Acute Downregulation of Zinc α2-Glycoprotein: Evidence from Human and HepG2 Cell Studies

Abstract

Zinc-alpha2-glycoprotein (ZAG) is a soluble glycoprotein primarily produced in adipocytes and the liver, with key roles in lipid metabolism, including lipolysis and the browning of adipose tissue. Despite extensive studies on its role in rodents, the relationship between ZAG and insulin in humans remains unclear. Given the emerging interest in ZAG's involvement in metabolic diseases such as metabolic-dysfunction-associated steatotic liver disease, this study aimed to investigate the acute effects of insulin on ZAG levels both in vivo and in vitro. We recruited 24 healthy, individuals who were non-obese and assessed the impact of oral glucose overload, a standardized liquid nutritional supplement, and intravenous glucagon on circulating ZAG levels. In parallel, we explored the effects of insulin on ZAG production in cultured HepG2 cells. Our findings revealed a consistent acute reduction in serum ZAG levels following all in vivo tests, coinciding with increased insulin levels. In vitro, insulin rapidly downregulated ZAG protein and mRNA levels in HepG2 cells, with significant reductions observed within 15 min, followed by partial recovery after 2 h. These results suggest a potential acute inhibitory effect of insulin on ZAG production, supporting its role in promoting energy storage by suppressing lipolysis postprandially. This study provides new insights into the complex interplay between insulin and ZAG in regulating energy balance and highlights the potential of ZAG as a therapeutic target in metabolic diseases.

Keywords: HepG2 cells; ZAG; adipose tissue; glucagon; glucose tolerance test; insulin; standard meal.

Figure 1

Acute insulin downregulation of ZAG production by HepG2 cells. HepG2 cells were treated in a 2 h experiment with vehicle or insulin (20 µIU/mL) at different time points. (A) ZAG media from HepG2 cells were measured over the course of a 2 h experiment, treated with vehicle or insulin (20 µIU/mL), measured by ELISA. Data are expressed as mean ± SD of triplicates. (B) Representative Western blot images of ZAG and CypA from extracts of HepG2 cells treated with insulin, as described in (A). No untreated control condition was included in this experiment. (C) ZAG mRNA levels from HepG2 cells treated with insulin, as in (A). Human 18S was amplified as internal control, and values are expressed as percentage relative to untreated cells. Data are expressed as mean ± SD of triplicates.