic ischemia. Mechanistically, specific means of reducing hepatic vulnerability to ischemia are of increasing clinical importance. In this study, we demonstrate that the glucagon-like peptide-1 receptor agonist Exendin 4 (Ex4) protects hepatocytes from ischemia reperfusion injury by mitigating necrosis and apoptosis. Importantly, this effect is more pronounced in steatotic livers, with significantly reducing cell death and facilitating the initiation of lipolysis. Ex4 treatment leads to increased lipid droplet fission, and phosphorylation of perilipin and hormone sensitive lipase – all hallmarks of lipolysis. Importantly, the protective effects of Ex4 are seen after a short course of perioperative treatment, potentially making this clinically relevant. Thus, we conclude that Ex4 has a role in protecting lean and fatty livers from ischemic injury. The rapidity of the effect and the clinical availability of Ex4 make this an attractive new therapeutic approach for treating fatty livers at the time of an ischemic insult. Nonalcoholic fatty liver disease is an increasingly prevalent spectrum of conditions characterized by excess fat deposition within hepatocytes. Affected hepatocytes are known to be highly susceptible to ischemic insults, responding to injury with increased cell death, and commensurate liver dysfunction. Numerous clinical circumstances lead to hepatic ischemia. Mechanistically, specific means of reducing hepatic vulnerability to ischemia are of increasing clinical importance. In this study, we demonstrate that the glucagon-like peptide-1 receptor agonist Exendin 4 (Ex4) protects hepatocytes from ischemia reperfusion injury by mitigating necrosis and apoptosis. Importantly, this effect is more pronounced in steatotic livers, with significantly reducing cell death and facilitating the initiation of lipolysis. Ex4 treatment leads to increased lipid droplet fission, and phosphorylation of perilipin and hormone sensitive lipase – all hallmarks of lipolysis. Importantly, the protective effects of Ex4 are seen after a short course of perioperative treatment, potentially making this clinically relevant. Thus, we conclude that Ex4 has a role in protecting lean and fatty livers from ischemic injury. The rapidity of the effect and the clinical availability of Ex4 make this an attractive new therapeutic approach for treating fatty livers at the time of an ischemic insult. The incidence of obesity and fatty liver disease is increasing worldwide. Non alcoholic fatty liver disease (NAFLD) includes a spectrum of liver abnormalities ranging from simple steatosis with preserved synthetic function to end-stage liver disease requiring transplantation.1Day C.P. Non-alcoholic fatty liver disease: a massive problem.Clinical Med. 2011; 11: 176-178Crossref PubMed Scopus (93) Google Scholar, 2Hanouneh I.A. Zein N.N. Metabolic syndrome and liver transplantation.Minerva Gastroenterol Dietol. 2010; 56: 297-304PubMed Google Scholar The cause of hepatic dysfunction related to steatosis remains incompletely defined.3Selzner M. Clavien P.A. Fatty liver in liver transplantation and surgery.Semin Liver Dis. 2001; 21: 105-113Crossref PubMed Scopus (373) Google Scholar However, it is known that a steatotic liver has increased susceptibility to ischemic insults, such as those induced during liver resections and liver surgery,4Gomez D. Malik H.Z. Bonney G.K. Wong V. Toogood G.J. Lodge J.P. Prasad K.R. Steatosis predicts postoperative morbidity following hepatic resection for colorectal metastasis.Br J Surg. 2007; 94: 1395-1402Crossref PubMed Scopus (102) Google Scholar, 5Vetelainen R. van Vliet A. Gouma D.J. van Gulik T.M. Steatosis as a risk factor in liver surgery.Ann Surg. 2007; 245: 20-30Crossref PubMed Scopus (218) Google Scholar, 6Chavin K.D. Fiorini R.N. Shafizadeh S. Cheng G. Wan C. Evans Z. Rodwell D. Polito C. Haines J.K. Baillie G.M. Schmidt M.G. Fatty acid synthase blockade protects steatotic livers from warm ischemia reperfusion injury and transplantation.Am J Transplant. 