Investigation of Adhesion G Protein-Coupled Receptor 56 and type III Collagen in 3T3-L1 Adipogenesis

Abstract

Obesity imposes a significant threat to global public health. The inappropriate expansion of adipose tissue leads to hypertrophic obesity, characterised by impaired adipogenesis, and adipocytes dysfunction, which are the major drivers of obesity-associated metabolic defects. Identifying new adipogenic regulators may recognise novel targets for treating obesity as a means of reducing the burden of the associated metabolic defects. Adhesion G-protein coupled receptor 56 (GPRS6) and type III collagen (COL3A1) are both expressed in adipocyte, but their roles have not been described before.

This study shows that GprS6 is differentially expressed during 3T3-L1 preadipocytes differentiation. In vivo, gprSG transcript is reduced (55%) in the abdominal visceral adipose depots of genetically obese Zucker rats compared to their lean littermates. The expression of both Gpr56 and Col3al are essential for adipocytes development in vitro. The ectopic expression ofGpr56 moderately suppressed adipogenesis, demonstrated by decreased in neutral lipid accumulation (33%) and reduced the abundance of adipogenic proteins, peroxisome proliferator-activated receptor-gamma 2 (Ppar-/2) (60%) and adipocytes lipid-binding protein 2 (aP2) (36%). Transient knockdown (KD) of eitherGprS6 or Co/3ol substantially suppressed adipogenesis, demonstrated by reduced lipid droplet accumulation (56-46%). Gpr56 KD significantly reduced the abundance of Ppary2 and aP2 at both transcripts (35-22%) and proteins (62-56%) levels. The importance of both Gpr56 and Co/3ol for adipogenesis in 3T3-L1 cells was further validated by genome editing. Genome-edited cell lines harbouring compound heterozygous frameshift mutations in either Gpr56 or Co/3ol were incapable of differentiation. BothGpr56~/~ and Col3al~/~ cells sustained significant levels of active P-catenin (600-400%), a negative adipogenic regulator. Transient KD of β-catenin
partially rescued adipogenesis in Col3al~/~ but not 6pr56 null cells.

Here, gain and loss of function studies supported the premise that Gpr56 is a positive regulator of the proliferation of 3T3-L1 preadipocytes. Additionally, Gpr56-/~ or Col3al~/~cells had reduced adhesion, aberrant actin cytoskeleton and altered expression profiles of the extracellular matrix (ECM)-associated genes. Cell adhesion was substantially reduced in Gpr56-/- (56-64%) and Co/3al-/- (41-50%) cells relative to the wild-type 3T3-LI cells. During adipogenic induction, the genome-edited cells had dramatically altered the expression of the ECM component gene transcripts. Fibronectin was upregulated inGprS6~/~ and Co/3ol7- cells (311-130%), whereas type IV collagen was reduced(74-58%).In both cell lines, the expression of filamentous actin (F-actin) reduced (25-57%) and the stress fibres were disassembled into thin cortical actin fibres. These results suggest that both GPR56 and type III collagen are required for optimal adipogenesis in 3T3-L1 cells. The phenotypic similarity of the GprS6~/~ and Col3al~/~ cell lines suggest a functional interplay between Gpr56 and type III collagen during adipogenesis in 3T3-L1 and that they may act on the same pathway. Their role in differentiation is currently unclear but is likely to be related to the regulation of P-catenin, cell proliferation, adhesion, actin cytoskeleton and ECM organisation. This study sets the stage for further analysis considering Gpr56 and type III collagen as potential targets for developing therapeutic strategies for reducing the burden of the adverse metabolic defects in obesity.

Date of Award	2019
Original language	English
Awarding Institution	Glasgow Caledonian University
Supervisor	Christopher Bartholomew (Supervisor) & Steven Patterson (Supervisor)