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TWIST1 and TWIST2 Regulate Glycogen Storage and Inflammatory

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TWIST1 and TWIST2 Regulate Glycogen Storage and Inflammatory J M MUDRY and others TWIST regulation of metabolism 224:3 303–313 Research in muscle TWIST1 and TWIST2 regulate glycogen storage and inflammatory genes in skeletal muscle Correspondence 1 1 2 1,2 Jonathan M Mudry , Julie Massart , Ferenc L M Szekeres and Anna Krook should be addressed to A Krook 1Section for Integrative Physiology, Department of Molecular Medicine and Surgery, and 2Section for Integrative Email Physiology, Department of Physiology and Pharmacology, Karolinska Institutet, SE-171 77 Stockholm, Sweden [email protected] Abstract TWIST proteins are important for development of embryonic skeletal muscle and play a role Key Words in the metabolism of tumor and white adipose tissue. The impact of TWIST on metabolism " TWIST in skeletal muscle is incompletely studied. Our aim was to assess the impact of TWIST1 " metabolism and TWIST2 overexpression on glucose and lipid metabolism. In intact mouse muscle, " glycogen overexpression of Twist reduced total glycogen content without altering glucose uptake. " skeletal muscle Expression of TWIST1 or TWIST2 reduced Pdk4 mRNA, while increasing mRNA levels of Il6, Tnfa, and Il1b. Phosphorylation of AKT was increased and protein abundance of acetyl CoA carboxylase (ACC) was decreased in skeletal muscle overexpressing TWIST1 or TWIST2. Glycogen synthesis and fatty acid oxidation remained stable in C2C12 cells overexpressing TWIST1 or TWIST2. Finally, skeletal muscle mRNA levels remain unaltered in ob/ob mice, type 2 diabetic patients, or in healthy subjects before and after 3 months of exercise training. Journal of Endocrinology Collectively, our results indicate that TWIST1 and TWIST2 are expressed in skeletal muscle. Overexpression of these proteins impacts proteins in metabolic pathways and mRNA level of cytokines. However, skeletal muscle levels of TWIST transcripts are unaltered in metabolic diseases. Journal of Endocrinology (2015) 224, 303–313 Introduction The transcription factors TWIST1 and TWIST2 (also TWIST1 and TWIST2 have overlapping but not known as DERMO1) are genes belonging to the basic identical effects, with TWIST1 being the more thoroughly helix–loop–helix (bHLH) transcription factor family, investigated gene. TWIST proteins play important roles in originally described in Drosophila and conserved in tissue differentiation. In skeletal muscle, TWIST1 blocks humans (Thisse et al. 1987). TWIST1 and TWIST2 form myogenesis via inhibition of MYOD transactivation homo- or heterodimers, which together with other (Hamamori et al. 1997), which may lead to myotube bHLH family members bind to the E-box DNA sequence dedifferentiation (Hjiantoniou et al. 2008). TWIST 50-NCANNTGN-30 (Castanon et al. 2001). The resulting proteins have also been implicated in cancer as they transcriptional activity depends on post-transcriptional block p53 and Myc-dependent apoptosis (Maestro et al. modifications, partner choice, and cellular context, thus 1999). Moreover TWIST1 facilitates the appearance of complicating the characterization of modes of action of metastasis by promoting cell migration (Yang et al. 2004), the TWIST proteins (Laursen et al. 2007). and its expression level correlates with metastasis and poor http://joe.endocrinology-journals.org Ñ 2015 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JOE-14-0474 Printed in Great Britain Downloaded from Bioscientifica.com at 09/28/2021 11:24:25AM via free access Research J M MUDRY and others TWIST regulation of metabolism 224:3 304 in muscle prognosis in cancer (Hosono et al. 2007, Ou et al. 2008). Table 1 Anthropometric measurements and metabolic para- Mutations in TWIST1 are responsible for Saethre–Chotzen meters of human volunteers. Data are presented as meansGS.E.M. syndrome (OMIM 101400) characterized by craniosynos- tosis and limb abnormalities. In mice, homozygous NGT T2D deletion of the Twist1 gene results in embryonic lethality, n 10 10 while mice with a deletion of the Twist2 gene die 3 days Sex (F/M) 5/5 4/6 Age (y) 58G263G1* after birth due to cachexia with high levels of pro- Height (cm) 171G3 170G4 inflammatory cytokines and a complete absence of Weight (kg) 84.8G3.6 90.8G5.8 G G glycogen stores (Sosic et al. 2003). Waist circumference (cm) 93.2 2 106.0 4* BMI (kg/m2) 29.1G0.6 31.3G1.2 A role for TWIST proteins in metabolism has been SBP (mmHg) 133G5 140G3 proposed in adipose tissue, where TWIST1 is reported DBP (mmHg) 79G378G2 G G to regulate cytokine expression (Pettersson et al. 2010). FBG (mmol/l) 5.5 0.0 7.9 0.2** 2-h BG (mmol/l) 6.6G0.4 14.5G1.4* In adipose cells, TWIST1 silencing reduces fatty acid HbA1c (%) 4.6G0.1 5.3G0.2* oxidation and modulates pro-inflammatory cytokine Insulin (pmol/l) 51.4G12.5 92.5G16.6* G G expression, in particular IL6 (Dobrian 2012). Furthermore, HDL (mmol/l) 1.45 0.33 1.24 0.05* LDL (mmol/l) 3.24G0.18 2.74G0.28 reduced TWIST1 expression in human white adipose TG (mmol/l) 51.4G12.8 92.5G16.9 tissue has been correlated with insulin-resistance and IL6 (pg/ml) 1.54G0.37 2.23G0.51 G G obesity (Pettersson et al. 2011). TWIST1 is also a negative HOMA-IR 1.97 0.10 3.48 0.30** feed-back regulator of PGC1a/PPARd-mediated signaling NGT, normal-glucose tolerant; T2D, type 2 diabetes; SBP, systolic blood in brown fat (Pan et al. 2009). The role of TWIST proteins pressure; DBP, diastolic blood pressure; FBG, fasting blood glucose; HbA1c, in mature skeletal muscle is less well explored. Based on glycated hemoglobin; TG, triglycerides; HOMA, homeostatic model assessment of insulin resistance. **P!0.01; *P!0.05. reported effects of TWIST1 in adipose cells, we hypothesi- zed that TWIST proteins may be involved in skeletal muscle Mice glucose and lipid metabolism, as well as in the regulation of expression of skeletal muscle-derived cytokines. C57BL/6J mice (12–14 weeks old) were purchased from Charles River (Sulzfeld, Germany) and acclimatized for at least 1 week before use. Mice were housed in a humidity Materials and methods and temperature-controlled environment with 12 h Journal of Endocrinology light:12 h darkness cycle and provided ad libitum access Human subjects to water and standard rodent chow (4% fat, 16.5% Male and female volunteers with type 2 diabetes mellitus protein, 58% carbohydrates, 3.0 kcal/g purchased from (T2D) or normal glucose tolerance (NGT) were matched Lantma¨nnen, Stockholm, Sweden). Tissue collection from C C for weight and BMI. Detailed clinical characteristics of C57BL/6J-ob/ob and lean / female mice at 14–16 weeks the patients are given in Table 1. As expected, fasting and of age was performed after anesthesia using 0.02 ml/g 2 h glucose levels post oral glucose tolerance test were body weight of Avertin (2.5% solution of 99% 2,2,2- increased in T2D patients. Patients on insulin treatment and tribromo ethanol and tertiary amyl alcohol purchased with symptomatic coronary heart disease were excluded. from Sigma–Aldrich). The muscles were immediately A muscle biopsy of the vastus lateralis and anthropomor- frozen in liquid nitrogen and stored at K80 8C until RNA phic measurements were taken at enrolment in the study extraction. The study protocols were approved by the (Barres et al.2012, Kulkarni et al.2012). In a separate cohort, animal ethics committee of Stockholm north. 13 sedentary volunteers participated in an exercise training program consisting of 60 min a day, 5 days/week for 3 weeks. Plasmid construct Biopsies of the vastus lateralis muscle were obtained before the first and 48 h after the last training bout. This exercise pZsGreen1-C1 construct was purchased from Clontech. training program increased VO2 max by 13% (P!0.05; The pZsGreen1 (GFP) sequence was removed and replaced Czepluch et al.2011). The clinical parameters and detailed with Twist1 or Twist2 sequences. The inserted sequences of the exercise protocol are reported elsewhere (Czepluch were species-optimized for mice by Geneart (Regensburg, et al.2011). All studies were performed according to the Germany). The Twist1 and Twist2 DNA sequences were declaration of Helsinki, with informed consent and created by GeneArt using the mice protein sequence for approved by the ethics committee at Karolinska Institute. TWIST1 (Swiss-Prot: P26687.1) and TWIST2 (Swiss-Prot: http://joe.endocrinology-journals.org Ñ 2015 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JOE-14-0474 Printed in Great Britain Downloaded from Bioscientifica.com at 09/28/2021 11:24:25AM via free access Research J M MUDRY and others TWIST regulation of metabolism 224:3 305 in muscle Q9D030.1) respectively. The optimization process con- (between passages 9 and 12) were seeded at 2!104 cells/cm2 sisted of DNA sequence adaptation to remove internal in a six-well plate (Corning, New York, NY, USA) for 24 h and TATA-boxes, c-sites and ribosomal entry sites, AT-rich then transfected with 2 mg Twist1 or Twist2 DNA construct or GC-rich sequence stretches, RNA instability motifs, (1 mg/ml) and 6 ml/ml of FuGENE HD (Promega) or FuGENE repeat sequences and RNA secondary structures, cryptic HD only (as control) in DMEM containing 10% FBS but splice donor, and acceptor sites in higher eukaryotes. without antibiotic or antimycotic additions for 24 h. Each condition was performed in triplicate. Gene transfer by electroporation in intact skeletal muscle and metabolic analysis mRNA extraction and analysis Male C57BL/6 mice (12–14 weeks old) were purchased from mRNA was extracted from 10 mg of skeletal muscle tissue Charles River and acclimatized for at least 1 week before homogenized in 1 ml of TRIzol reagent (Invitrogen) use. Mice were housed in a humidity- and temperature- and was purified according to the manufacturer’s rec- controlled environment with 12 h light:12 h darkness ommendations.
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