2004; 4: 1440-1447Crossref PubMed Scopus (53) Google Scholar heart failure,7Marcos A. Ham J.M. Fisher R.A. Olzinski A.T. Posner: Single-center analysis of the first 40 adult-to-adult living donor liver transplants using the right lobe.Liver Transpl. 2000; 6: 296-301Crossref PubMed Scopus (262) Google Scholar and shock.8Matheson P.J. Hurt R.T. Franklin G.A. McClain C.J. Garrison R.N. Obesity-induced hepatic hypoperfusion primes for hepatic dysfunction after resuscitated hemorrhagic shock.Surgery. 2009; 146: 739-748Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar In addition, steatotic livers are known to weather the ischemic insult of transplantation poorly,9Selzner M. Clavien P.A. Failure of regeneration of the steatotic rat liver: disruption at two different levels in the regeneration pathway.Hepatology. 2000; 31: 35-42Crossref PubMed Scopus (215) Google Scholar resulting in increased rates of primary nonfunction and initial graft dysfunction.10Clavien P.A. Selzner M. Hepatic steatosis and transplantation.Liver Transpl. 2002; 8: 980Crossref PubMed Scopus (11) Google Scholar, 11Sharkey F.E. Lytvak I. Prihoda T.J. Speeg K.V. Washburn W.K. Halff G.A. High-grade microsteatosis and delay in hepatic function after orthotopic liver transplantation.Hu Pathol. 2011; 42: 1337-1342Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar As such, fatty livers are routinely turned down for transplantation and this impacts transplant wait list morbidity and mortality.12Cheng Y. Chen C. Lai C.Y. Chen T. Huang T. Lee T. Lin C. Lord R. Chen Y. Eng H. Pan T.L. Lee T.H. Wang Y.H. Iwashita Y. Kitano Goto S. Assessment of donor fatty livers for liver transplantation.Transplantation. 2001; 71: 1206-1207Crossref PubMed Scopus (3) Google Scholar Thus, liver steatosis contributes to the public health burden and methods to mollify the adverse effects of liver steatosis are relevant across a large spectrum of hepatic diseases. The inability of a steatotic liver to withstand ischemic insult is directly related to increased post ischemic cell death, which can occur through necrosis and apoptosis. The fundamental connection between intracellular fat and poor hepatic cell survival13Sorrentino P. Terracciano L. D'Angelo S. Ferbo U. Bracigliano A. Tarantino L. Perrella A. Perrella O. De Chiara G. Panico L. De Stefano N. Lepore M. Mariolina Vecchione R. Oxidative stress and steatosis are cofactors of liver injury in primary biliary cirrhosis.J Gastroenterol. 2010; 45: 1053-1062Crossref PubMed Scopus (32) Google Scholar is incompletely understood. However, it has been suggested that methods that decrease intracellular fat reverse this susceptibility and the use of glucagon-like peptide-1 (GLP-1) analogues is one such approach. GLP-1 is secreted from the L cells of the small intestine and its cognate receptor (GLP-1R) is present in several organs, such as the pancreas, brain, heart, kidney, and liver. Although it is well known for its incretin action,14Thorens B. Waeber G. Glucagon-like peptide-I and the control of insulin secretion in the normal state and in NIDDM.Diabetes. 1993; 42: 1219-1225Crossref PubMed Scopus (122) Google Scholar it also has pleotropic effects.15Acitores A. Gonzalez N. Sancho V. Valverde I. Villanueva-Penacarrillo M.L. Cell signalling of glucagon-like peptide-1 action in rat skeletal muscle.J Endocrinol. 2004; 180: 389-398Crossref PubMed Scopus (53) Google Scholar, 16Drucker D.J. Biological actions and therapeutic potential of the glucagon-like peptides.Gastroenterology. 2002; 122: 531-544Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 17Ban K. Noyan-Ashraf M.H. Hoefer J. Bolz S.S. Drucker D.J. Husain M. Cardioprotective and vasodilatory actions of glucagon-like peptide 1 receptor are mediated through both glucagon-like peptide 1 receptor-dependent and -independent pathways.Circulation. 2008; 117: 2340-2350Crossref PubMed Scopus (832) Google Scholar, 18Baggio L.L. Holland D. Wither J. Drucker D.J. Lymphocytic infiltration and immune activation in metallothionein promoter-exendin-4 (MT-Exendin) transgenic mice.Diabetes. 2006; 55: 1562-1570Crossref PubMed Scopus (17) Google Scholar, 19Alvarez E. Roncero I. Chowen J.A. Thorens B. Blazquez E. Expression of the glucagon-like peptide-1 receptor gene in rat brain.J Neurochem. 