Ptrf Transgenic Mice Exhibit Obesity and Fatty Liver

Received: 26 April 2017 | Revised: 16 January 2018 | Accepted: 18 January 2018 DOI: 10.1111/1440-1681.12920

ORIGINAL ARTICLE

Ptrf transgenic mice exhibit obesity and fatty liver

1Institute of Aging Research, Hangzhou Normal University School of Medicine, Summary Hangzhou, China Polymerase I and transcript release factor (Ptrf, also known as Cavin1) is an essential 2 Key Laboratory of Human Disease component in the biogenesis and function of caveolae. Ptrf knockout mice or patients Comparative Medicine of the Ministry of Health, Institute of Laboratory Animal with PTRF mutations exhibit numerous pathologies including markedly aberrant fuel Science, Chinese Academy of Medical metabolism, lipodystrophy and muscular dystrophy. In this study, we generated Ptrf Sciences and Comparative Medical Center, Peking Union Medical College, transgenic mice to explore its function in vivo. Compared with wild-type (WT) mice, Beijing, China we found that the Ptrf transgenic mice showed obesity with an increased level of ALT 3Center of Metabolic Disease Research, (alanine aminotransferase) and AST (aspartate transaminase). Ptrf transgenic mice Nanjing Medical University, Nanjing, China 4National Laboratory of Biomacromolecules, exhibited severe fat degeneration and a higher degree of fat accumulation in the liver Institute of Biophysics, Chinese Academy of compared with WT mice. Consistently, we found that the expression of the fat syn- Sciences, Beijing, China thesis gene, Fasn, was increased in the liver of Ptrf transgenic mice. Thus, Ptrf trans- Correspondence genic mice would be a good model for investigating the molecular mechanism and Miao Wang, Institute of Aging Research, Hangzhou Normal University School of therapeutic targets of obesity and fatty liver associated diseases. Medicine, Hangzhou, China. Email: [email protected] KEYWORDS and Fasn, fatty liver, obesity, Ptrf Lin Bai, Key Laboratory of Human Disease Comparative Medicine of the Ministry of Health, Chinese Academy of Medical Sciences and Comparative Medical Center, Peking Union Medical College, Beijing, China. Email: [email protected]

Funding information National Natural Science Foundation of China, Grant/Award Number: 31171320, 31201038, 31672374

1 | INTRODUCTION formation. CAV1 is a marker protein for caveolae organelles and plays an important role in caveolae function. Cavins are another group of Caveolae are specialized invaginations of the plasma membrane that proteins that have been documented recently as being essential ca- 3 have been found in many cell types and are most abundant in adi- veolar components. There are 4 cavins which are recruited to caveo- pocytes, endothelial and smooth muscle cells. Caveolae play funda- lae. CAVIN1 was first described as polymerase I transcription release mental roles in a variety of cellular processes, including endocytosis, factor (PTRF), which enhances ribosomal RNA synthesis by dissoci- signal transduction, lipid trafficking and cholesterol transport.1,2 ating the ternary complex of RNA polymerase I, and this function is 4,5 CAV1 was the first protein discovered that is required for caveolae independent of caveolae. PTRF was recently demonstrated to be an essential component of caveolae.6,7 CAVIN2 was discovered as a 8 Qian Li and Lin Bai contributed equally to this work. serum deprivation response gene (SDPR), and CAVIN3 was named

