Hepatic PLIN5 signals via SIRT1 to promote autophagy and prevent inflammation during fasting

Abstract

Lipid droplets (LDs) are energy-storage organelles that are coated with hundreds of proteins, including members of the perilipin (PLIN) family. PLIN5 is highly expressed in oxidative tissues, including the liver, and is thought to play a key role in uncoupling LD accumulation from lipotoxicity; however, the mechanisms behind this action are incompletely defined. We investigated the role of hepatic PLIN5 in inflammation and lipotoxicity in a murine model under both fasting and refeeding conditions and in hepatocyte cultures. PLIN5 ablation with antisense oligonucleotides triggered a pro-inflammatory response in livers from mice only under fasting conditions. Similarly, PLIN5 mitigated lipopolysaccharide- or palmitic acid-induced inflammatory responses in hepatocytes. During fasting, PLIN5 was also required for the induction of autophagy, which contributed to its anti-inflammatory effects. The ability of PLIN5 to promote autophagy and prevent inflammation were dependent upon signaling through sirtuin 1 (SIRT1), which is known to be activated in response to nuclear PLIN5 under fasting conditions. Taken together, these data show that PLIN5 signals via SIRT1 to promote autophagy and prevent FA-induced inflammation as a means to maintain hepatocyte homeostasis during periods of fasting and FA mobilization. Lipid droplets (LDs) are energy-storage organelles that are coated with hundreds of proteins, including members of the perilipin (PLIN) family. PLIN5 is highly expressed in oxidative tissues, including the liver, and is thought to play a key role in uncoupling LD accumulation from lipotoxicity; however, the mechanisms behind this action are incompletely defined. We investigated the role of hepatic PLIN5 in inflammation and lipotoxicity in a murine model under both fasting and refeeding conditions and in hepatocyte cultures. PLIN5 ablation with antisense oligonucleotides triggered a pro-inflammatory response in livers from mice only under fasting conditions. Similarly, PLIN5 mitigated lipopolysaccharide- or palmitic acid-induced inflammatory responses in hepatocytes. During fasting, PLIN5 was also required for the induction of autophagy, which contributed to its anti-inflammatory effects. The ability of PLIN5 to promote autophagy and prevent inflammation were dependent upon signaling through sirtuin 1 (SIRT1), which is known to be activated in response to nuclear PLIN5 under fasting conditions. Taken together, these data show that PLIN5 signals via SIRT1 to promote autophagy and prevent FA-induced inflammation as a means to maintain hepatocyte homeostasis during periods of fasting and FA mobilization. Intracellular lipid droplets (LDs) are energy-storage organelles composed of a core of neutral lipids surrounded by a phospholipid monolayer embedded with hundreds of proteins. LD accumulation in non-adipose tissue is tightly linked to numerous metabolic diseases, including type 2 diabetes (1Greenberg A.S. Coleman R.A. Kraemer F.B. McManaman J.L. Obin M.S. Puri V. Yan Q.W. Miyoshi H. Mashek D.G. The role of lipid droplets in metabolic disease in rodents and humans.J. Clin. Invest. 2011; 121: 2102-2110Crossref PubMed Scopus (456) Google Scholar), and is the defining characteristic of nonalcoholic fatty liver disease (NAFLD) (2Mashek D.G. Khan S.A. Sathyanarayan A. Ploeger J.M. Franklin M.P. Hepatic lipid droplet biology: getting to the root of fatty liver.Hepatology. 2015; 62: 964-967Crossref PubMed Scopus (80) Google Scholar). While LDs themselves are often considered inert, a growing body of literature shows that dysregulation of lipid metabolic pathways leading to the accumulation of intermediates in triacylglycerol (TAG) metabolism can trigger lipotoxicity and impair cellular homeostasis. In addition, the presence of specific proteins on the surface of LDs can couple/uncouple LD accumulation and cellular dysfunction. Perilipins (PLINs) are a family of constitutive LD proteins comprised of five members. PLIN2 is the most abundant isoform in the liver, and its expression is tightly correlated with LD abundance and NAFLD (3Fujii H. Ikura Y. Arimoto J. Sugioka K. Iezzoni J.C. Park S.H. Naruko T. Itabe H. Kawada N. Caldwell S.H. et al.Expression of perilipin and adipophilin in nonalcoholic fatty liver disease; relevance to oxidative injury and hepatocyte ballooning.J. Atheroscler. Thromb. 