GDF11 rapidly increases lipid accumulation in liver cancer cells through ALK5-dependent signaling

Jan Frohlich,Tommaso Mazza,Cyril Sobolewski,Michelangelo Foti,Manlio Vinciguerra

doi:10.1016/j.bbalip.2021.158920

Abstract

Hepatocellular carcinoma (HCC) is one of the fastest-growing causes of cancer-related mortalities worldwide and this trend is mimicked by the surge of non-alcoholic fatty liver disease (NAFLD). Altered hepatic lipid metabolism promotes HCC development through inflammation and activation of oncogenes. GDF11 is a member of the TGF-β superfamily and recent data have implicated GDF11 as an anti-aging factor that can alleviate high-fat diet induced obesity, hyperglycemia, insulin resistance and NAFLD. However, its role in hepatic lipid metabolism is still not fully delineated. The aim of the present study was to characterize the role of GDF11 in hepatic and HCC cells lipid accumulation. To achieve this, we performed imaging, biochemical, lipidomic, and transcriptomic analyses in primary hepatocytes and in HCC cells treated with GDF11 to study the GDF11-activated signaling pathways. GDF11 treatment rapidly triggered ALK5-dependent SMAD2/3 nuclear translocation and elevated lipid droplets in HCC cells, but not in primary hepatocytes. In HCC cells, ALK5 inhibition hampered GDF11-mediated SMAD2/3 signaling and attenuated lipid accumulation. Using ultra-high-performance liquid chromatography/mass spectrometry, we detected increased accumulation of longer acyl-chain di/tri-acylglycerols and glycerophospholipids. Unbiased transcriptomic analysis identified TGF-β and PI3K-AKT signaling among the top pathways/cellular processes activated in GDF11 treated HCC cells.In summary, GDF11 supplementation promotes pro-lipogenic gene expression and lipid accumulation in HCC cells. Integration of our “omics” data pointed to a GDF11-induced upregulation of de novo lipogenesis through activation of ALK5/SMAD2/3/PI3K-AKT pathways. Thus, GDF11 could contribute to metabolic reprogramming and dysregulation of lipid metabolism in HCC cells, without effects on healthy hepatocytes.

Highlights

Under physiological conditions, the liver is a central hub for carbo hydrate and lipid metabolism, with uptake, esterification, oxidation and secretion of fatty acids (FAs), all occurring in hepatocytes
Flasks/plates with at least 60–70% confluence were treated with the following compounds: 25–50–100 ng/ ml recombinant Growth differentiation factor 11 (GDF11) (PeproTech, NJ, US), 100 μM free fatty acid solution (OA/LA; comprising of oleic and linoleic acids in 1:1 ratio, L9655 Sigma-Aldrich) or 100 μM free fatty acid solution enriched with palmitic acid (OA/LA/PA, oleic:linoleic:palmitic acids in 1:1:1 ratio); ALK5 inhibitors: SB431542 (25 μM) or Repsox, PI3K in hibitors: Wortmannin (1 μM or LY294002 (1 μM) (Selleckchem, TX, USA); human Insulin solution (50 nM, I9278, Sigma-Aldrich)
The addition of high glucose/OA in the cell media triggered substantial accumulation of intracellular neutral lipids in the primary mouse hepatocytes (PMH) that was reflected by a proportional in crease in BODIPY staining of lipid droplets (LDs) (Fig. 1A–C)

Summary

Introduction

The liver is a central hub for carbo hydrate and lipid metabolism, with uptake, esterification, oxidation and secretion of fatty acids (FAs), all occurring in hepatocytes. Fatty acids present in the liver are stored in the hepatocytes in the form of lipid droplets (LDs), which are dynamic cytosolic organelles with the hydrophobic core that contains and stores newly synthesized or dietary acquired neutral lipids [1,2]. Those can later serve as an energy reser voir during deprivation [2]. The number and size of LDs are tightly regulated and too many lipid droplets formed during excess can lead to serious pathologic conditions [4]

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