Diabetes Metab J.  2019 Oct;43(5):683-699. 10.4093/dmj.2019.0112.

PF-04620110, a Potent Antidiabetic Agent, Suppresses Fatty Acid-Induced NLRP3 Inflammasome Activation in Macrophages

• Jo SI
• Bae JH
• Kim SJ
• Lee JM
• Jeong JH
• Moon JS

• KMID: 2460961
• DOI: http://doi.org/10.4093/dmj.2019.0112

Abstract

BACKGROUND
Chronic inflammation has been linked to insulin resistance and type 2 diabetes mellitus (T2DM). High-fat diet (HFD)-derived fatty acid is associated with the activation of chronic inflammation in T2DM. PF-04620110, which is currently in phase 1 clinical trials as a selective acyl-CoA:diacylglycerol acyltransferase-1 (DGAT1) inhibitor, is a potent anti-diabetic agent that may be important for the regulation of chronic inflammation in T2DM. However, the mechanisms by which PF-04620110 regulates fatty acid-induced chronic inflammation remain unclear.
METHODS
PF-04620110 was used in vitro and in vivo. DGAT1-targeting gRNAs were used for deletion of mouse DGAT1 via CRISPR ribonucleoprotein (RNP) system. The activation of NLRP3 inflammasome was measured by immunoblot or cytokine analysis in vitro and in vivo.
RESULTS
Here we show that PF-04620110 suppressed fatty acid-induced nucleotide-binding domain, leucine-rich-repeat-containing receptor (NLR), pyrin-domain-containing 3 (NLRP3) inflammasome activation in macrophages. In contrast, PF-04620110 did not change the activation of the NLR family, CARD-domain-containing 4 (NLRC4), or the absent in melanoma 2 (AIM2) inflammasomes. Moreover, PF-04620110 inhibited K⁺ efflux and the NLRP3 inflammasome complex formation, which are required for NLRP3 inflammasome activation. PF-04620110 reduced the production of interleukin 1β (IL-1β) and IL-18 and blood glucose levels in the plasma of mice fed HFD. Furthermore, genetic inhibition of DGAT1 suppressed fatty acid-induced NLRP3 inflammasome activation.
CONCLUSION
Our results suggest that PF-04620110 suppresses fatty acid-induced NLRP3 inflammasome activation.

Keyword

Diabetes mellitus, type 2; Diacylglycerol O-acyltransferase; Fatty acids; Inflammasomes; NLR family, pyrin domain-containing 3 protein; PF-04620110

MeSH Terms

Animals
Blood Glucose
Clinical Trials, Phase I as Topic
Clustered Regularly Interspaced Short Palindromic Repeats
Diabetes Mellitus, Type 2
Diacylglycerol O-Acyltransferase
Diet, High-Fat
Fatty Acids
Humans
In Vitro Techniques
Inflammasomes*
Inflammation
Insulin Resistance
Interleukin-18
Interleukins
Macrophages*
Melanoma
Mice
Plasma
Ribonucleoproteins
RNA, Guide
Blood Glucose
Diacylglycerol O-Acyltransferase
Fatty Acids
Inflammasomes
Interleukin-18
Interleukins
RNA, Guide
Ribonucleoproteins

Figure

• 

  Fig. 1 PF-04620110 suppresses fatty acid-induced nucleotide-binding domain, leucine-rich-repeat-containing receptor (NLR), pyrin-domain-containing 3 (NLRP3) inflammasome activation. (A) Quantification of interleukin 1β (IL-1β; left), IL-18 (middle), and tumor necrosis factor α (TNF-α; right) secretion from wild-type (WT) bone marrow-derived macrophages (BMDMs) were pretreated with PF-04620110 (50 µM, 2 hours) or dimethyl sulfoxide (DMSO) (control), followed by incubation with palmitate-bovine serum albumin (PA-BSA) after lipopolysaccharide (LPS) stimulation (n=10 mice per group). (B) Quantification of IL-1β secretion from WT BMDMs that were pretreated with PF-04620110 in a dose-dependent manner (12.5, 25, 50, or 100 µM, 2 hours) or DMSO (control), followed by incubation with PA-BSA after LPS stimulation (n=10 mice per group). (C) Quantification of IL-1β secretion from WT BMDMs that were pretreated with PF-04620110 (50 µM, 2 hours) or DMSO (control), followed by incubation with poly(dA:dT) or flagellin after LPS stimulation (n=10 mice per group). (D) Representative immunoblot analysis for caspase-1 and IL-1β (left), and densitometry quantification of caspase-1 p10 and IL-1β p17 levels (normalized to levels of β-actin) (right), from WT BMDMs that were pretreated with PF-04620110 (50 µM, 2 hours) or DMSO, followed by incubation with PA-BSA after LPS stimulation. For immunoblots, β-actin was used as loading control (n=6 mice per group). Data are mean±standard deviation. aP<0.001, bP<0.01, cP<0.05 by two-tailed t-test or analysis of variance.