2009; 16: 893-901Crossref PubMed Scopus (69) Google Scholar). Ablation of PLIN2 reduces hepatic LD accumulation and prevents NAFLD and related complications, suggesting a role for PLIN2 in linking LDs to cellular dysfunction (4Najt C.P. Senthivinayagam S. Aljazi M.B. Fader K.A. Olenic S.D. Brock J.R. Lydic T.A. Jones A.D. Atshaves B.P. Liver-specific loss of perilipin 2 alleviates diet-induced hepatic steatosis, inflammation, and fibrosis.Am. J. Physiol. Gastrointest. Liver Physiol. 2016; 310: G726-G738Crossref PubMed Scopus (70) Google Scholar, 5Imai Y. Varela G.M. Jackson M.B. Graham M.J. Crooke R.M. Ahima R.S. Reduction of hepatosteatosis and lipid levels by an adipose differentiation-related protein antisense oligonucleotide.Gastroenterology. 2007; 132: 1947-1954Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). In contrast, the presence of PLIN5 on LDs can uncouple LD accumulation from metabolic dysregulation. PLIN5 overexpression in liver, muscle, or heart promotes LD accumulation but prevents insulin resistance (6Trevino M.B. Mazur-Hart D. Machida Y. King T. Nadler J. Galkina E.V. Poddar A. Dutta S. Imai Y. Liver perilipin 5 expression worsens hepatosteatosis but not insulin resistance in high fat-fed mice.Mol. Endocrinol. 2015; 29: 1414-1425Crossref PubMed Scopus (28) Google Scholar, 7Pollak N.M. Schweiger M. Jaeger D. Kolb D. Kumari M. Schreiber R. Kolleritsch S. Markolin P. Grabner G.F. Heier C. et al.Cardiac-specific overexpression of perilipin 5 provokes severe cardiac steatosis via the formation of a lipolytic barrier.J. Lipid Res. 2013; 54: 1092-1102Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar); whereas, liver-specific ablation of PLIN5 causes insulin resistance despite reduced LD accumulation (8Keenan S.N. Meex R.C. Lo J.C.Y. Ryan A. Nie S. Montgomery M.K. Watt M.J. Perilipin 5 deletion in hepatocytes remodels lipid metabolism and causes hepatic insulin resistance in mice.Diabetes. 2019; 68: 543-555Crossref PubMed Scopus (37) Google Scholar). Both PLIN2 and PLIN5 affect LD metabolism, at least in part, through their inhibition of cytosolic lipases (9Sztalryd C. Brasaemle D.L. The perilipin family of lipid droplet proteins: gatekeepers of intracellular lipolysis.Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2017; 1862: 1221-1232Crossref PubMed Scopus (258) Google Scholar), which may explain why ablation of either PLIN2 or PLIN5 lowers hepatic LD accumulation. However, PLIN5 also serves a critical role in signaling through sirtuin 1 (SIRT1), a protein deacylase that influences a myriad of cellular functions. Specifically, following PKA-mediated phosphorylation, PLIN5 translocates to the nucleus where it interacts with and promotes the activity of SIRT1 and the transcriptional complex of PGC1-α/PPAR-α to increase the expression of downstream target genes governing mitochondrial biogenesis and oxidative metabolism (10Gallardo-Montejano V.I. Saxena G. Kusminski C.M. Yang C. McAfee J.L. Hahner L. Hoch K. Dubinsky W. Narkar V.A. Bickel P.E. Nuclear perilipin 5 integrates lipid droplet lipolysis with PGC-1α/SIRT1-dependent transcriptional regulation of mitochondrial function.Nat. Commun. 2016; 7: 12723Crossref PubMed Scopus (82) Google Scholar). This increase in mitochondrial number and function may be a key factor in uncoupling LD accumulation from insulin resistance and cellular dysfunction. Hepatic inflammation is associated with the majority of acute and chronic liver diseases. Lipid accumulation in response to high fat feeding leads to subacute hepatic inflammation via NF-κB activation with increased downstream cytokines such as TNF-α, interleukin (IL)-6, and IL-1β (11Del Campo J.A. Gallego P. Grande L. Role of inflammatory response in liver diseases: therapeutic strategies.World J. Hepatol. 