Adult mice are unresponsive to AAV8-Gremlin1 gene therapy targeting the liver

PLoS One. 2021 Feb 19;16(2):e0247300. doi: 10.1371/journal.pone.0247300. eCollection 2021.

Abstract

Objective: Gremlin 1 (GREM1) is a secreted BMP2/4 inhibitor which regulates commitment and differentiation of human adipose precursor cells and prevents the browning effect of BMP4. GREM1 is an insulin antagonist and serum levels are high in type 2 diabetes (T2D). We here examined in vivo effects of AAV8 (Adeno-Associated Viral vectors of serotype eight) GREM 1 targeting the liver in mature mice to increase its systemic secretion and also, in a separate study, injected recombinant GREM 1 intraperitoneally. The objective was to characterize systemic effects of GREM 1 on insulin sensitivity, glucose tolerance, body weight, adipose cell browning and other local tissue effects.

Methods: Adult mice were injected with AAV8 vectors expressing GREM1 in the liver or receiving regular intra-peritoneal injections of recombinant GREM1 protein. The mice were fed with a low fat or high fat diet (HFD) and followed over time.

Results: Liver-targeted AAV8-GREM1 did not alter body weight, whole-body glucose and insulin tolerance, or adipose tissue gene expression. Although GREM1 protein accumulated in liver cells, GREM1 serum levels were not increased suggesting that it may not have been normally processed for secretion. Hepatic lipid accumulation, inflammation and fibrosis were also not changed. Repeated intraperitoneal rec-GREM1 injections for 5 weeks were also without effects on body weight and insulin sensitivity. UCP1 was slightly but significantly reduced in both white and brown adipose tissue but this was not of sufficient magnitude to alter body weight. We validated that recombinant GREM1 inhibited BMP4-induced pSMAD1/5/9 in murine cells in vitro, but saw no direct inhibitory effect on insulin signalling and pAkt (ser 473 and thr 308) activation.

Conclusion: GREM1 accumulates intracellularly when overexpressed in the liver cells of mature mice and is apparently not normally processed/secreted. However, also repeated intraperitoneal injections were without effects on body weight and insulin sensitivity and adipose tissue UCP1 levels were only marginally reduced. These results suggest that mature mice do not readily respond to GREMLIN 1 but treatment of murine cells with GREMLIN 1 protein in vitro validated its inhibitory effect on BMP4 signalling while insulin signalling was not altered.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3-L1 Cells
  • Animals
  • Body Weight
  • Cell Line
  • Dependovirus / genetics*
  • Diet, High-Fat / adverse effects*
  • Disease Models, Animal
  • Genetic Therapy
  • Genetic Vectors / administration & dosage
  • Glucose Tolerance Test
  • Humans
  • Injections, Intraperitoneal
  • Insulin Resistance / genetics*
  • Intercellular Signaling Peptides and Proteins / administration & dosage
  • Intercellular Signaling Peptides and Proteins / genetics*
  • Intercellular Signaling Peptides and Proteins / metabolism*
  • Liver / metabolism*
  • Male
  • Mice
  • Recombinant Proteins / administration & dosage

Substances

  • Grem1 protein, mouse
  • Intercellular Signaling Peptides and Proteins
  • Recombinant Proteins

Grants and funding

Ulf Smith received financial support from the Swedish Research Council, the Novo Nordisk Foundation, the Torsten Söderberg Foundation, the Swedish ALF Funds (no. 718601) and the Swedish Diabetes Association. Co-authors SH, CC and JB are employees of Astra Zeneca. However, the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors’ salaries. The specific roles of these authors are articulated in the ‘author contributions’ section.