• 

  Fig. 2 PF-04620110 inhibits K+ efflux and the formation of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks during nucleotide-binding domain, leucine-rich-repeat-containing receptor (NLR), pyrin-domain-containing 3 (NLRP3) inflammasome activation. (A) Quantification of triglyceride levels from wild-type (WT) bone marrow-derived macrophages (BMDMs) that were pretreated with PF-04620110 (50 µM, 2 hours) or dimethyl sulfoxide (DMSO; control), followed by incubation with palmitate-bovine serum albumin (PA-BSA) after lipopolysaccharide (LPS) stimulation (n=10 mice per group). (B) Quantification of triglyceride levels from WT BMDMs that were pretreated with PF-04620110 in a dose-dependent manner (12.5, 25, 50, or 100 µM, 2 hours) or DMSO (control), followed by incubation with PA-BSA after LPS stimulation (n=10 mice per group). (C) Intracellular Ca2+ flux assays from WT BMDMs that were pretreated with PF-04620110 (50 µM, 2 hours) or DMSO (control), followed by incubation with PA-BSA after LPS stimulation (n=3 mice per group). (D) Quantification of interleukin 1β (IL-1β; left), IL-18 (middle), and tumor necrosis factor α (TNF-α; right) secretion from WT BMDMs that were pretreated with PF-04620110 (50 µM, 2 hours), KCl (100 mM, 1 hour), or DMSO (control), followed by incubation with PA-BSA after LPS stimulation (n=6 mice per group). (E) Representative immunoblot analysis for caspase-1 and IL-1β (left), and densitometry quantification of caspase-1 p10 and IL-1β p17 levels (normalized to levels of β-actin) (right), from WT BMDMs that were pretreated with PF-04620110 (50 µM, 2 hours), KCl (100 mM, 1 hour), or DMSO, followed by incubation with PA-BSA after LPS stimulation. For immunoblots, β-actin was used as loading control (n=6 mice per group). (F) Representative immunofluorescence images (total 100 cells in 15 individual images per group; left), and quantification (right), of ASC speck formation (white arrows) (the percent of ASC speck positive cells for each mouse) in WT BMDMs that were pretreated with PF-04620110 (50 µM, 2 hours) or DMSO, followed by incubation with adenosine triphosphate (ATP) after LPS stimulation. Scale bars, 20 µm (n=5 mice per group). Data are mean±standard deviation. Data are representative of three independent experiments, and each was done in triplicate. aP<0.001, bP<0.01, cP<0.05 by two-tailed t-test or analysis of variance.

• 

  Fig. 3 Genetic inhibition of diacylglycerol acyltransferase-1 (DGAT1) suppresses fatty acid-induced nucleotide-binding domain, leucine-rich-repeat-containing receptor (NLR), pyrin-domain-containing 3 (NLRP3) inflammasome activation. (A) Representative immunoblot analysis for DGAT1, caspase-1, and interleukin 1β (IL-1β; left), and densitometry quantification of DGAT1, caspase-1 p10, and IL-1β p17 levels (normalized to levels of β-actin; right), from wild-type (WT) bone marrow-derived macrophages (BMDMs) were transduced with two independent Dgat1-targeting gRNAs (Dgat1 gRNA #1 and Dgat1 gRNA #2), or with a control plasmid (control), and were stimulated with lipopolysaccharide (LPS) and palmitate-bovine serum albumin (PA-BSA). For immunoblots, β-actin was used as loading control (n=5 mice per group). (B) Quantification of IL-1β (left), IL-18 (middle), and tumor necrosis factor α (TNF-α; right) secretion from WT BMDMs were transduced with two independent Dgat1-targeting gRNAs (Dgat1 gRNA #1 and Dgat1 gRNA #2), or with a control plasmid (control), and were stimulated with LPS and PA-BSA (n=10 mice per group). (C) Quantification of IL-1β and IL-18 secretion from WT BMDMs that were transduced with two independent Dgat1-targeting gRNAs (Dgat1 gRNA #1 and Dgat1 gRNA #2), or with a control plasmid (control), and were incubated with poly(dA:dT) or flagellin after LPS stimulation (n=10 mice per group). Data are mean±standard deviation. aP<0.01, bP<0.05, by two-tailed t-test or analysis of variance.

