Abstract
Glucagon regulates glucose homeostasis by controlling glycogenolysis and gluconeogenesis in the liver. Exaggerated and dysregulated glucagon secretion can exacerbate hyperglycemia contributing to type 2 diabetes (T2D). Thus, it is important to understand how glucagon receptor (GCGR) activity and signaling is controlled in hepatocytes. To better understand this, we sought to identify proteins that interact with the GCGR to affect ligand-dependent receptor activation. A Flag-tagged human GCGR was recombinantly expressed in Chinese hamster ovary (CHO) cells, and GCGR complexes were isolated by affinity purification (AP). Complexes were then analyzed by mass spectrometry (MS), and protein-GCGR interactions were validated by co-immunoprecipitation (Co-IP) and Western blot. This was followed by studies in primary hepatocytes to assess the effects of each interactor on glucagon-dependent glucose production and intracellular cAMP accumulation, and then in immortalized CHO and liver cell lines to further examine cell signaling. Thirty-three unique interactors were identified from the AP-MS screening of GCGR expressing CHO cells in both glucagon liganded and unliganded states. These studies revealed a particularly robust interaction between GCGR and 5 proteins, further validated by Co-IP, Western blot and qPCR. Overexpression of selected interactors in mouse hepatocytes indicated that two interactors, LDLR and TMED2, significantly enhanced glucagon-stimulated glucose production, while YWHAB inhibited glucose production. This was mirrored with glucagon-stimulated cAMP production, with LDLR and TMED2 enhancing and YWHAB inhibiting cAMP accumulation. To further link these interactors to glucose production, key gluconeogenic genes were assessed. Both LDLR and TMED2 stimulated while YWHAB inhibited PEPCK and G6Pase gene expression. In the present study, we have probed the GCGR interactome and found three novel GCGR interactors that control glucagon-stimulated glucose production by modulating cAMP accumulation and genes that control gluconeogenesis. These interactors may be useful targets to control glucose homeostasis in T2D.
Highlights
Glucagon, released from pancreatic islet alpha cells, promotes glycogenolysis and gluconeogenesis in the liver to elevate blood glucose levels during fasting
We recently identified a set of novel GLP-1R interactors in Chinese hamster ovary (CHO) and MIN6 β cells expressing GLP-1R using a similar affinity purification (AP)-mass spectrometry (MS) method which revealed 99 potential interactors [17]
By Western blot, we showed that the Flag-tagged glucagon receptor (GCGR) was detected in total lysate and following affinity purification using anti-Flag affinity gel (Fig 1B)
Summary
Glucagon, released from pancreatic islet alpha cells, promotes glycogenolysis and gluconeogenesis in the liver to elevate blood glucose levels during fasting. This effect is mediated via its cognate receptor, GCGR. Upon receptor activation by glucagon, Gs alpha is released to activate adenylate cyclase and increase intracellular cAMP levels, subsequently activating protein kinase A (PKA) [2]. The first GCGR antagonist identified was the small molecule skyrin, a fungal bisanthroquinone, which was found to inhibit glucagon-stimulated cAMP formation and glucose output from rat and human hepatocytes [9].Later, another GCGR antagonist, Cpd-A, was shown in preclinical models to lower blood glucose, but circulating glucagon and glucagon-like peptide 1 (GLP-1) levels were moderately elevated [10]. To facilitate the discovery of novel GCGR antagonists there is a need for a comprehensive understanding of factors/proteins involved in the regulation of its activity and cell signaling
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