The Identification of Novel Protein-Protein Interactions in Liver that Affect Glucagon Receptor Activity

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

Characterization of Zinc Influx Transporters (ZIPs) in pancreatic beta cells: roles in regulating cytosolic zinc homeostasis and insulin secretion

Zinc plays an essential role in the regulation of pancreatic beta cell function, affecting important processes including proinsulin biosynthesis, glucose-stimulated insulin secretion, and cell viability. Mutations in zinc efflux transport protein ZnT8, have been linked with both type 1 and type 2 diabetes, further supporting an important role for zinc in glucose homeostasis. However, very little is known about how cytosolic zinc is controlled by zinc influx proteins (ZIPs). In the current study, we have examined the beta cell and islet ZIP transcriptome and show consistent high expression of ZIP6 (Slc39a6) and ZIP7 (Slc39a7) genes across human, mouse islets and MIN6 beta cells. Modulation of ZIP6 and ZIP7 expression significantly altered cytosolic zinc influx in pancreatic beta cells, indicating an important role for ZIP6 and ZIP7 in regulating cellular zinc homeostasis. Functionally, this deregulated cytosolic zinc homeostasis led to impaired insulin exocytosis and insulin secretion. In parallel studies, we identified both ZIP6 and ZIP7 as potential interacting proteins with GLP-1R by a membrane yeast-two-hybrid (MYTH) assay. Knock-down of ZIP6 but not ZIP7 in MIN6 beta cells impaired the protective effects of GLP-1 on fatty acid-induced cell death possibly via reduced p-ERK pathway. Thus, our data suggests that ZIP6 and ZIP7 function as two important zinc influx transporters to regulate cytosolic zinc concentrations and insulin secretion in beta cells. In particular, ZIP6 is also capable of directly interacting with GLP-1R to facilitate the protective effect of GLP-1 on beta cell survival

The identification of novel protein-protein interactions in liver that affect glucagon receptor activity

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

Hart Hall Student Council

This editorial summarizes and comments on the papers published in issue 11(4) so as to raise the bar in applied spatial economic research and highlight new trends. The first paper deals with common factors and spatial dependence in the error term specification of a production function model. The second paper sets forth a New Economic Geography (NEG) model with production activities that vary in their complexity, so as to analyse the impact on specialization patterns across different regions. The third paper measures the efficiency of local public investments using a relatively unknown econometric technique in which the time span over which the variables in the regression equation are measured is increased by one time period every run. The fourth paper adopts a conditional quantile regression approach to determine the impact of people employed in informal jobs on the wage distribution in Colombia and five of its regions. Finally, the last paper proposes and tests two new Bayesian variable selection approaches for spatial econometric models

Progesterone receptor membrane component 1 is a functional part of the glucagon-like peptide-1 (GLP-1) receptor complex in pancreatic beta cells

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that regulates glucose homeostasis. Because of their direct stimulation of insulin secretion from pancreatic cells, GLP-1 receptor (GLP-1R) agonists are now important therapeutic options for the treatment of type 2 diabetes. To better understand the mechanisms that control the insulinotropic actions of GLP-1, affinity purification and mass spectrometry (AP-MS) were employed to uncover potential proteins that functionally interact with the GLP-1R. AP-MS performed on Chinese hamster ovary cells or MIN6 cells, both expressing the human GLP-1R, revealed 99 proteins potentially associated with the GLP-1R. Three novel GLP-1R interactors (PGRMC1, Rab5b, and Rab5c) were further validated through co-immunoprecipitation/immunoblotting, fluorescence resonance energy transfer, and immunofluorescence. Functional studies revealed that overexpression of PGRMC1, a novel cell surface receptor that associated with liganded GLP-1R, enhanced GLP-1-induced insulin secretion (GIIS) with the most robust effect. Knockdown of PGRMC1 in cells decreased GIIS, indicative of positive interaction with GLP-1R. To gain insight mechanistically, we demonstrated that the cell surface PGRMC1 ligand P4-BSA increased GIIS, whereas its antagonist AG-205 decreased GIIS. It was then found that PGRMC1 increased GLP-1-induced cAMP accumulation. PGRMC1 activation and GIIS induced by P4-BSA could be blocked by inhibition of adenylyl cyclase/EPAC signaling or the EGF receptor-PI3K signal transduction pathway. These data reveal a dual mechanism for PGRMC1-increased GIIS mediated through cAMP and EGF receptor signaling. In conclusion, we identified several novel GLP-1R interacting proteins. PGRMC1 expressed on the cell surface of cells was shown to interact with the activated GLP-1R to enhance the insulinotropic actions of GLP-1

A) The network of GCGR interactors and their functional clusters revealed by AP-MS.

<p>Biological functions were retrieved from the Uniprot database. Each gray line indicates known protein-protein interactions extracted from the String database. Green nodes denote those interactors only identified under unliganded state. Blue nodes represent exclusively liganded interactors. Yellow nodes are interactors that were found in both liganded and unliganded states. B) Biological process enrichment analysis showed GCGR interactors are enriched with respect to specific biological functions, as indicated by the DAVID bioinformatics tool. Shown here are biological processes that have an EASE Score <0.05 (a modified Fisher Exact p-Value).</p

Effects of overexpression of selected GCGR interactors on glucose production in primary mouse hepatocytes.

<p>A) Transfection of GFP in primary hepatocytes. B) Dose-response curve of glucagon induced glucose production in primary hepatocytes. 100 nM glucagon treatment significantly increased glucose production in primary hepatocytes (**p<0.01). Readings were normalized to protein amount. Results are presented as mean ± S.E. of three independent experiments. C) Overexpression of CAV1 and GALK1 increased glucose production significantly at basal level (*p< 0.05, **p<0.01 vs cells transfected with pcDNA3.1); D) Overexpression of LDLR and TMED2 increased 100 nM glucagon-stimulated glucose production while YWHAB decreased glucagon-stimulated glucose production significantly (*p< 0.05,**p<0.01 vs cells transfected with pcDNA3.1, N = 3 per group).</p

Validation of novel GCGR interactors by Co-IP/WB in CHO cells.

<p>HA-tagged interactors and Flag-tagged GCGR were co-transfected into CHO cells, while cells only transfected with HA-tagged interactors were used as a control. Anti-Flag co-immunoprecipitation (Co-IP) and anti-HA Western blot were performed. A) Representative gels from the Co-IP/WB. I = Input proteins before Co-IP, W3 = 3^rd wash, E = co-IP elutes. N = 3 per group. B) The relative binding strength for each interactor. For each of the interacting proteins, intensity of the elute/input band was quantified and expressed as a ratio to the lysate band (E:I ratio). The E:I ratio was used to estimate the percentage of input interactors binding to GCGR.</p

Assessment of the mechanism through which YWHAB decreases cAMP production.

<p>A) The affinity and B) radioactive counts of glucagon/GCGR binding in CHO cells transfected with YWHAB (N = 3 per group). C) YWHAB overexpression did not change levels of total serine phosphorylation in HepG2-GCGR cells.</p