• 

  Fig. 4 Deficiency of diacylglycerol acyltransferase-1 (DGAT1) suppressed K+ efflux and the formation of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks during nucleotide-binding domain, leucine-rich-repeat-containing receptor (NLR), pyrin-domain-containing 3 (NLRP3) inflammasome activation. (A) Quantification of triglyceride levels from wild-type (WT) bone marrow-derived macrophages (BMDMs) that were transduced with Dgat1-targeting gRNAs (Dgat1 gRNA), or with a control plasmid (control), and that were stimulated with lipopolysaccharide (LPS) and palmitate-bovine serum albumin (PA-BSA) (n=10 mice per group). (B) Intracellular Ca2+ flux assays from WT BMDMs that were transduced with Dgat1-targeting gRNAs (Dgat1 gRNA), or with a control plasmid (control), and that were stimulated with PA-BSA stimulation after LPS incubation (n=3 mice per group). (C) Representative immunofluorescence images (total 100 cells in 15 individual images per group) (left), and quantification (right), of ASC speck formation (white arrows) (the percent of ASC speck positive cells for each mouse) in WT BMDMs that were transduced with Dgat1-targeting gRNAs (Dgat1 gRNA), or with a control plasmid (control), and that were stimulated with LPS and PA-BSA. Scale bars, 20 µm (n=5 mice per group). Data are mean±standard deviation. aP<0.01, by two-tailed t-test or analysis of variance.

• 

  Fig. 5 PF-04620110 suppressed high-fat diet (HFD)-induced interleukin 1β (IL-1β) and IL-18 production in mice. Quantification of (A) IL-1β (left), (B) IL-18 (right) levels in plasma from wild-type (WT) mice fed HFD or regular diet (RD) for 12 weeks, and then treated with PF-04620110 or vehicle control (dimethyl sulfoxide [DMSO]) once a day at the dose of 3 mg/kg for another 4 weeks (RD n=5, HFD n=10; RD+PF-04620110 n=5, HFD+PF-04620110 n=10). (C) Fasting and (D) fed blood glucose levels from WT mice fed HFD or RD for 12 weeks, and then treated with PF-04620110 or vehicle control (DMSO) once a day at the dose of 3 mg/kg for another 4 weeks (RD n=5, HFD n=10, HFD+PF-04620110 n=10). Data are mean±standard deviation. aP<0.01, bP<0.05, by two-tailed t-test or analysis of variance.

Reference

1. Franchi L, Eigenbrod T, Munoz-Planillo R, Nunez G. The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis. Nat Immunol. 2009; 10:241–247. PMID: 19221555.

2. Sutterwala FS, Ogura Y, Szczepanik M, Lara-Tejero M, Lichtenberger GS, Grant EP, Bertin J, Coyle AJ, Galan JE, Askenase PW, Flavell RA. Critical role for NALP3/CIAS1/cryopyrin in innate and adaptive immunity through its regulation of caspase-1. Immunity. 2006; 24:317–327. PMID: 16546100.

3. Tschopp J, Schroder K. NLRP3 inflammasome activation: the convergence of multiple signalling pathways on ROS production? Nat Rev Immunol. 2010; 10:210–215. PMID: 20168318.

4. Latz E, Xiao TS, Stutz A. Activation and regulation of the inflammasomes. Nat Rev Immunol. 2013; 13:397–411. PMID: 23702978.

5. Hotamisligil GS. Inflammation and metabolic disorders. Nature. 2006; 444:860–867. PMID: 17167474.

6. Wen H, Gris D, Lei Y, Jha S, Zhang L, Huang MT, Brickey WJ, Ting JP. Fatty acid-induced NLRP3-ASC inflammasome activation interferes with insulin signaling. Nat Immunol. 2011; 12:408–415. PMID: 21478880.