SRBC for the SDR-related gene that binds to PKC.9 CAVIN4 is also 2 | RESULTS known as muscle-related coiled-coil protein (MURC) and is found only 2.1 | Generation and identification of Ptrf in cardiac and skeletal muscle cells.10 Cavins act as regulators of cave- transgenic mice olin function and organization, and each has been assigned different roles based on caveolae morphology and cell type.11-13 The molecular To investigate the physiological role of Ptrf in vivo, we generated mechanisms of the cavin proteins in the regulation of the formation, Ptrf transgenic mice in a C57BL/6 J genetic background (Figure 1A). structure, and function of caveolae remain elusive. Four Ptrf transgenic founders were genotyped by PCR (Figure 1B). In vitro, Ptrf knockdown decreased the release of glycerol in re- The expression levels of PTRF in the control or transgenic mice were sponse to the beta-2-agonist isoproterenol, and overexpression of characterized by western blot (Figure 1C,D) in the heart, liver and Ptrf increased glycerol release.14 Ptrf knockout mice had no morpho- lung. Then, the expression pattern of Ptrf in the transgenic mice was logically detectable caveolae in any cell type and exhibited higher further confirmed by immunohistochemistry (Figure 1E). These data circulating triglyceride levels, significantly reduced adipose tissue demonstrated that Ptrf was stably overexpressed in transgenic mice mass, glucose intolerance, and hyperinsulinaemia.15 Ptrf knockout compared to the control mice. mice present a mildly fatty liver on a normal diet, and the level of liver fat is not exacerbated by high fat feeding.16,17 Human PTRF mu- 2.2 | Ptrf transgenic mice display obesity tations have been associated with congenital generalized lipodystrophy, insulin resistance and dyslipidemia.18-20 These observations Ptrf knockout mice were previously reported to have lower body suggest that PTRF has an important role in lipogenesis. weights than WT littermates after 5 months of age and to also dis- In this report, we generated Ptrf transgenic mice to study the play a leaner body mass than wild-type animals.15,21 We examined effect of Ptrf overexpression in vivo. We showed that Ptrf transgenic the body weight of Ptrf transgenic mice compared with control mice are obese with an increased level of ALT and AST and a higher mice from 1 to 13 months of age. We found that Ptrf transgenic degree of fat accumulation in the liver compared with WT mice. mice were more obese than WT mice beginning at 6 months of age

FIGURE 1 Generation of Ptrf transgenic mice and detection of Ptrf expression in mice tissues. A, The construct was generated by inserting the murine Ptrf cDNA into a vector with a CMV promoter. The transgenic mice were created by the microinjection method. B, The genotype of the Ptrf transgenic founders was identified by PCR analysis. C, Total protein from the heart, liver and lung of wild-type (WT) and transgenic mice (Ptrf) was examined by western blotting with anti-PTRF and anti-β -actin antibodies. D, Quantitative expression of PTRF in the indicated tissues. The experiment was repeated 3 times, and the results represent the mean ± SD. *P < .05; **P < .01. E, Immunohistochemical analysis of PTRF in the liver and heart tissues from Ptrf transgenic mice and wild-type mice 706 | LI et al.

FIGURE 2 The phenotype of Ptrf transgenic mice. A, Photos of Ptrf transgenic mice and control mice. B, Body weight of Ptrf transgenic mice and control mice at the indicated times. C, Magnetic resonance imaging was performed at 11 months of age Ptrf transgenic mice and wild-type mice (n = 3) to measure total body fat. D, The volume of total body fat quantified by software ImageJ. E, H&E staining of the adipose tissue from Ptrf transgenic mice and control mice at the indicated times