1996; 66: 920-927Crossref PubMed Scopus (147) Google Scholar In the liver we have shown that GLP-1 or its homologue Exendin 4 (Ex4) acts directly on steatotic hepatocytes to decrease their lipid content.20Gupta N.A. Mells J. Dunham R.M. Grakoui A. Handy J. Saxena N.K. Anania F.A. Glucagon-like peptide-1 receptor is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway.Hepatology. 2010; 51: 1584-1592Crossref PubMed Scopus (347) Google Scholar, 21Ding X. Saxena N.K. Lin S. Gupta N.A. Anania F.A. Exendin-4, a glucagon-like protein-1 (GLP-1) receptor agonist, reverses hepatic steatosis in ob/ob mice.Hepatology. 2006; 43: 173-181Crossref PubMed Scopus (435) Google Scholar In addition, a cytoprotective action of Ex4 with improvement in cell survival has also been reported.22Li L. El-Kholy W. Rhodes C.J. Brubaker P.L. Glucagon-like peptide-1 protects beta cells from cytokine-induced apoptosis and necrosis: role of protein kinase B.Diabetologia. 2005; 48: 1339-1349Crossref PubMed Scopus (165) Google Scholar Thus, we hypothesize that anti-steatotic effects of Ex4 in hepatocytes and cytoprotective effects in other organs make it a rational target for investigation in steatotic livers undergoing ischemia reperfusion injury (IRI), a common clinical scenario in people with NAFLD. In this study, we explore the role of Ex4 in protecting against necrosis and apoptosis, the two forms of cell death encountered in hepatic IRI, and we provide evidence to show that Ex4 stimulates lipolysis with a short course of treatment. To our knowledge, this is the first study showing a direct and rapid action of Ex4 in acutely reversing the vulnerability of a steatotic liver to ischemic insults, supporting the investigation of Ex4 as a potential therapeutic agent for treatment of people with NAFLD undergoing ischemic injury and at the time of procurement of a fatty liver for transplantation. The Institutional Animal Care and Use Committee (IACUC) of Emory University approved all procedures performed on animals and all experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, published by the United States Public Health Service. C57BL/6 male mice were obtained from Jackson Research Laboratories (Bar Harbor, ME) at 4 weeks of age and were maintained on a 12-hour dark-light cycle and allowed free access to food and water under conditions of controlled temperatures (25 ± 2°C). Animals were divided into two groups. Half the animals were fed a regular diet and the other half received a high fat diet (60% fat; Research Diets, Inc, NJ) ad libitum for 12 weeks. Body weights were monitored during this period. After 12 weeks of feeding, mice on a lean and a high fat diet were subdivided into control and IRI groups with and without Ex4 (n = 8 per group). Sham controls were also included in the study. Ex4 (20 μg/kg; Sigma-Aldrich, St. Louis, MO) was given 2 hours before surgery and 2 hours after surgery through the tail vein. Hepatic IRI was performed under general anesthesia, induced, and maintained by ketamine/xylazine. A vertical incision was made through the skin and peritoneum, exposing the porta hepatis. A small clamp was applied to the portal vein and hepatic artery, and ischemia was induced for 20 minutes. The clamp was then removed and blood flow was restored (reperfusion). After closing the abdomen, the mice were placed in a recovery cage and allowed free access to food and water. Sham surgery animals underwent a laparotomy and closure without hepatic vascular clamping. All animals were sacrificed 24 hours after closure. Serum samples were collected for serum alanine aminotransferase (ALT) measurement and liver tissues were frozen or fixed in buffered formalin. Serum ALT was used as a measure of hepatocellular damage and was determined by using Infinity ALT reagent (Thermo Scientific, Middletown, VA). Paraffin sections of liver were stained with H&E. Six random images were taken from each slide and necrotic areas were quantified using Metamorph software (version 7.5.6) (Molecular Devices, Sunnyvale, CA). Percent area of