7. Youm YH, Nguyen KY, Grant RW, Goldberg EL, Bodogai M, Kim D, D'Agostino D, Planavsky N, Lupfer C, Kanneganti TD, Kang S, Horvath TL, Fahmy TM, Crawford PA, Biragyn A, Alnemri E, Dixit VD. The ketone metabolite β-hydroxybutyrate blocks NLRP3 inflammasome-mediated inflammatory disease. Nat Med. 2015; 21:263–269. PMID: 25686106.

8. Moon JS, Hisata S, Park MA, DeNicola GM, Ryter SW, Nakahira K, Choi AMK. mTORC1-induced HK1-dependent glycolysis regulates NLRP3 inflammasome activation. Cell Rep. 2015; 12:102–115. PMID: 26119735.

9. Moon JS, Nakahira K, Chung KP, DeNicola GM, Koo MJ, Pabon MA, Rooney KT, Yoon JH, Ryter SW, Stout-Delgado H, Choi AM. NOX4-dependent fatty acid oxidation promotes NLRP3 inflammasome activation in macrophages. Nat Med. 2016; 22:1002–1012. PMID: 27455510.

10. Spranger J, Kroke A, Mohlig M, Hoffmann K, Bergmann MM, Ristow M, Boeing H, Pfeiffer AF. Inflammatory cytokines and the risk to develop type 2 diabetes: results of the prospective population-based European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam Study. Diabetes. 2003; 52:812–817. PMID: 12606524.

11. Mandrup-Poulsen T. The role of interleukin-1 in the pathogenesis of IDDM. Diabetologia. 1996; 39:1005–1029. PMID: 8877284.

12. Maedler K, Sergeev P, Ris F, Oberholzer J, Joller-Jemelka HI, Spinas GA, Kaiser N, Halban PA, Donath MY. Glucose-induced beta cell production of IL-1beta contributes to glucotoxicity in human pancreatic islets. J Clin Invest. 2002; 110:851–860. PMID: 12235117.

13. Jager J, Gremeaux T, Cormont M, Le Marchand-Brustel Y, Tanti JF. Interleukin-1beta-induced insulin resistance in adipocytes through down-regulation of insulin receptor substrate-1 expression. Endocrinology. 2007; 148:241–251. PMID: 17038556.

14. Lagathu C, Yvan-Charvet L, Bastard JP, Maachi M, Quignard-Boulange A, Capeau J, Caron M. Long-term treatment with interleukin-1beta induces insulin resistance in murine and human adipocytes. Diabetologia. 2006; 49:2162–2173. PMID: 16865359.

15. Boden G. Interaction between free fatty acids and glucose metabolism. Curr Opin Clin Nutr Metab Care. 2002; 5:545–549. PMID: 12172479.

16. Dow RL, Li JC, Pence MP, Gibbs EM, LaPerle JL, Litchfield J, Piotrowski DW, Munchhof MJ, Manion TB, Zavadoski WJ, Walker GS, McPherson RK, Tapley S, Sugarman E, Guzman-Perez A, DaSilva-Jardine P. Discovery of PF-04620110, a potent, selective, and orally bioavailable inhibitor of DGAT-1. ACS Med Chem Lett. 2011; 2:407–412. PMID: 24900321.

17. Enayetallah AE, Ziemek D, Leininger MT, Randhawa R, Yang J, Manion TB, Mather DE, Zavadoski WJ, Kuhn M, Treadway JL, des Etages SA, Gibbs EM, Greene N, Steppan CM. Modeling the mechanism of action of a DGAT1 inhibitor using a causal reasoning platform. PLoS One. 2011; 6:e27009. PMID: 22073239.

18. King AJ, Segreti JA, Larson KJ, Souers AJ, Kym PR, Reilly RM, Zhao G, Mittelstadt SW, Cox BF. Diacylglycerol acyltransferase 1 inhibition lowers serum triglycerides in the Zucker fatty rat and the hyperlipidemic hamster. J Pharmacol Exp Ther. 2009; 330:526–531. PMID: 19478132.

19. Lu A, Magupalli VG, Ruan J, Yin Q, Atianand MK, Vos MR, Schroder GF, Fitzgerald KA, Wu H, Egelman EH. Unified polymerization mechanism for the assembly of ASC-dependent inflammasomes. Cell. 2014; 156:1193–1206. PMID: 24630722.

20. Yu JW, Wu J, Zhang Z, Datta P, Ibrahimi I, Taniguchi S, Sagara J, Fernandes-Alnemri T, Alnemri ES. Cryopyrin and pyrin activate caspase-1, but not NF-kappaB, via ASC oligomerization. Cell Death Differ. 2006; 13:236–249. PMID: 16037825.