(Figure 2A). No significant difference in body weight was found and NEFA (Figure 3I) were increased in the Ptrf transgenic mice between the control and Ptrf transgenic mice before 3 months of compared with control mice. Because Ptrf null mice have impaired age, while a significant difference in body weight became apparent whole body glucose tolerance compared to WT mice,15 we also beginning at 6 months of age (Figure 2B). The distribution of fat in measured the serum glucose level at 15, 30, 45, 60, 75, 90, 105 the control and Ptrf transgenic mice was then examined by MRI. The and 120 minutes after D-glucose administration. We found that results showed that the interscapular and axillary WAT, inguinal the Ptrf transgenic mice had normal glucose tolerance compared WAT, perigonadal WAT and retroperitoneal WAT were increased with WT mice. Thus, we proposed that the Ptrf transgenic mice in the Ptrf transgenic mice compared with WT mice (Figure 2C,D). will be a good model for the obesity but not diabetes. We detected the adipocytes morphology by the hematoxylin and eosin (H&E) staining. The adipocytes of Ptrf transgenic mice were 2.4 | Pathological changes in the liver of Ptrf obviously larger than the control mice at 13 months of age, but transgenic mice there was no significant difference at 2 months of age between Ptrf transgenic and control mice (Figure 2E). In the knockout mice, Alanine aminotransferase (ALT) and aspartate transaminase (AST) the epididymal, subcutaneous, and perirenal white fat depot are sensitive and widely used liver enzymes. The increased levels weights were reduced by 60%-70%.21 This reduced adiposity in of ALT and AST suggest that Ptrf transgenic mice may have liver Ptrf knockout mice is in contrast to that in Ptrf transgenic mice. injury. To assess any pathological changes, we analyzed the livers These observations further confirmed a role of Ptrf in adipocyte from 8-month-old control and transgenic mice by H&E staining. lipid storage and release. Compared with WT mice, the liver of the Ptrf transgenic mice exhibited obviously severe fatty degeneration of liver cells (Figure 4A). By using Oil Red O staining, we found Ptrf transgenic mice exhibited 2.3 | Ptrf transgenic mice have increased levels of a higher degree of fat accumulation in the liver (Figure 4B). We next ALT and AST examined the expression levels of genes associated with fat syn- Given the obesity observed in Ptrf transgenic mice and lipodys- thesis and transport in the liver. The levels of Acc (acetyl CoA car- trophy in Ptrf null mice and in humans with a PTRF mutation, we boxylase), Cd36 and Scd1 were not significantly different (Figure 4C) then measured a number of metabolic parameters associated with between the two groups of mice; however, the expression level of metabolism in the blood serum at 4, 8 and 20 months from Ptrf Fasn was markedly increased in the liver of Ptrf transgenic mice transgenic mice and WT mice. The results showed that the level compared with WT mice (Figure 4C). Fasn is a key regulator of fat of ALT and AST was increased in the Ptrf transgenic mice com- synthesis, and down-regulated expression of Fasn is associated with pared with control mice (Figure 3A,B), but no difference in the lev- reduced fatty liver. Therefore, the up-regulation of Fasn may lead to els of CHO, TBIL, HDL-C, and LDL-C between the Ptrf transgenic an accumulation of fat in Ptrf transgenic mice, thereby inducing the mice and control mice were observed (Figure 3C-F). The level of fatty liver phenotype. Further work will investigate the regulatory GLU was decreased (Figure 3G), and the levels of TG (Figure 3H) mechanism of Fasn expression by Ptrf or caveolae. LI et al. | 707

FIGURE 3 Serum biochemical examination of Ptrf transgenic mice. A-I, Serum alanine aminotransferase (ALT), glutamic oxalacetic transaminase (AST), total cholesterol (CHO), total bilirubin (TBIL), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), blood glucose (GLU), triglyceride (TG) and free fatty acid (NEFA) were measured in Ptrf transgenic mice and wild-type mice (n = 8) as described in Experimental Procedures. The results are presented as the means ± SE. The statistical significance of differences was determined by the Student’s t-test (*P < .05, **P < .01. J, 2 months of age Ptrf transgenic mice and WT mice (n = 5 for each group) were administered with D-glucose, and serum glucose levels were determined at the indicated times after glucose administration 708 | LI et al.

FIGURE 4 Pathological changes in the liver of Ptrf transgenic mice. A, H&E staining and (B) Oil Red O Staining of liver tissue from 4 months of age Ptrf transgenic mice and control mice. C, RT-PCR analysis of the liver associated gene expression in Ptrf transgenic mice and control mice. Gapdh expression was used for normalization. The experiment was repeated three times, and the results represent the mean ± SD. *P < .05; **P < .01

