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Ptrf Transgenic Mice Exhibit Obesity and Fatty Liver

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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 Qian Li1 | Lin Bai2 | Guiying Shi2 | Lianfeng Zhang2 | Yifan Dai3 | Pingsheng Liu4 | Yu-Sheng Cong1 | Miao Wang1 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- 1,2 signal transduction, lipid trafficking and cholesterol transport. 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 704 | © 2018 John Wiley & Sons Australia, Ltd wileyonlinelibrary.com/journal/cep Clin Exp Pharmacol Physiol. 2018;45:704–710. LI ET AL. | 705 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 lipodys- trophy, 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 ex- hibited obviously severe fatty degeneration of liver
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