21. Horwood NJ, Mahon T, McDaid JP, Campbell J, Mano H, Brennan FM, Webster D, Foxwell BM. Bruton's tyrosine kinase is required for lipopolysaccharide-induced tumor necrosis factor alpha production. J Exp Med. 2003; 197:1603–1611. PMID: 12810683.

22. Horwood NJ, Page TH, McDaid JP, Palmer CD, Campbell J, Mahon T, Brennan FM, Webster D, Foxwell BM. Bruton's tyrosine kinase is required for TLR2 and TLR4-induced TNF, but not IL-6, production. J Immunol. 2006; 176:3635–3641. PMID: 16517732.

23. Schroder K, Tschopp J. The inflammasomes. Cell. 2010; 140:821–832. PMID: 20303873.

24. Coll RC, Robertson AA, Chae JJ, Higgins SC, Munoz-Planillo R, Inserra MC, Vetter I, Dungan LS, Monks BG, Stutz A, Croker DE, Butler MS, Haneklaus M, Sutton CE, Nunez G, Latz E, Kastner DL, Mills KH, Masters SL, Schroder K, Cooper MA, O'Neill LA. A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases. Nat Med. 2015; 21:248–255. PMID: 25686105.

25. Gordon R, Albornoz EA, Christie DC, Langley MR, Kumar V, Mantovani S, Robertson AAB, Butler MS, Rowe DB, O'Neill LA, Kanthasamy AG, Schroder K, Cooper MA, Woodruff TM. Inflammasome inhibition prevents α-synuclein pathology and dopaminergic neurodegeneration in mice. Sci Transl Med. 2018; 10:eaah4066. PMID: 30381407.

26. Duncan JA, Bergstralh DT, Wang Y, Willingham SB, Ye Z, Zimmermann AG, Ting JP. Cryopyrin/NALP3 binds ATP/dATP, is an ATPase, and requires ATP binding to mediate inflammatory signaling. Proc Natl Acad Sci U S A. 2007; 104:8041–8046. PMID: 17483456.

27. Marchetti C, Swartzwelter B, Koenders MI, Azam T, Tengesdal IW, Powers N, de Graaf DM, Dinarello CA, Joosten LAB. NLRP3 inflammasome inhibitor OLT1177 suppresses joint inflammation in murine models of acute arthritis. Arthritis Res Ther. 2018; 20:169. PMID: 30075804.

28. Huang Y, Jiang H, Chen Y, Wang X, Yang Y, Tao J, Deng X, Liang G, Zhang H, Jiang W, Zhou R. Tranilast directly targets NLRP3 to treat inflammasome-driven diseases. EMBO Mol Med. 2018; 10:e8689. PMID: 29531021.

29. He H, Jiang H, Chen Y, Ye J, Wang A, Wang C, Liu Q, Liang G, Deng X, Jiang W, Zhou R. Oridonin is a covalent NLRP3 inhibitor with strong anti-inflammasome activity. Nat Commun. 2018; 9:2550. PMID: 29959312.

30. Cases S, Smith SJ, Zheng YW, Myers HM, Lear SR, Sande E, Novak S, Collins C, Welch CB, Lusis AJ, Erickson SK, Farese RV Jr. Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis. Proc Natl Acad Sci U S A. 1998; 95:13018–13023. PMID: 9789033.

31. Cao J, Zhou Y, Peng H, Huang X, Stahler S, Suri V, Qadri A, Gareski T, Jones J, Hahm S, Perreault M, McKew J, Shi M, Xu X, Tobin JF, Gimeno RE. Targeting Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) with small molecule inhibitors for the treatment of metabolic diseases. J Biol Chem. 2011; 286:41838–41851. PMID: 21990351.

32. Koliwad SK, Streeper RS, Monetti M, Cornelissen I, Chan L, Terayama K, Naylor S, Rao M, Hubbard B, Farese RV Jr. DGAT1-dependent triacylglycerol storage by macrophages protects mice from diet-induced insulin resistance and inflammation. J Clin Invest. 2010; 120:756–767. PMID: 20124729.

33. Zhou YP, Grill V. Long term exposure to fatty acids and ketones inhibits B-cell functions in human pancreatic islets of Langerhans. J Clin Endocrinol Metab. 1995; 80:1584–1590. PMID: 7745004.