Over-expression of Follistatin-like 3 attenuates fat accumulation and improves insulin sensitivity in mice☆
Introduction
It is becoming increasingly evident that not only endurance exercise but also resistance exercise confers protection against metabolic disorders, including type 2 diabetes and cardiovascular diseases [1]. Resistance training improves insulin sensitivity and elevates the basal metabolic rate through muscle hypertrophy [1], [2]. During muscle contractions, skeletal muscles release a number of proteins that via the circulation may affect metabolism in other organs [3]. One such muscle-derived protein is follistatin like 3 (fstl3) that is expressed [4] and secreted from skeletal muscle [5]. During resistance exercise, fstl3 expression is elevated both in plasma and locally in the muscle [4], [6], [7].
The major identified role of extracellular fstl3 is formation of high-affinity complexes with members of the TGF-β super-family, thereby inhibiting the functions of the latter [8], [9]. In the blood, fstl3 is primarily bound to myostatin [10], and plasma fstl3 levels correlate with plasma myostatin levels in young men [7]. Thus, the systemic role of fstl3 is believed to be inhibition of circulating myostatin. However, biochemical studies have also shown that fstl3 forms complexes with activin A with almost equal affinity as with myostatin [9], [11]. Yet, no in vivo data exist on fstl3 binding to activin A.
Global knockout of myostatin or pharmacological blockade of the myostatin receptor, ActRIIB, protects against high-fat (HF) diet-induced obesity [12], [13], [14]. In continuation, increased plasma and muscle myostatin levels are associated with obesity and insulin resistance in humans [15], [16], [17]. Similar to myostatin, circulating activin A levels are associated with insulin resistance in humans [18] and loss of activin A in mice also confers resistance to HF-induced weight gain and improvements in insulin sensitivity in mice [19].
Interestingly, when relieving the blockade of myostatin and activin A through fstl3 knock-down (KO), no effect could be shown on body weight or muscle mass, but it improved glucose tolerance and insulin tolerance, while at the same time suppressed glucose levels for at least 120 min post insulin injection [20]. The fstl3 KO mice had increased beta cell mass, a reduction in alpha cell number and a redistribution of fat from the visceral adipose compartment to the liver [20]. As fstl3 mainly functions through blockade of myostatin and activin A, we hypothesized that fstl3 over-expression through systemic blockade of these would protect against HF diet-induced obesity and insulin resistance.
Therefore, we investigated the metabolic effects of systemic fstl3 over-expression using DNA electrotransfer in mouse hindlimb muscle.
Section snippets
Mice
For all the studies, 8–10 week old female C57BL/C mice, bred in-house (Copenhagen University Hospital Herlev, Denmark) were used. Breeding pairs were bought from Harlan (the Netherlands). All animal experiments were conducted in accordance with the recommendations of the European Convention for the Protection of Vertebrate Animals used for Experimentation and upon permission from the Danish Animal Experiments Inspectorate. Mice were kept at 23 °C at a 12:12 h light–dark cycle with free access to
Systemically elevated fstl3 lowers central adiposity
We generated an fstl3 over-expression mouse model (fstl3) by DNA electrotransfer of an fstl3 encoding plasmid into muscles of the hind legs. This resulted in a 2.8-fold increase in serum fstl3 one week after transfection (vector: 4677 ± 2614 pg/ml; fstl3: 13016 ± 5209 pg/ml, P < 0.001, n = 10). After 12 weeks, serum fstl3 remained significantly elevated (vector: 3985 ± 782 pg/ml; fstl3: 5997 ± 970 pg/ml, P < 0.01, n = 9).