necrosis was calculated out of the total area. Steatosis was assessed in frozen liver sections using Oil Red O (ORO) stain, as represented by the red color. HuH7 cells were cultured using Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS, Hyclone, Logan, UT). After overnight serum starvation, the cells were treated with free fatty acids: palmitic and oleic acid (400 nmol/L), along with 10% essential free fatty acids free bovine serum albumin (BSA, Sigma Aldrich) for 10 hours. Steatosis was assessed using ORO stain. The steatotic hepatocytes were kept in a hypoxia chamber (1% O2, 9% CO2, and 95% N2) for 30 minutes, followed by a 2-hour reperfusion by adding serum containing medium and placing the cells in the regular incubator at normal atmospheric conditions (20% O2, 5% CO2). Ex4 was added 30 minutes before and after hypoxia, and the monolayers were tested for apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. TUNEL staining was performed according to the manufacturer's instructions (Roche Applied Sciences, Indianapolis, IN) both in the liver tissue from mice undergoing IRI and in in vitro steatotic hepatocytes with appropriate control groups. Nuclei were stained with DAPI. Images were scanned and processed as immunofluorescent samples (described as follows). HuH7 cells were plated on chamber slides, and on reaching confluence they were treated with palmitic and oleic acid for 10 hours followed by treatment with Ex4 (20 nmol/L) for 10 minutes (controls were treated with Ex4 free media), washed, permeabilized, and fixed in 4% paraformaldehyde. These cells were further blocked in 2% bovine serum albumin and incubated with phospho-perilipin mouse monoclonal antibody (Ser 497, Vala Sciences, San Diego, CA) and rabbit phospho-hormone-sensitive lipase (p-HSL Ser563, Cell Signaling Technology, Inc., Danvers, MA) overnight at 4°C. They were then incubated with Alexa fluor 594 secondary antibody (Cell Signaling Technology, Inc) for 60 minutes, at 37°C. Nuclei were stained with DAPI. Samples were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA) and scanned using an inverted laser confocal microscope (Olympus Fluoview FV1000, IX81; Olympus America Inc; Center Valley, PA). Each image was acquired three-dimensionally (x, y, and z), sequentially with appropriate lasers at 800 × 800 pixels, 12 bits with the speed of 4 μs/pixel). Six random fields were captured using ×40 objective into z stacks (1 micron each) for each sample. All of the independent experiments were acquired similarly with same settings. Image processing and analysis were performed using FV10-ASW Version 2.1 (Olympus America Inc., Center Valley, PA). Briefly, the following arithmetic numbers were obtained from independent experiments and fields (3 z-stacks from each field) of all samples to perform statistical analysis: intensity, area, overlay, graphics, co-localization, and file conversions. These experiments were repeated three times. 3T3-L1 cells were grown on chamber slides and differentiated into adipocytes by placing in a differentiation medium (Dulbecco's modified Eagle's medium with 10% fetal bovine serum supplemented with dexamethasone (1 μmol/L), isobutylmethylxanthine (500 μmol/L), and insulin (10 μg/mL). The differentiated adipocytes were then treated with Ex4 and forskolin (positive control) for 10 minutes. Immunofluorescence was performed with phospho-perilipin and phospho-HSL antibodies, as previously described. The cells were stained with a fluorescent lipid staining kit (Vala Sciences, San Diego, CA) for 1 hour at 37°C followed by nuclear staining with DAPI. Liver tissue triglycerides were measured using a triglyceride assay according to the manufacturer's instructions (Biovision, CA) and were expressed as nmol/mg liver tissue. Liver tissues were fixed in glutaraldehyde and electron microscopy was performed by the Electron Microscopy Core facility, Emory University. Statistical analysis was performed by Student's t-test (Graph Pad Prism 4.0, San Diego, CA). A probability value of P < 0.05 was regarded as significant. One-way analysis of variance was used where more than two