Page 1 of 41
Diabetes
Prmt7 deficiency causes reduced skeletal muscle oxidative metabolism and age-related obesity Hyeon-Ju Jeong1*, Hye-Jin Lee1*, Tuan Anh Vuong1, Kyu-Sil Choi3, Dahee Choi4, Sung-Hoi Koo4, Sung Chun Cho5, Hana Cho2, Jong-Sun Kang1#
1
Department of Molecular Cell Biology, 2Department of Physiology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suwon, 440-746, 3 Samsung Biomedical Research Institute, Samsung medical center, Seoul 135-710, 4Division of Life Science, Korea University, Seoul 136-713, 5Well Aging Research Center, Samsung Advanced Institute of Technology, Samsung Electronics Co. Ltd, Suwon, 446-712, Korea
Running title: Prmt7 regulates skeletal muscle oxidative metabolism
* These authors have contributed equally to this work
# Corresponding author: Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, 2066, Seobu-Ro, Jangan-gu, Suwon, Gyunggi-do, Korea 440-746, Phone: +82-31-299-6135, Fax: +82-31-299-6157. E-mail address:
[email protected]
The word count : 4,400 Number of tables and figures : 2tables and 8figures.
Diabetes Publish Ahead of Print, published online April 26, 2016
Diabetes
Abstract
Maintenance of skeletal muscle function is critical for metabolic health and the disruption of
which exacerbates many chronic diseases such as obesity and diabetes. Skeletal muscle responds to exercise or metabolic demands by a fiber-type switch regulated by signalingtranscription networks that remains to be fully defined. Here, we report that protein arginine
methyltransferase 7 (Prmt7) is a key regulator for skeletal muscle oxidative metabolism. Prmt7 is expressed with the highest levels in skeletal muscle and decreased in skeletal muscles with age or obesity. Prmt7-/- muscles exhibit decreased oxidative metabolism with decreased expression of genes involved in muscle oxidative metabolism, including PGC-1α. Consistently, Prmt7-/- mice exhibited significantly reduced endurance exercise capacities. Furthermore, Prmt7-/- mice exhibit decreased energy expenditure, which might contribute to the exacerbated age-related obesity of Prmt7-/- mice. Similarly to Prmt7-/- muscles, Prmt7 depletion in myoblasts also reduces PGC-1α expression and PGC-1α-promoter driven reporter activities. Prmt7 regulates PGC-1α expression through interaction with and activation of p38MAPK which in turn activates ATF2, an upstream transcriptional activator for PGC-1α. Taken together, Prmt7 is a novel regulator for muscle oxidative metabolism via activation of p38MAPK/ATF2/PGC-1α.
Introduction Skeletal muscle aging (called sarcopenia) exhibits decreased muscle mass and exercise capacities accompanied by increased fatigability and muscle atrophy leading to a reduced quality of life (1-3). Skeletal muscle exhibits a remarkable plasticity in energy metabolism and contractile functions in response to various stimuli like exercise, hormones or nutritional states (4). Defects in the oxidative metabolism and function of skeletal muscle have been implicated in numerous metabolic pathologies, including insulin resistance, glucose intolerance, and obesity (5-8). Skeletal muscle adaptation toward oxidative metabolism promoted by stimuli like endurance exercise involves multiple signaling pathways, including CAMKs, AMPKs and p38MAPK which induce the expression and activation of a key transcriptional coactivator PGC-1α (9-13). PGC-1α is a critical regulator for mitochondrial function and oxidative muscle metabolism (9; 14; 15). Consistently, mice
Page 2 of 41
Page 3 of 41
Diabetes
lacking PGC-1α exhibit declined muscle oxidative metabolism and exercise capacity (16; 17). Furthermore, PGC-1α expression in skeletal muscle is reduced in obesity, diabetes or denervation associated with the fiber type switch toward glycolytic fibers (15; 18-21). PGC1α is preferentially expressed in oxidative fibers, and its ectopic expression in skeletal muscle drives oxidative fiber formation, at least in part through MEF2 interactions thereby directly activating oxidative fiber-specific gene expression (22). Among signaling pathways, p38MAPK (p38) plays a critical role for metabolic adaptations of skeletal muscle induced by exercise-triggered muscle contraction which is mediated through regulation of PGC-1α expression and activity. p38 can phosphorylate the transcription factors, such as ATF2 and MEF2c to stimulate the transcription of PGC-1α gene (23-25). In addition, p38 