Insulin resistance causes excess hepatic glucose production (HGP), glucose intolerance and type 2 diabetes mellitus. Now, new research by Morris White and colleagues shows that disruption of the function of follistatin — a protein that regulates the expression of members of the transforming growth factor-β (TGFβ) superfamily — improves insulin sensitivity of white adipose tissue (WAT) and systemic glucose tolerance in mice.

Credit: Liver sections from LDKO and control mice immunostained with an antibody against follistatin (green). DAPI (red) is used to visualize nuclei. Reproduced from Tao, R. et al. Nat. Med. https://doi.org/10.1038/s41591-018-0048-0 (2018), Macmillan Publishers Limited.

“Ten years ago, we developed the LDKO mouse model, where hepatic expression of insulin receptor substrate 1 (Irs1) and Irs2 were specifically knocked out, to test whether IRS1 and IRS2 mediated insulin signalling in the murine liver,” explains White. “Our experiments confirmed that IRS1 and IRS2 in liver are important mediators of insulin signalling, because LDKO mice developed systemic insulin resistance marked by hyperinsulinaemia and hyperglycaemia.”

Interestingly, White and colleagues also showed that hepatic deletion of the transcription factor forkhead box protein O1 (FOXO1) in LDKO mice (LTKO mice) corrected the hepatic and systemic metabolic dysregulation, despite being unable to restore hepatic insulin signalling. The underlying mechanism of this restoration in LTKO mice via FOXO1 disruption is unclear, leading the team to the current study.

In the present study, the researchers show that normalization of HGP by FOXO1 disruption was partially due to restoring insulin sensitivity in WAT. “We reasoned that the insulin-resistant liver must secrete some FOXO1-dependent factor into the circulation that causes systemic insulin resistance, glucose intolerance and unregulated hepatic gluconeogenesis,” noted White. To understand this mechanism, the researchers interrogated previously published microarray data from LDKO mice to find hepatokines that promoted systemic insulin resistance. The search identified several candidate hepatokines, and by overexpressing individual candidates in wild-type mice fed a high-fat diet (HFD), the authors found that overexpression of follistatin substantially impaired glucose tolerance when compared with other candidates. “We settled upon follistatin as the factor secreted from the insulin-resistant liver that promotes systemic insulin resistance,” explains White.

overexpression of follistatin substantially impaired glucose tolerance

The team showed that the livers of LDKO mice had higher mRNA levels of follistatin than livers from control mice; however, follistatin levels in LTKO mice showed no such difference. Promoter-binding studies revealed that FOXO1 binds directly to the promoter region of follistatin, which confirms that FOXO1 is a regulator of follistatin expression.

Next, the researchers used targeted CRISPR–Cas9 technology to knock down hepatic follistatin expression in LDKO mice to determine whether follistatin caused insulin resistance in WAT. Follistatin knockdown LDKO mice showed increased insulin sensitivity in epigonadal WAT (eWAT) and improved glucose tolerance compared with LDKO mice with intact follistatin expression. This effect is similar to Foxo1 knockout in LDKO mice. Similarly, viral vector-mediated knockdown of hepatic follistatin expression in C57BL6 mice fed a HFD also improved glucose tolerance. The results suggest that follistatin causes glucose intolerance in genetic and physiological models of type 2 diabetes mellitus.

Finally, the team overexpressed follistatin in the livers of C57BL6 mice fed a HFD to investigate whether follistatin causes insulin resistance in WAT from wild-type mice. Overexpression of follistatin caused insulin resistance in eWAT, inguinal WAT and skeletal muscle. Increasing follistatin levels also increased HGP and reduced the ability of insulin to suppress HGP and circulating levels of free fatty acids. The researchers showed that follistatin inhibits lipolysis suppression via WAT insulin resistance.

“We will continue to investigate the use of follistatin interference to improve hyperglycaemia in diabetes mellitus,” concludes White. “This might include blocking antibodies or other proteins or peptides that reduce the effect of follistatin to increase circulating glucose.”