groups were compared. To investigate the effect of Ex4 in IRI of lean and steatotic livers, we used an established in vivo model of hepatic steatosis. Body weights were monitored at regular intervals and mice were fed a high fat diet, which showed a significant increase in weight relative to regular chow fed controls (high fat diet 37.1 ± 1.0 grams versus lean 25.2 ± 0.5 grams; P < 0.0001) (Figure 1A). The presence of hepatic steatosis was verified by ORO staining (Figure 1B, top panel, red staining). After IRI, an extensive area of necrosis was evident in the steatotic livers as compared to livers of lean mice (31.0% ± 3.0 versus 5.2% ± 0.1) (Figure 1, G versus D). When treated with Ex4, before and after IRI, a protective effect was seen leading to significantly decreased necrosis in steatotic mice (3.2% ± 0.9; P < 0.0001) (Figure 1H). In addition, lean mice treated with Ex4 also demonstrated minimal to no necrosis (Figure 1E). Hepatocellular damage was also assessed by measuring serum ALT levels. As expected, the baseline serum ALT was numerically, but not statistically significantly higher in steatotic mice as compared to the lean mice. However, after IRI and 24 hours of reperfusion, serum ALT levels were markedly increased in the steatotic mice as compared to the lean mice (700 ± 43.5 IU/L versus 370 ± 33.7 IU/L; P < 0.01) (Figure 1J), representing more extensive hepatocellular affliction in the steatotic mice. On treatment with Ex4, a protective effect was seen with significant reduction of serum ALT levels in both steatotic and lean mice undergoing IRI. A fourfold decrease in serum ALT was demonstrated in the Ex4 treated steatotic mice undergoing IRI (180 ± 37.16 IU/L versus 700 ± 43.5 IU/L; P < 0.008) (Figure 1J). Improvement in serum ALT was also evident in Ex4-treated lean mice undergoing IRI (95.76 ± 24.05 versus 370 ± 33.72 IU/L; P < 0.001), but the effect was twofold. These data show that a short course of Ex4 treatment has a protective effect on the liver undergoing IRI, as evidenced by significantly less necrosis and reduced elevations in post ischemic serum ALT, and this effect is particularly evident in the steatotic liver. One of the mechanisms by which hepatocyte death occurs during IRI is apoptosis.23Cursio R. Colosetti P. Saint-Paul M.C. Pagnotta S. Gounon P. Iannelli A. Auberger P. Gugenheim J. Induction of different types of cell death after normothermic liver ischemia-reperfusion.Transplant Proc. 2010; 42: 3977-3980Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar To determine whether Ex4 had an anti-apoptotic effect, H&E stained liver sections were assessed by a pathologist blinded to the treatment groups. Apoptotic bodies were identified by the presence of pyknotic nuclei and various stages of karyorrhexis and karyolysis. The total number of apoptotic bodies were counted in each field and normalized to the live cells. We observed that on treatment with Ex4, there was a significantly lower number of apoptotic bodies in the steatotic liver as compared to the nontreated steatotic mice undergoing IRI (6.9 ± 3.06 versus 25.3 ± 4.3; P < 0.03) (Figure 2, F versus E). Although after IRI, the apoptotic bodies were not significantly increased in the lean liver (as compared to the steatotic liver), Ex4 treatment lead to lower apoptosis as compared to nontreated lean IRI (1.5 ± 0.6 versus 6.0 ± 1.4; P < 0.003) (Figure 2, C versus B), To further verify these findings, we performed a TUNEL assay on frozen liver tissues. Treatment with Ex4 led to a fourfold reduction in TUNEL-positive cells in liver sections of steatotic mice undergoing IRI (23.67 ± 2.9 versus 9.0 ± 0.7; P < 0.0008) (Figure 2, M versus L). This result, along with direct histological evidence shows that Ex4 protects the steatotic liver from cell death not only by necrosis as previously shown, but also from apoptosis, resulting in overall improvement in resilience of the liver to IRI, in particular in the steatotic liver. To verify that the effect of Ex4 on steatosis was indeed a direct action, we performed TUNEL assays in HuH7 cells that were made steatotic in vitro and exposed