3 | DISCUSSION into triacylglycerol and phosphatidylserine.22,23 Liver function in Cav1 knockout mice appears to be protective, and these mice show In this report, we generated Ptrf transgenic mice and characterized reduced levels of steatosis.24 Ptrf knockout mice had mildly fatty their body weight, fat distribution and metabolic parameters com- liver on a normal diet, and the level of liver fat was not exacerbated pared with WT controls. We found that Ptrf overexpression in vivo by high fat feeding. In contrast, we found that the liver of the Ptrf induced obesity, fatty degeneration and a higher degree of fat ac- transgenic mice exhibited severe fatty liver compared with WT mice. cumulation in the liver compared to control mice. Consistent with this observation, we found that Ptrf transgenic mice Accumulating evidence suggests an evolving role of obesity in met- exhibited a higher degree of fat accumulation in the liver, suggesting abolic syndrome and metabolic disorders. The liver is a central organ that overexpression of Ptrf in vivo induced the accumulation of liver in lipogenesis, gluconeogenesis and cholesterol metabolism. Fatty liver fat. disease is defined by fat accumulation in the liver cells, which uncon- Fasn, a key regulator of carbohydrate and lipid metabolism, was trolled can lead to parenchymal fibrosis and cell death. An initial step found to associate transiently with lipid raft membranes following al- in liver damage is a simple blood test to determine the presence of terations in signal transduction within the Src, Akt and Egfr pathways. certain liver enzymes in the blood. Under normal conditions, the en- In human and murine prostate cancer cells, endogenous Fasn forms a zymes reside within the cells of the liver, but when the liver is injured, complex with Cav1, suggesting that colocalization of Fasn and Cav1 enzymes including AST and ALT are released into the blood. is dependent on activation of upstream signalling mediators.25,26 Caveolae play a particular role in fatty acid metabolism. Caveolar Interestingly, our results showed that the expression level of Fasn was proteins are able to bind and sequester fatty acids and convert them markedly increased in the liver of Ptrf transgenic mice compared with LI et al. | 709

WT mice. Therefore, we hypothesize that Ptrf may be involved in the 4.3 | Western blot interaction of Fasn with Cav1, and thereby regulates the Src, Akt and Egfr signalling pathways. Our data show that Ptrf transgenic mice ex- Cell lysates were prepared in RIPA buffer (50 mmol/L Tris–Cl, pH hibit obesity and fatty liver. Overexpression of Ptrf in the liver may 8.0, 100 mmol/L NaCl, 0.1% SDS, 0.5% sodium deoxycholate and 1% increase the interaction between Fasn and Cav1, thereby regulating NP-40) containing a protease inhibitor cocktail (Roche Diagnostics, the Src, Akt and Egfr signalling pathways. Future investigation of me- Indianapolis, IN, USA). Total protein concentrations were measured tabolisms and signalling pathways using the PTRF transgenic mice and using a BCA kit, and immunoblotting was performed by running PTRF knockout mice would provide insights into the role and regula- clarified cell extracts through 8% SDS–polyacrylamide gels. The tion of PTRF in lipodystrophy and its consequences. Ptrf transgenic proteins were transferred to NC membranes (Millipore, Darmstadt, mice may represent a good model of obesity and fatty liver for investi- Germany) and incubated at 4°C overnight with an anti-PTRF anti- gating the physiological processes, molecular mechanisms and thera- body. An HRP-conjugated anti-rabbit secondary antibody was used peutic targets of obesity and fatty liver associated diseases. for detection using a chemiluminescent detection system (Invitrogen Life Technologies).

4 | METHODS 4.4 | Serum biochemistry 4.1 | Animals Whole blood was centrifuged at 3000 g for 10 minutes at 4°C to The full-length mouse Ptrf cDNA (GeneID: 19285) was cloned by PCR. obtain the serum for measurements of total alanine aminotrans- The transcript was then confirmed by sequencing analysis. The PCR ferase (ALT), aspartate transaminase (AST), cholesterol (CHO), product was subcloned into the pCDNA3.1 vector using the EcoR I total bilirubin (TBIL), high density lipoprotein (HDL), low density site. The Ptrf transgenic mice were generated using the microinjec- lipoprotein (LDL), glucose (Glu), triglycerides (TG) and nonesteri- tion method. Genotyping of the Ptrf transgenic mice was performed fied fatty acid (NEFA) detection using a HITACHI 7100 Automatic using PCR with the primers 5′-GTCAATGGGTGGAGTATTTACG-3′ Analyzer. and 5′-GCTTATATAGACCTCCCACCGT-3′, Tm = 60°C, 30 cycles. The Ptrf transgenic mice were maintained in a C57BL/6J genetic 4.5 | Histological analysis background. The mice were maintained in a pathogen-free environment Heart and liver were fixed in 4% formaldehyde and mounted in par- and fed a standard diet. The use of the animals was approved by affin blocks. The sections were stained with H&E and analyzed using the Animal Care and Use Committee of the Institute of Laboratory the Aperio Image Scope v8.2.5 software. Animal Science at the Peking Union Medical College.