can also derepress PGC-1α by phosphorylation and in turn PGC-1α can autoregulate via interaction with MEF2c (24; 26). The ectopic activation of p38 by expression of a constitutive active form of MKK6 (MKK6EE) enhanced oxidative metabolism by the upregulation of PGC-1α and mitochondrial proteins in skeletal muscles (24). ATF2 binds to the CRE (cAMP response element) sequence in the PGC-1α promoter region in addition to CREB transcription factors to stimulate gene expression (23; 24). Recent studies suggest the crosstalk and redundancy of signaling pathways aforementioned in regulation of fiber type switch and oxidative metabolism in response to exercise. Even though PGC-1α was long believed to be indispensible for the muscle adaptation in response to exercise (27; 28), mice lacking PGC1α still showed the exercised-induced fiber type switch with the reduced expression of mitochondrial genes, suggesting that PGC-1α is specifically required for mitochondrial function involved in muscle adaptation (29). These studies suggest that the signalingtranscription network promoting skeletal muscle adaptation induced by various stimuli including exercise remains to be fully defined. Protein arginine methyltransferases (Prmts) catalyze symmetric or asymmetric dimethylations of arginine residues on both histone and non-histone substrates linked to gene regulation in diverse biological processes including glucose metabolism and myoblast differentiation (30; 31). Based on dimethylation characteristics, Prmts can be classified as either the type I catalyzing asymmetric arginine dimethylation (Prmt1, Prmt2, Prmt3, Prmt4, Prmt6, Prmt8) or the type 2 subfamily generating symmetric dimethyl-arginine residues (Prmt5 and Prmt7) (30; 32). Among these, Prmt1, 4 and 5 are relatively well studied and known to play critical roles in various biological processes. Recent studies have shown that
Diabetes
Prmt1 and Prmt5 are involved in regulation of hepatic gluconeogenesis through inhibition of FOXO (33) or through activation of CREB (34). In addition, Prmt1 has been shown to methylate PGC-1α thereby stimulates the nuclear receptor-mediated expression of mitochondrial genes in non-muscle cells (35). Prmt4 and Prmt5 have been implicated in promotion of myoblast differentiation through interaction with Mef2c or MyoD, respectively (36; 37). Recent studies with muscle stem cell-specific knockout mouse models have suggested that Prmt4 and Prmt5 play critical roles in control of muscle stem cell functions during muscle regeneration (38; 39). In this study, we characterized the role of Prmt7 in skeletal muscle metabolism by using Prmt7 deficient mouse models. Prmt7 is highly expressed in skeletal muscle and its expression declined in skeletal muscles with age and obesity. Interestingly, Prmt7 deficiency results in reduced oxidative metabolism and diminished endurance exercise capacity. Furthermore, Prmt7-deficient mice exhibit agerelated obesity with excessive body fat accumulation and hyperglycemia. This function of Prmt7 appears to be partly mediated through regulation of PGC-1α expression by interaction with and activation of p38. Taken together, our data supports that Prmt7 is important for muscle function maintenance thereby contributing to the balanced body metabolism.
Experimental Procedures
Animal studies Male and female Prmt7 mice were purchased from Sanger Institute. All animal experiments were approved by the Institutional Animal Care and Research Advisory Committee at Sungkyunkwan University School of Medicine Laboratory Animal Research Center. Mice were backcrossed onto C57BL/6J background for at least 6 generations and maintained in C57BL6/J background and littermate wildtype controls were used for comparison with Prmt7-/- mice in all experiments. To assess the age-related effect of Prmt7 deficiency on obesity, Prmt7+/+ or Prmt7-/- mice (n=20-25) were used. The blood glucose levels were measured after fasting for 16 hours with free water. For the glucose and pyruvate tolerance test, mice were fasted for 16 hours prior to intraperitoneal injection with 1.5g/kg body weight of 20% D-(+) glucose or 20% pyruvate (Sigma), followed by measurement of blood glucose levels. For the insulin tolerance test, mice were fasted for 5 hours and injected intraperitoneally with 0.5 IU/kg body weight of insulin (Sigma), followed by measurement of blood glucose levels.