HF feeding of both vector and fstl3 transfected mice led to a higher body weight due to
Discussion
In line with our hypothesis, we observed that systemically increased fstl3 levels in mice decreased fat gain and improved insulin sensitivity in response to high-fat feeding without changes in body weight and muscle mass. In contrast, fstl3 over-expression did not improve glucose tolerance, which could be explained by altered pancreatic insulin/glucagon ratio, enhanced hepatic glucagon sensitivity and glucose output. These adaptations helped maintain blood glucose levels during prolonged
Author Contributions
Concept and design of the study (CB, BKP, PH), acquisition and analysis of data (CB, RHH, JBH, CHO, PG, JG, PH), drafting the article (CB, PH), critically revision of the manuscript (CB, TMP, BKP). All authors approved the final version of the manuscript.
Funding
The Centre of Inflammation and Metabolism (CIM) is supported by a grant from the Danish National Research Foundation (DNRF55). The Centre for Physical Activity Research (CFAS) is supported by a grant from Trygfonden. This study was further supported by the Danish Council for Independent Research — Medical Sciences and by grants from the Novo Nordisk Foundation, Lundbeck Foundation and Fabrikant Einar Willumsens legat. CIM is part of the UNIK Project: Food, Fitness & Pharma for Health and
Disclosure Statement
The authors have no conflicts of interest to declare.
Acknowledgments
Anne Boye, Lone Christensen, Ruth Rovsing, Hanne Villumsen and Noemi James are acknowledged for their technical assistance and Henriette Pilegaard for providing primers and helping setting up the glycogen and lactate measurements.
References (46)
- et al.
Exercise metabolism and the molecular regulation of skeletal muscle adaptation
Cell Metab
(2013) - et al.
Dynamics of the skeletal muscle secretome during myoblast differentiation
Mol Cell Proteomics
(2010) - et al.
Identification and characterization of a novel follistatin-like protein as a binding protein for the TGF-beta family
J Biol Chem
(2000) - et al.
The myostatin propeptide and the follistatin-related gene are inhibitory binding proteins of myostatin in normal serum
J Biol Chem
(2002) - et al.
Structure of myostatin.follistatin-like 3: N-terminal domains of follistatin-type molecules exhibit alternate modes of binding
J Biol Chem
(2012) - et al.
An enzymic method for measurement of glycogen
Anal Biochem
(1967) - et al.
Divalent metal transporter 1 regulates iron-mediated ROS and pancreatic beta cell fate in response to cytokines
Cell Metab
(2012) - et al.
Muscle-specific interleukin-6 deletion influences body weight and body fat in a sex-dependent manner
Brain Behav Immun
(2014 Aug) - et al.
Initiation of insulin secretion in glucose-free medium by activin A
Mol Cell Endocrinol
(1995) - et al.
Metabolic benefits of resistance training and fast glycolytic skeletal muscle
Am J Physiol Endocrinol Metab
(2011)
Muscle as a secretory organ
Compr Physiol
Strength training with blood flow restriction diminishes myostatin gene expression
Med Sci Sports Exerc
Effects of heavy resistance training on myostatin mRNA and protein expression
Med Sci Sports Exerc
Effects of concentric and eccentric muscle actions on myostatin and follistatin-like related gene
J Sports Sci Med
Biological activity of follistatin isoforms and follistatin-like-3 is dependent on differential cell surface binding and specificity for activin, myostatin, and bone morphogenetic proteins
Endocrinology
The effects of a soluble activin type IIB receptor on obesity and insulin sensitivity
Int J Obes (Lond)
Myostatin inhibition in muscle, but not adipose tissue, decreases fat mass and improves insulin sensitivity
PLoS One
Suppression of body fat accumulation in myostatin-deficient mice
J Clin Invest
Plasma and muscle myostatin in relation to type 2 diabetes
PLoS One
Increased secretion and expression of myostatin in skeletal muscle from extremely obese women
Diabetes
Myostatin decreases with aerobic exercise and associates with insulin resistance
Med Sci Sports Exerc
Correlation between blood activin levels and clinical parameters of type 2 diabetes
Exp Diabetes Res
Activin signaling: effects on body composition and mitochondrial energy metabolism
Endocrinology
Cited by (0)
- ☆
The authors have no conflicts of interest to declare.