to a hypoxia chamber. As shown in Figures 3, C and F, there was a significantly lower number of TUNEL-positive cells in lean and steatotic hepatocytes exposed to Ex4 as compared to nontreated lean and steatotic cells (5.8 ± 0.29 versus 26.6 ± 2.2 in lean cells; P < 0.001 and 13.53 ± 0.94 versus 34.52 ± 5.3 in steatotic cells; P < 0.001) (Figure 3G). This mitigation of apoptosis by Ex4 in hepatocytes undergoing ischemia-hypoxia-reperfusion in vitro provides support to the in vivo protective effect of Ex4 seen in our previous experiment in decreasing cell death in hepatocytes undergoing IRI. Although the reduction in apoptosis is seen in both lean and steatotic cells, it is several-fold higher in the steatotic hepatocytes. The effectiveness of Ex4 in normal and steatotic livers undergoing IRI broadens the potential of its clinical applicability. To assess the effect of Ex4 on lipid droplets, we examined liver histology by H&E staining. In steatotic mice undergoing IRI without Ex4, there was an increase in coalescence of fat droplets or lipid droplet “fusion” leading to macrovesicular steatosis (Figures 4, B and E), a striking difference from the predominantly microvesicular fat before IRI (Figures 4, A and D). In the Ex4 treated mice, this effect was ameliorated with a significant reduction in large fat droplets (Figures 4, C and F). To study these lipid droplets at a subcellular level, we performed electron microscopy. In the Ex4-treated mice undergoing IRI, electron microscopic images showed that the vast majority of lipid droplets in a state of “fission” in which the otherwise smooth contour of lipid droplets had become irregular, showing multiple pseudopodia-like protrusions (Figures 4, I and L). These results clearly demonstrated a direct effect on the lipid droplet, leading to fission, a known precursor of lipolysis.24Marcinkiewicz A. Gauthier D. Garcia A. Brasaemle D.L. The phosphorylation of serine 492 of perilipin a directs lipid droplet fragmentation and dispersion.J Biol Chem. 2006; 281: 11901-11909Crossref PubMed Scopus (153) Google Scholar In addition, total liver triglycerides were significantly reduced in the Ex4-treated steatotic mice undergoing IRI (49.51 ± 4.85 versus 95.75 ± 15.32 nmol/mg of liver tissue; P < 0.009). The baseline triglyceride level of the control steatotic mice (without IRI and Ex4) was similar to that of the steatotic mice undergoing IRI without Ex4 (53.06 ± 8.7 nmol/mg) (Figure 4M). The reduction in liver triglyceride content by Ex4 provides further evidence for the lipolytic effects of Ex4. To confirm that the lipolytic action was indeed a direct effect of Ex4, we performed in vitro studies and exposed steatotic HuH7 cells to ischemia and hypoxia followed by reperfusion, with and without Ex4 treatment. Ex4-treated cells showed substantially less steatosis than the nontreated steatotic HuH7 cells. ORO staining confirmed the decrease steatosis in Ex4-treated steatotic HuH7 cells (Figure 5) (P < 0.006). This provides evidence for the direct action of Ex4 on hypoxic hepatocytes causing a reduction in hepatic steatosis. To investigate the underlying mechanism of the effects of Ex4 on the lipid droplet, we investigated proteins that have a crucial role in lipid droplet stabilization by using adipocytes as our experimental model. Perilipin is one such protein that is bound to the lipid droplet preventing its degradation from hormone sensitive lipases (HSL). When perilipin is phosphorylated, access of HSL to the lipid droplet is facilitated, leading to lipolysis. To investigate this action, adipocytes and steatotic HuH7 cells (Ex4-treated and nontreated controls) were probed with phospho-perilipin (Ser 497) and phospho-HSL (Ser 563) antibodies and examined by confocal microscopy. As shown in Figure 6, A–C, perilipin was phosphorylated in the Ex4 treated adipocytes as compared to nontreated controls, an effect that was similar to that seen with forskolin, a positive control for lipolysis.25McDonough P.M. Ingermanson R.S. Loy P.A. Koon E.D. Whittaker R. Laris C.A. Hilton J.M. Nicoll J.B. Buehrer B.M. Price J.H. Quantification of hormone sensitive lipase phosphorylation and colocalization with lipid droplets in murine 3T3L1 and human subcutaneous adipocytes via automated digital microscopy and high-content analysis.Assay Drug Dev Technol. 