4.6 | Oil Red O staining 4.2 | RT-PCR Frozen sections of liver were stained with Oil Red O. Sections were Total RNA was extracted from the cells using TRIzol reagent (Invitrogen fixed in 10% (vol/vol) formalin, rinsed with PBS and 60% (vol/vol) Life Technologies, Carlsbad, CA, USA) according to the manufactur- isopropanol, incubated with Oil Red O in 60% isopropanol, and then er’s instructions. The genes of interest were amplified from DNase rinsed in PBS. I-treated total RNA using M-MLV Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) and poly dT primers. The primers used 4.7 | MRI for PCR were as follows: Ptrf (5′-TGCCTGAGAAGGAGGGTGAC-3′ and 5′-GTGTGCCGTGTCTTCTCCAG-3′, Tm = 60°C, 26 cycles), Magnetic resonance imaging was performed on 11-month-old mice. Cav1 (5′- GACCCCAAGCATCTCAACGAC-3′ and 5′- GGATCGCA The mice were anesthetized with 2% isoflurane, then placed in a coil GAAGGTATGGACG-3′, Tm = 62°C, 26 cycles), Acc (5′-CGAGCAGCCC with a pneumatic pillow for respiration monitoring and maintained ATTCTCATCTATATC-3′ and 5′-ACTGTGTGTGCTCGTGGTTCAGCT at 1.5%-2% isoflurane guided by respiratory rate and heart rate. We C-3′, Tm = 63°C, 30 cycles), Fasn (5′-TACTTTGTGGCCTTCTCCTCTG used a Varian 7 T MRI system with a 160-mm vertical bore. The TAA-3′ and 5′-CTTCCACACCCATGAGCGAGTCCAGGCCGA-3′, Tm = body temperature was maintained at approximately 37°C with hot 62°C, 30 cycles), Cd36 (5′-GATGACGTGGCAAAGAACAG-3′ and 5′- wind. AAAGGAGGCTGCGTCTGTG-3′, Tm = 56°C, 30 cycles), Scd1 ( 5 ′ - GCC AGACCGGGCTGAACACC-3′ and 5′-GGCCTCCCAAGTGCAGC 4.8 | Statistics AGG-3′, Tm = 65°C, 30 cycles) and Gapdh (5′-GAGCGAGACCCC ACTAACAT-3′ and 5′-TTCACACCCATCACAAACAT-3′, Tm = 60°C, The data were analyzed using Microsoft Excel and GraphPad Prism 25 cycles). Real-time PCR using SYBR Premix Ex Taq II (Takara, software. Differences were considered to be significant at P < .05 Dalian, China) was carried out using the ABI StepOne detection sys- as analyzed using the Student’s t-test. The error bars represent the tem (Applied Biosystems). standard deviation in all of the figures. 710 | LI et al.