2018; 10: 1-7Crossref PubMed Scopus (183) Google Scholar). It is recognized that high levels of FAs, especially saturated FAs such as palmitate, induce lipotoxicity and metabolic dysfunction and are major contributors to hepatic inflammation (12Malhi H. Gores G.J. Molecular mechanisms of lipotoxicity in nonalcoholic fatty liver disease.Semin. Liver Dis. 2008; 28: 360-369Crossref PubMed Scopus (427) Google Scholar). The increased inflammatory signaling is both a hallmark and driver of more advanced liver diseases, including nonalcoholic steatohepatitis. Autophagy is a catabolic pathway involving the degradation of cellular components during nutrient deprivation. Since the initial discovery that autophagy can modulate LD catabolism, in a process termed lipophagy (13Singh R. Kaushik S. Wang Y. Xiang Y. Novak I. Komatsu M. Tanaka K. Cuervo A.M. Czaja M.J. Autophagy regulates lipid metabolism.Nature. 2009; 458: 1131-1135Crossref PubMed Scopus (2659) Google Scholar), a rapidly growing body of literature has further defined this pathway and highlighted the importance of autophagy in the development of numerous diseases, including NAFLD. It is well-known that autophagy is suppressed in NAFLD, and numerous studies utilizing genetic approaches or dietary phytochemicals to promote autophagy have shown beneficial effects on the prevention or treatment of NAFLD in animal models (14Khambu B. Yan S. Huda N. Liu G. Yin X.M. Autophagy in non-alcoholic fatty liver disease and alcoholic liver disease.Liver Res. 2018; 2: 112-119Crossref PubMed Scopus (50) Google Scholar, 15Lin C.W. Zhang H. Li M. Xiong X. Chen X. Chen X. Dong X.C. Yin X.M. Pharmacological promotion of autophagy alleviates steatosis and injury in alcoholic and non-alcoholic fatty liver conditions in mice.J. Hepatol. 2013; 58: 993-999Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). Because alterations in LD dynamics, autophagy, and inflammation are key components of NAFLD, we sought to further investigate the mechanism through which PLIN5 regulates these factors under normal feeding conditions. Herein, we report that PLIN5 promotes fasting-induced autophagy and prevents FA-induced inflammation as a means to uncouple LD accumulation from metabolic dysfunction. Eight-week-old male C57BL/6N mice were purchased from Charles River Laboratories and housed under controlled temperature and lighting (20–22°C; 10:14 h light-dark cycle) and with free access to water. All mice were maintained at the University of Minnesota Animal Facilities in accordance with the Institutional Animal Care Guidelines and all experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Minnesota. All mice were fed with the control purified diet (TD.94045; Harlan Teklad, Madison, WI). Control and PLIN5 antisense oligonucleotides (ASOs; Ionis Pharmaceuticals) were given via intraperitoneal injection twice per week at a dose of 40 mg/kg. After 3 weeks, all mice were euthanized for liver tissue and serum collection after overnight fasting or overnight fasting followed by 4 h refeeding. Where noted, leupeptin (40 mg/kg) was administered through intraperitoneal injection 4 h prior to euthanization to inhibit autophagy. All cells were maintained in a humidified incubator at 37°C, 5% CO2. AML-12 cells were obtained from the ATCC (Manassas, VA). Cells were grown in DMEM:F12 (1:1) supplemented with 10% FBS and ITS (10 μg/ml insulin, 5.5 μg/ml transferrin, 5 ng/ml selenium). Mouse primary hepatocytes were isolated by collagenase perfusion method from 10- to 12-week-old male C57BL/6 mice with free access to water and chow diet. Hepatocytes were plated on collagen-coated multi-well tissue culture plates for 4 h with M199 plating medium that 10% insulin, and M199 medium 5.5 and primary hepatocytes were with either control or PLIN5 by with the for h in or was by the and at the University of Minnesota. The from was as the for the of the and were for the with AML-12 cells were for h followed by to the PLIN5 was with from liver followed by with expression was and a and an were the all expression was to protein