2011; 9: 262-280Crossref PubMed Scopus (10) Google Scholar In addition, HSL, which in a nonstimulated state, has a cytoplasmic distribution that becomes phosphorylated and aligned along the lipid droplet surface, indicating initiation of the lipolytic process (Figures 6, D–F). In adipocytes, Ex4 treatment leads to a significant increase in co-localization of lipid droplet with phospho-perilipin and phospho-HSL, as quantified by confocal microscopy (Figure 6, G and H). This was also evident in the steatotic HuH7 cells (Figure 6, I–K). This provides evidence for the direct effect of Ex4 through phosphorylation of HSL enzymes and perilipin, in the process of lipolysis. With the rising incidence of obesity, there is an increase in the number of people with fatty liver disease. A fatty liver is vulnerable to ischemic insults which are encountered during shock, heart failure, liver resection, and transplantation, and manifest during the ensuing reperfusion.4Gomez D. Malik H.Z. Bonney G.K. Wong V. Toogood G.J. Lodge J.P. Prasad K.R. Steatosis predicts postoperative morbidity following hepatic resection for colorectal metastasis.Br J Surg. 2007; 94: 1395-1402Crossref PubMed Scopus (102) Google Scholar, 10Clavien P.A. Selzner M. Hepatic steatosis and transplantation.Liver Transpl. 2002; 8: 980Crossref PubMed Scopus (11) Google Scholar Thus, IRI results in extensive damage to the fatty liver, increasing morbidity in this burgeoning population and further escalating obesity-related health care costs. The effects of Ex4 are pleotropic and have been described in several organs, including the pancreas,26Ali S. Lamont B.J. Charron M.J. Drucker D.J. Dual elimination of the glucagon and GLP-1 receptors in mice reveals plasticity in the incretin axis.J Clin Invest. 2011; 121: 1917-1929Crossref PubMed Scopus (83) Google Scholar, 27Lamont B.J. Li Y. Kwan E. Brown T.J. Gaisano H. Drucker D.J. Pancreatic GLP-1 receptor activation is sufficient for incretin control of glucose metabolism in mice.J ClinI Invest. 2012; 122: 388-402Crossref PubMed Scopus (125) Google Scholar hypothalamus,28Cabou C. Vachoux C. Campistron G. Drucker D.J. Burcelin R. 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The effect of endogenously released glucose, insulin, glucagon-like peptide 1, ghrelin on cardiac output, heart rate, stroke volume, and blood pressure.Cardiovasc Ultrasound. 2011; 9: 43Crossref PubMed Scopus (20) Google Scholar and we have recently demonstrated its role in the reduction of hepatic steatosis.20Gupta N.A. Mells J. Dunham R.M. Grakoui A. Handy J. Saxena N.K. Anania F.A. Glucagon-like peptide-1 receptor is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway.Hepatology. 2010; 51: 1584-1592Crossref PubMed Scopus (347) Google Scholar In the present study, we show that Ex4 protects the liver from cell death induced during IRI, an effect that is significantly more pronounced in the fatty liver. We also show that Ex4 initiates lipolysis through perilipin and HSL, and we postulate that this is likely the reason for increased protection seen from cell death after IRI in the fatty liver. These effects make clinical applicability of Ex4 in patients with fatty livers at the time of liver surgery or organ procurement for transplantation of an exciting therapeutic option worth additional investigation. The exact mechanism for the inability of a steatotic liver to withstand ischemic insults is unclear, but it is known that t" @default.
• W2078570369 created "2016-06-24" @default.
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• W2078570369 date "2012-11-01" @default.
• W2078570369 modified "2023-10-18" @default.
• W2078570369 title "The Glucagon-Like Peptide-1 Receptor Agonist Exendin 4 Has a Protective Role in Ischemic Injury of Lean and Steatotic Liver by Inhibiting Cell Death and Stimulating Lipolysis" @default.
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