Elsevier

Metabolism

Volume 64, Issue 2, February 2015, Pages 283-295
Metabolism

Over-expression of Follistatin-like 3 attenuates fat accumulation and improves insulin sensitivity in mice

https://doi.org/10.1016/j.metabol.2014.10.007Get rights and content

Abstract

Objective

Follistatin-like 3 (fstl3), a natural inhibitor of members of the TGF-β family, increases during resistance training in human plasma. Fstl3 primarily binds myostatin and activin A, and thereby inhibits their functions. We hypothesize that blocking myostatin and activin A signalling through systemic fstl3 over-expression protects against diet-induced obesity and insulin resistance.

Methods

Fstl3 was over-expressed by DNA electrotransfer in tibialis anterior, quadriceps and gastrocnemius muscles in female C57BL/C mice, and the mice were subsequently randomized to chow or high-fat feeding. Body weight, food intake, fat accumulation by MR scanning, and glucose, insulin and glucagon tolerance were evaluated, as was the response in body weight and metabolic parameters to 24 h fasting. Effects of fstl3 on pancreatic insulin and glucagon content, and pancreatic islet morphology were determined.

Results

Fstl3 over-expression reduced fat accumulation during high-fat feeding by 16%, and liver fat by 50%, as determined by MRI. No changes in body weight were observed, while the weight of the transfected muscles increased by 10%. No transcriptional changes were found in the subcutaneous adipose tissue. Fstl3 mice displayed improved insulin sensitivity and muscle insulin signalling. In contrast, glucose tolerance was impaired in high-fat fed fstl3 mice, which was explained by increased hepatic glucagon sensitivity and glucose output, as well as a decrease in the pancreatic insulin/glucagon ratio. Accordingly, fstl3 transfection improved counter-regulation to 24 h fasting.

Conclusion

Fstl3 over-expression regulates insulin and glucagon sensitivities through increased muscular insulin action, as well as increased hepatic glucagon sensitivity and pancreatic glucagon content.

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.

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    The authors have no conflicts of interest to declare.

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