ACKNOWLEDEGMENTS lipolysis via a phosphorylation-dependent mechanism. Diabetes. 2011;60:757‐765. This work was supported by grants from the National Natural 15. Liu L, Brown D, McKee M, et al. Deletion of Cavin/PTRF causes Science Foundation of China (31201038, 31171320, 31672374). global loss of caveolae, dyslipidemia, and glucose intolerance. Cell Metab. 2008;8:310‐317. 16. Karbalaei MS, Rippe C, Albinsson S, et al. Impaired contractility and ORCID detrusor hypertrophy in cavin-1-deficient mice. Eur J Pharmacol. 2012;689:179‐185. Yu-Sheng Cong http://orcid.org/0000-0002-1307-379X 17. Perez-Diaz S, Johnson LA, DeKroon RM, et al. Polymerase I and transcript release factor (PTRF) regulates adipocyte differen- tiation and determines adipose tissue expandability. FASEB J. 2014;28:3769‐3779. REFERENCES 18. Patni N, Garg A. Congenital generalized lipodystrophies– 1. Parton RG, Simons K. The multiple faces of caveolae. Nat Rev Mol new insights into metabolic dysfunction. Nat Rev Endocrinol. Cell Biol. 2007;8:185‐194. 2015;11:522‐534. 2. Cheng JP, Nichols BJ. Caveolae: one function or many? Trends Cell 19. Jelani M, Ahmed S, Almramhi MM, et al. Novel nonsense mutation Biol. 2016;26:177‐189. in the PTRF gene underlies congenital generalized lipodystrophy in 3. Nassar ZD, Parat MO. Cavin family: new players in the biology of a consanguineous Saudi family. Eur J Med Genet. 2015;58:216‐221. caveolae. Int Rev Cell Mol Biol. 2015;320:235‐305. 20. Low JY, Nicholson HD. Emerging role of polymerase-1 and tran- 4. Jansa P, Burek C, Sander EE, Grummt I. The transcript release factor script release factor (PTRF/ Cavin-1) in health and disease. Cell PTRF augments ribosomal gene transcription by facilitating reinitia- Tissue Res. 2014;357:505‐513. tion of RNA polymerase I. Nucleic Acids Res. 2001;29:423‐429. 21. Ding SY, Lee MJ, Summer R, Liu L, Fried SK, Pilch PF. Pleiotropic 5. Jansa P, Grummt I. Mechanism of transcription termination: PTRF effects of cavin-1 deficiency on lipid metabolism. J Biol Chem. interacts with the largest subunit of RNA polymerase I and disso- 2014;289:8473‐8483. ciates paused transcription complexes from yeast and mouse. Mol 22. Ortegren U, Yin L, Ost A, Karlsson H, Nystrom FH, Stralfors P. Gen Genet. 1999;262:508‐514. Separation and characterization of caveolae subclasses in the 6. Hill MM, Bastiani M, Luetterforst R, et al. PTRF-Cavin, a conserved plasma membrane of primary adipocytes; segregation of specific cytoplasmic protein required for caveola formation and function. proteins and functions. FEBS J. 2006;273:3381‐3392. Cell. 2008;132:113‐124. 23. Ost A, Ortegren U, Gustavsson J, Nystrom FH, Stralfors P. 7. Liu L, Pilch PF. A critical role of cavin (polymerase I and transcript Triacylglycerol is synthesized in a specific subclass of caveolae in release factor) in caveolae formation and organization. J Biol Chem. primary adipocytes. J Biol Chem. 2005;280:5‐8. 2008;283:4314‐4322. 24. Razani B, Combs TP, Wang XB, et al. Caveolin-1-deficient 8. Hansen CG, Bright NA, Howard G, Nichols BJ. SDPR induces mem- mice are lean, resistant to diet-induced obesity, and show hy- brane curvature and functions in the formation of caveolae. Nat Cell pertriglyceridemia with adipocyte abnormalities. J Biol Chem. Biol. 2009;11:807‐814. 2002;277:8635‐8647. 9. McMahon KA, Zajicek H, Li WP, et al. SRBC/cavin-3 is a cave- 25. Pandey V, Vijayakumar MV, Ajay AK, Malvi P, Bhat MK. Diet- olin adapter protein that regulates caveolae function. EMBO J. induced obesity increases melanoma progression: involvement of 2009;28:1001‐1015. Cav-1 and FASN. Int J Cancer. 2012;130:497‐508. 10. Bastiani M, Liu L, Hill MM, et al. MURC/Cavin-4 and cavin fam- 26. Di Vizio D, Adam RM, Kim J, et al. Caveolin-1 interacts with a ily members form tissue-specific caveolar complexes. J Cell Biol. lipid raft-associated population of fatty acid synthase. Cell Cycle. 2009;185:1259‐1273. 2008;7:2257‐2267. 11. Chidlow JH Jr, Sessa WC. Caveolae, caveolins, and cavins: complex control of cellular signalling and inflammation. Cardiovasc Res. 2010;86:219‐225. How to cite this article: Li Q, Bai L, Shi G, et al. Ptrf transgenic 12. Hansen CG, Nichols BJ. Exploring the caves: cavins, caveolins and mice exhibit obesity and fatty liver. Clin Exp Pharmacol Physiol. caveolae. Trends Cell Biol. 2010;20:177‐186. 13. Briand N, Dugail I, Le Lay S. Cavin proteins: new players in the cav- 2018;45:704–710. https://doi.org/10.1111/1440-1681.12920 eolae field. Biochimie. 2011;93:71‐77. 14. Aboulaich N, Chui PC, Asara JM, Flier JS, Maratos-Flier E. Polymerase I and transcript release factor regulates