was on all to were with and protein were by After proteins were to a and with primary and The were and with a were with which was by and for all are shown and is from Liver were in water and twice with The was under and in were to liver AML-12 cells were with palmitic to or overnight and with followed by with and for Liver tissue was with and embedded in The tissue was for and liver tissue was for of was for AML-12 cells were with from T. H. Y. S. T. Y. A. T. T. N. that an 2016; Full Text Full Text PDF PubMed Scopus Google for h with to the After cells were with followed by AML-12 cells were at 1 cells per in plates the All were with to the Cells were with of expression the of murine NF-κB and after were the and to All were in was from liver tissue the purchased from of from 4 treatment were to the University of Minnesota for and were a The was 1 with a of or to were to of was purified and The was and to and of was and and via were and to a were to a and were by on the the was on the and of an and and was for of were by and were the per were with the 3 from the control on data for were with was via the as was via for and for expressed genes were the in The was on the of a and genes were to for pathway that a 1 and an were to The is in was are expressed as were or where was considered the of hepatic PLIN5 in metabolism, we to PLIN5 expression in with PLIN5 for 3 reduced the expression of PLIN5 to levels and fasting increased PLIN5 expression PLIN5 ablation on body or liver during the 3 week treatment PLIN5 reduced adipose PLIN5 but in of the adipose were fasting increased hepatic LD accumulation and with refeeding as and PLIN5 mice reduced abundance of hepatic LDs and in both and conditions with reduced LD PLIN5 ablation reduced and overexpression increased FA LD suggesting that these alterations in FA may to the hepatic in mice PLIN5 in the effects of PLIN5 under the feeding we of liver of the data was to the key pathways in response to PLIN5 We a of genes in the in the mice administered PLIN5 with the majority of these In contrast, PLIN5 ablation under fasting conditions the expression of with and the genes as a of PLIN5 only were under both feeding conditions that the were control and PLIN5 mice under fasting conditions further was on the effects of PLIN5 during of the that was a increase of pro-inflammatory in PLIN5 livers with control with pathways hepatic were in response to PLIN5 during fasting further the of PLIN5 in inflammation we inflammatory in the liver including TNF-α, and which were not in the in the of All of the were in livers following PLIN5 the was increased with PLIN5 suggesting increased hepatic of isolated primary hepatocytes with reduced PLIN5 which is with PLIN5 an anti-inflammatory role In with the studies in PLIN5 in primary hepatocytes increased the pro-inflammatory response to as by increased expression of TNF-α, and in cells with PLIN5 In contrast, PLIN5 overexpression inflammatory expression these data show that PLIN5 or fasting-induced While autophagy was not a pathway that was to the we that autophagy genes expression in response to PLIN5 were the further this we expression of protein of autophagy were Ablation of PLIN5 reduced the of and all of which are with reduced autophagy the effects of PLIN5 on we administered leupeptin to mice 4 h prior to the abundance of and as but PLIN5 this increase In addition, PLIN5 ablation reduced autophagy in hepatocytes as by the We PLIN5 to its expression was to autophagy. PLIN5 overexpression increased and reduced and treatment with leupeptin that these increased Taken together, these data show for the that PLIN5 a key role in fasting-induced autophagy. that liver-specific PLIN5 promote acute inflammatory responses and inhibit autophagy we these signals were we primary hepatocytes with and of autophagy and We that treatment for both and h accumulation and reduced the inhibition of autophagy However, h treatment with not the expression of known inflammation suggesting that PLIN5 suppressed autophagy prior to further the autophagy and inflammation we control and AML-12 cells