Page 4 of 41
Page 5 of 41
Diabetes
For assessment of metabolic parameters, five month-old Prmt7+/+ and Prmt7-/- mice were analyzed with metabolic cages (Harvard Apparatus: Panlab). Measurements were performed for 48 hours, during which animals had a free access to food and water. To assess muscle endurance capacity, Prmt7+/+ and Prmt7-/- mice (n =10 per group) were tested for the treadmill running and grip strength. For the treadmill running, a Columbus exer-6M treadmill was used. Prior to exercise, mice were accustomed to the treadmill with 5minute run at 7m/min once per day for 5 days. The exercise test was performed on a 10% incline for 8m min-1 for 20 minutes, followed by increased toward 9m min-1 until exhaustion. The grip strength of forelimb was measured by a grip strength meter (Bioseb). All grip strength readings (measured in g) were normalized to body weight. The exercise was assessed every 15 minutes for total 1 hour.
Measurement of metabolite and lipids Mice tissues and whole blood were collected at the end of experiments. To assess metabolite content, blood serum was separated and plasma insulin was measured by mouse insulin elisa kits (U-type, Shibayagi Corp.). Triglyceride and nonesterified fatty acid (NEFA) in blood serum were measured by colorimetric assay kits (Wako). To measure the hepatic triglyceride content, frozen livers were digested in ethanolic KOH at 55oC for overnight followed by the extraction of organic layer with 50% ethanol and centrifugation. After adding 1µM MgCl2, the collected organic layer was used for analysis of the triglyceride content. The blood lactate levels were measured with Prmt7+/+ and Prmt7-/- mice (n=9) before or after a single bout of treadmill running with 10m/min for 2 hours by using a Lactate Pro2 kit (Arkray).
Cryosections, histology and immunostaining analysis Liver were processed through a fixation with 4% paraformaldehyde (PFA) and sucrose series followed by cryo-embedding and sectioning with 10µm thickness on cryostat microtome (Leica). Freshly dissected muscles were snap-frozen in OCT and sectioned with 7µm thickness. NADH dehydrogenase activity was determined by incubation for 30 minutes with 0.9 mM NADH and 1.5 mM Nitro blue tetrazolium (Sigma) in 3.5mM phosphate buffer (pH7.4). Succinate dehydrogenase activity was determined by incubation for 1 hour with 50uM sodium succinate and 0.3mM Nitro blue tetrazolium in 114 mM phosphate buffer containing K-EGTA (Sigma). For Myh immunostaining, muscle sections were fixed,
Diabetes
permeabilized and processed for incubation with primary antibodies against MyhI, MyhIIa and MyhIIb (DSHB) and secondary antibodies. Images were captured under Nikon ECLIPS TE-2000U and NIS-Elements F software (Nikon). Myofibers were traced and their area was measured using Image J software. For hematoxylin and eosin staining, cryosections were processed for staining with Mayer's hematoxylin and eosin (BBC Biomedical). For Periodic Acid Schiff staining, liver sections were fixed with Carnoy's fixative for 10 minutes, followed by incubation with 0.5% periodic acid solution (Sigma) for 10 minutes and counterstaining with Mayer's hematoxylin. For oil-red O staining, liver sections were fixed and washed with 60% isoprophanol, followed by staining with oil-red O working solution.
