Metabolic profiling of follistatin overexpression: a novel therapeutic strategy for metabolic diseases

Rajan Singh1 Department of Obstetrics and Gynecology, UCLA School of Medicine, Los Angeles, CA, USA;2 Division of Endocrinology and Metabolism, Charles R. Drew University of Medicine and Science, Los Angeles, CA, USACorrespondence[email protected]

Shehla Pervin1 Department of Obstetrics and Gynecology, UCLA School of Medicine, Los Angeles, CA, USA;2 Division of Endocrinology and Metabolism, Charles R. Drew University of Medicine and Science, Los Angeles, CA, USA

Se-Jin Lee3 The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA;4 Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, CT, USA

Alan Kuo5 Department of Biology, California State University Dominguez Hills, CA, USA

Victor Grijalva6 Department of Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, CA, USA

John David7 Department of Comparative Medicine, Pfizer Inc, San Diego, CA, USA

Laurent Vergnes8 Department of Human Genetics, UCLA School of Medicine, Los Angeles, CA, USA

Srinivasa T Reddy1 Department of Obstetrics and Gynecology, UCLA School of Medicine, Los Angeles, CA, USA;6 Department of Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, CA, USA

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Abstract

Background

Follistatin (Fst) promotes brown adipocyte characteristics in adipose tissues.

Methods

Abdominal fat volume (CT scan), glucose clearance (GTT test), and metabolomics analysis (mass spectrometry) of adipose tissues from Fst transgenic (Fst-Tg) and wild type (WT) control mice were analyzed. Oxygen consumption (Seahorse Analyzer) and lipidomics (gas chromatography) was analyzed in 3T3-L1 cells.

Results

Fst-Tg mice show significant decrease in abdominal fat content, increased glucose clearance, improved plasma lipid profiles and significant changes in several conventional metabolites compared to the WT mice. Furthermore, overexpression of Fst in 3T3-L1 cells resulted in up regulation of key brown/beige markers and changes in lipidomics profiles.

Conclusion

Fst modulates key factors involved in promoting metabolic syndrome and could be used for therapeutic intervention.

Keywords:

Acknowledgments

The authors would like to acknowledge Metabolon Inc. (Morrisville, NC, USA) (www.metabolon.com) and UC Davis Metabolomics Core Services (http://metabolomics.ucdavis.edu/core-services/metabolomics-central-service-core) for adipose tissue metabolomics profiling and cell lysate lipidomics profiling, respectively. They would also like to thank the expert technical assistance from Melissa Braga and Dr Meher Parveen. This work was supported by National Institute of Health Grants SC1AG049682 (RS) and SC1CA1658650 (SP), and in part by RO1HL071776 (STR), R01AR060636 (SJL), and S21MD000103 (RS, SP) grants. CT scanning was done at UCLA Molecular Imaging services core.

Disclosure

The authors report no conflicts of interest in this work.

Supplementary materials

Figure S1 (A) Vector map of PiggyBac-CMV-Fst-Puro. (B) Polymerase chain reaction analysis confirming Fst insertion in the PiggyBac-CMV-Fst-Puro construct.

Abbreviation: Fst, follistatin.

Figure S2 Analysis of Mup1 expression in liver tissues from WT and Fst-Tg mice. Immunohistochemical analysis (A) and quantitative image analysis (B) of tissues sections. Integrated optical density (immunopositive area × intensity). Quantitation was performed using 20 images from 4 different areas as described previously.^1–3 Analysis of Mup1 protein (C) and gene expression (D) by Western blot and quantitative real-time polymerase chain reaction analysis using standard procedures. Data are expressed as mean ± SD. *P≤0.05; **P≤0.01.

Abbreviations: WT, wild type; Fst-Tg, follistatin transgenic; IHC, immunohistochemistry; Mup, major urinary protein; IOD, integrated optical density; GADPH, glyceraldehyde 3-phosphate dehydrogenase.

Figure S3 Real-time quantitative polymerase chain reaction analysis of C2C12 and Fst overexpressing (C2C12-Fst) myoblast cells to compare gene expression profiles of key BCAA metabolizing enzymes. Data are expressed as mean ± SD. *P≤0.05; **P≤0.01 (n=3).

Abbreviations: Fst, follistatin transgenic; BCAA, branched chain amino acid.

Figure S4 Analysis of FGF21-associated markers in BAT and liver tissues from 6-weeks-old male WT and Fst-Tg mice. (A) Western blot analysis of FGF21, PGC-1α, and IL-6 proteins. (B to C) Quantitative gene expression analysis of FGF21, its receptors (Fgfr1-3), and beta-klotho (Klb) in BAT and liver tissues from WT and Fst-Tg mice. Data are expressed as mean ± SD. *P≤0.05; **P≤0.01; ***P≤0.001 (n=3).

Abbreviations: WT, wild type; Fst-Tg, follistatin transgenic; FGF, fibroblast growth factor; PGC, peroxisome proliferator-activated receptor-gamma coactivator; IL, interleukin; GADPH, glyceraldehyde 3-phosphate dehydrogenase; BAT, brown adipose tissue.

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