with for h We that overexpression of PLIN5 increased levels of protein of autophagy and the in autophagy following as shown by the and we NF-κB and expression of inflammatory genes in response an autophagy in primary hepatocytes treatment with either or NF-κB which was further and were PLIN5 increased and PLIN5 overexpression reduced activity in response to or but the effects of PLIN5 were with the of both the ability of PLIN5 overexpression to expression of pro-inflammatory genes was in the of suggesting that the induction of autophagy may to the anti-inflammatory effects of PLIN5 It is that saturated FAs, especially promote inflammation and are of lipotoxicity Lipid signaling and lipotoxicity in for metabolic disease and Lipid Res. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). that PLIN5 inflammation during fasting, FA levels are we investigated PLIN5 expression was of to or of PLIN5 increased expression of TNF-α, and in primary hepatocytes as However, a more increase in inflammatory expression PLIN5 was with these overexpression of PLIN5 the increase in inflammatory expression in response to in AML-12 cells data that the presence of which is increased upon fasting, is critical to FA-induced The effects of on autophagy in the liver are with studies that it either promotes or autophagy N. X. Y. K. S. Chen X. Yang H. Y. L. Autophagy in PubMed Scopus Google Scholar, S. H. T. R. Y. S. Y. T. Y. N. et autophagy and hepatocyte and lipid accumulation in nonalcoholic fatty liver disease in 2016; PubMed Scopus Google Scholar). of primary hepatocytes to reduced however, PLIN5 overexpression was to these effects of PLIN5 on accumulation in response to PLIN5 the effects of on in AML-12 by abundance and protein was by as but PLIN5 overexpression these effects the presence of PLIN5 the effects of on autophagy inflammation, and SIRT1 is recognized as a major of autophagy through its of proteins in autophagy as a of factors that autophagy L. R. Liu J. M. T. role for the in the regulation of 2008; PubMed Scopus Google Scholar). PLIN5 interacts with SIRT1 under conditions such as fasting that promote signaling to increase SIRT1 activity (10Gallardo-Montejano V.I. Saxena G. Kusminski C.M. Yang C. McAfee J.L. Hahner L. Hoch K. Dubinsky W. Narkar V.A. Bickel P.E. Nuclear perilipin 5 integrates lipid droplet lipolysis with PGC-1α/SIRT1-dependent transcriptional regulation of mitochondrial function.Nat. Commun. 2016; 7: 12723Crossref PubMed Scopus (82) Google Scholar). investigate the PLIN5 and we primary hepatocytes with an to PLIN5 and cells with a specific SIRT1 we an increase in the and with PLIN5 of increased however, treatment with these we SIRT1 to the role of SIRT1 on autophagy PLIN5 overexpression in increased and the and but these effects were not in SIRT1 cells the of SIRT1 in the inhibition of inflammation, we the NF-κB in and SIRT1 with PLIN5 overexpression or inflammation in but not in SIRT1 cells Because SIRT1 to its we the of to control PLIN5 treatment increased in mice with reduced signaling via SIRT1 these data show that PLIN5 signals via SIRT1 to promote autophagy and prevent inflammation fasting and PKA-mediated phosphorylation, PLIN5 translocates to the nucleus where it signals via SIRT1 to transcriptional known to autophagy and a fasting-induced autophagy is and inflammation is PLIN5 to play an role in the response to inflammatory signals to maintain studies the function and of PLIN5 in the liver under dietary conditions. The most effects of PLIN5 ablation were in response to fasting, with only in expression upon refeeding. data are with the of PLIN5 expression during fasting and increased PLIN5 in response to such as fasting, that promote signaling P.E. proteins, an family of lipid droplet proteins that cellular lipid Biophys. 