Cgref1, a novel hepatokine regulated by CREB-H, promotes insulin resistance and obesity by suppressing glucose uptake in white adipose tissue

Abstract

Transcription factor cAMP-responsive element-binding protein-H (CREB-H) is known to regulate many lipid and glucose metabolism genes and its deficiency is associated with dyslipidemia, atherosclerosis, and fatty liver. This study presents a new regulatory target of CREB-H – cell growth regulator with EF-hand domain 1 (Cgref1). Cgref1 is an extracellular calcium binding protein that is highly conserved among species and has been shown to inhibit cell growth previously. However, details of its regulation and functions are limited. Here, I have identified the presence of Cgref1 in mouse serum, which suggested it was freely available in the circulation. I have also found the upregulation of Cgref1 in mouse livers during fasting and the provision of high fat diet. Phenotypic analyses revealed that Cgref1-knockout (Cgref1-/-) mice had a lower fat to lean mass ratio compared to wild type (WT) mice, as well as lower levels of blood glucose, triglycerides, and total cholesterol (TCHO). They also performed better in glucose and insulin tolerance tests. Fundamentally, Cgref1-/- mice showed stronger glucose uptake specifically in white adipose tissues (WAT). This finding was complemented by further investigations using cell culture models. In 3T3-L1 adipocytes, the treatment of recombinant Cgref1 protein severely suppressed the expression of glucose transporter 4 (Glut4). Simultaneously, the phosphorylation of Akt-S473 in 3T3-L1 adipocytes was attenuated, which suggested impaired insulin sensitivity. These differences, however, were not observed in mouse liver cell line Hepa1-6 when the same conditions applied and pinpointed that Cgref1 had effects specific to WAT. Moreover, the distinctive phenotypic differences between WT and Cgref1-/- mice have indicated that Cgref1 essentially had a systemic impact. I propose that, the suppression of insulin sensitivity and glucose uptake in WAT by Cgref1 would alter metabolism, at least, in major glucose-consuming organs. In the liver, de novo lipogenesis (DNL) was increased as indicated by different lines of evidence. Firstly, serum TG and TCHO levels, the hallmarks of fatty liver disease, were raised in WT mice that had normal Cgref1 expression. Secondly, fatty acid synthesis genes including acetyl-CoA carboxylases 1 and 2 (Acc1 and Acc2) and stearoyl-CoA desaturase 1 (Scd1) were also upregulated in WT mouse livers. Separately, in skeletal muscles, glycogen stores were saturated by higher blood glucose and became less sensitive to insulin. Besides, lipids derived from hepatic DNL posed higher chances of obesity development. From an evolutionary point of view, Cgref1 serves as a hepatokine that is beneficial during food shortage by diverting glucose, an important energy source, to other organs. However, in today’s society where food is surplus with a reduced need for physical activity, Cgref1 becomes a natural cause of metabolic imbalance.