Electron microscopy and in vivo microCT imaging For electron microscopy (EM), TA muscle samples were fixed overnight with 2.5% glutaraldehyde/4% PFA solution at 4 °C. After incubation for 1 hour in 1% OsO4, the specimens were dehydrated in ethanol series, passed through propylene oxide, and embedded in epoxy resin (Epok). Ultrathin sections (70 nm) were collected on 200 mesh nickel grids and stained for 20 minutes in 2% uranyl acetate and reynolds lead citrate. The specimens were observed with a Hitachi HT7700 electron microscope at 100 kV. EM was performed by the Research Electron Microscopy Core at Samsung Biomedical Institute. Micro-computed tomography was performed on a preclinical scanner (Inveon Preclinical CT, Siemens Healthcare) at 200 µm slice thickness, with exposure time of 500 msec, photon energy of 60 keV and current of 400 µA. The projection images were reconstructed into three-dimensional image with IRW software. Total abdominal fat volumes including subcutaneous and visceral fat were using Siemens Inveon Software. RNA, Protein analysis and Mitochondrial DNA Contents Quantitative RT-PCR analysis was carried out as described previously (40), tissues were homogenized by FastPrepR-24 (MP Biomedicals) and extracted with Easy-spin total RNA extract kit (iNtRON). All data are normalized to expression of a ribosomal gene L32. The primer sequences are shown in Supplementary Table 1. Western blot analysis was performed as previously described (41; 42). Briefly, cells were lyzed in cell extraction buffer (10mM Tris-HCl, pH8.0, 150mM NaCl, 1mM EDTA, 1% Triton X-100) containing complete protease inhibitor cocktail (Roche), followed by SDSPAGE and incubation with primary and secondary antibodies. Tissue extracts were prepared
Page 6 of 41
Page 7 of 41
Diabetes
by cryo-pulverization with liquid nitrogen and lyzed in cell extraction buffer. Primary antibodies used are Prmt7, ATF2, pATF2, Hsp90 (Santa Cruz), CREB, pCREB, p38MAPK, pp38MAPK, pFOXO1, GAPDH, Prmt5, Prmt1 (Cell signaling), pFoxo1 (Cell signaling), PGC-1α, Prmt1, SYM10 (Millipore), β-tubulin (Zymed), HA (AbFrontier). Immunoprecipitation were performed as described previously (43). Briefly, precleared cell extracts were incubated with primary antibodies overnight at 4oC, followed by incubation with protein G-agarose beads (Roche) for 1 hour and washing three times with cell extraction buffer. Precipitates were analyzed by western blotting. For mitochondrial DNA contents, total DNA was extracted from TA muscles, hearts or BATs using a DNeasy Blood and Tissue kit (QIAGEN). The amount of mitochondrial DNA was quantified by the ratio of Mtco2 to β-actin by quantitative PCR.
Cell culture, constructs, luciferase assay and Chromatin immunoprecipitation (ChIP) C2C12 myoblasts (44), 293T and 10T1/2 cells were cultured as previously described (41). For the generation of stable cell lines, C2C12 cells were transfected with pSuper or pSuper/Prmt7 shRNA and selected with 1µg/ml puromycin followed by pooling the colonies and analyzed. 5 different small hairpin RNAs (shRNAs) against Prmt7 as listed in Supplementary Table 1 were tested and screened for the knockdown effectiveness in P19 embryonal carcinoma cells (Supplementary Fig. 7). Prmt7-shRNA1 and 2 are cloned into pSuper-puro vector based on the reproducibility and utilized interchangeably. Luciferase assays were performed as previously described (45). All experiments were carried out as triplicates and repeated at least three times. Constructs used in this study are as following: pCMV6-Prmt7 (Origene), CMV-βgalactosidase (33), CRE-Luc (46), PGC-1α-Luc (Addgene). Chip assay was carried out as previously described (46). To generate HA-tagged Prmt7 construct, Prmt7 was subcloned into a pCDNA3.1-HA vector (Clonetech). Antibodies used for ChIP included Rabbit IgG (Millipore), Prmt7 (GeneTex) and ATF2 (Cell signaling). The primers used to amplify the PGC-1α promoter region between -260 to -54 are listed in Supplementary Table 1. All primer sequences are listed in Supplementary Table 1.
Oxygen consumption Analysis Oxygen probe analysis was performed as previously described (47). C2C12/pSuper and C2C12/ shPrmt7 were induced to differentiate for 2 day and incubated with Mito-ID O2
Diabetes
Probe kit (Enzo) for 4 hours followed by washing and analysis by using a fluorescent plate reader in 100 µL of DMEM without serum or phenol red. To induce or inhibit ATP production, cells were preincubated for 30 minutes in serum-free DMEM containing 1µM FCCP (Sigma) or 1µM antimycin A (Sigma) and covered in mineral oil, followed by analysis using a fluorescent plate reader.
Statistical Analyses Values are expressed as means ± SD or ± SEM, as indicated in the figure legends. Statistical significance was calculated using paired or unpaired two-tailed Student's t test. Differences were considered statistically significant at or under values of P