2009; PubMed Scopus Google Scholar). We also that PLIN5 ablation reduced LD accumulation in both fed and data are in with studies that PLIN5 lipolysis C. Y. X. Li L. Y. Liu Zhang L. J. P. Zhang X. et 5 hepatic lipotoxicity by 2015; PubMed Scopus Google via and inhibition of H. M. H. Liu J. K. C. T. Coleman R. et regulation of adipose by perilipin a lipid Biol. 2011; J. Biol. Full Text Full Text PDF PubMed Scopus Google and promotes FA cellular (8Keenan S.N. Meex R.C. Lo J.C.Y. Ryan A. Nie S. Montgomery M.K. Watt M.J. Perilipin 5 deletion in hepatocytes remodels lipid metabolism and causes hepatic insulin resistance in mice.Diabetes. 2019; 68: 543-555Crossref PubMed Scopus (37) Google Scholar, M.B. Machida Y. A. Dutta S. Y. Imai Y. Perilipin 5 regulates lipid metabolism and insulin in a of its role in the insulin 2015; PubMed Scopus (28) Google Scholar). PLIN5 ablation in a of inflammation in livers from PLIN5 ablation is to increase insulin resistance and mitochondrial which often with inflammation (8Keenan S.N. Meex R.C. Lo J.C.Y. Ryan A. Nie S. Montgomery M.K. Watt M.J. Perilipin 5 deletion in hepatocytes remodels lipid metabolism and causes hepatic insulin resistance in mice.Diabetes. 2019; 68: 543-555Crossref PubMed Scopus (37) Google Scholar, R. M. A. G.M. N. Watt M.J. PLIN5 deletion remodels intracellular lipid and causes insulin resistance in PubMed Scopus Google Scholar, M.B. Mazur-Hart D. Machida Y. King T. Nadler J. Galkina E.V. Poddar A. Dutta S. Imai Y. Liver perilipin 5 expression worsens hepatosteatosis but not insulin resistance in high fat-fed mice.Mol. Endocrinol. 2015; 29: 1414-1425Crossref PubMed Scopus Google Scholar). In addition, downstream signaling of such as and have anti-inflammatory effects A. T. J. K. A. NF-κB and SIRT1 in the regulation of inflammation and metabolic 2013; PubMed Scopus Google Scholar). data show that PLIN5 regulates inflammation under the most effects are in response to inflammatory such as or The ability of PLIN5 to inflammation in response to also that the increase in fasting-induced inflammation in livers may be to an to to the of FAs or the signaling required to metabolism of the PLIN5 expression was also suppressed following suggesting that its may be a critical to for inflammatory major of the is that PLIN5 suppressed fasting-induced autophagy. While this is the to a PLIN5 and autophagy, that PLIN5 to the regulation of autophagy. PLIN5 and in response to its to the nucleus to promote SIRT1 activity leading to increased signaling and mitochondrial biogenesis (10Gallardo-Montejano V.I. Saxena G. Kusminski C.M. Yang C. McAfee J.L. Hahner L. Hoch K. Dubinsky W. Narkar V.A. Bickel P.E. Nuclear perilipin 5 integrates lipid droplet lipolysis with PGC-1α/SIRT1-dependent transcriptional regulation of mitochondrial function.Nat. Commun. 2016; 7: 12723Crossref PubMed Scopus (82) Google Scholar). However, SIRT1 is also to be a of autophagy L. R. Liu J. M. T. role for the in the regulation of 2008; PubMed Scopus Google and is required for fasting-induced autophagy in the liver Y. M. Yan H. Y. Zhang A. Liu et hepatic steatosis and in mice by autophagy and factor J. 2018; PubMed Scopus Google Scholar). these studies are together, a PLIN5 and autophagy the not only the critical role of PLIN5 on fasting-induced autophagy but also shows that these effects are dependent upon as either or genetic inhibition of SIRT1 the effects of with a role for autophagy as a of inflammation X. Wang Y. Li H. J. J. X. Y. H. autophagy NF-κB signaling to inflammatory response to Dis. 2019; 10: PubMed Scopus Google Scholar), we also that autophagy is required for the anti-inflammatory effects of PLIN5 in response to or the regulation of both autophagy and inflammation to play to the effects of In these data a critical role for PLIN5 in fasting-induced autophagy and inflammation in the SIRT1 as a key downstream of PLIN5 that PLIN5 to alterations in both autophagy and inflammatory data the mechanism the beneficial effects of PLIN5 and further for that the signaling as a means to prevent or by lipid metabolism and metabolic dysfunction. The to the University of Minnesota and from the University of Minnesota for and the and at the University of Minnesota for with the with antisense interleukin lipid droplet nonalcoholic fatty liver disease palmitic perilipin sirtuin 1 triacylglycerol

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