Global Transcriptomic Analysis of Zebrafish Glucagon Receptor Mutant Reveals Its Regulated Metabolic Network

Abstract

The glucagon receptor (GCGR) is a G-protein-coupled receptor (GPCR) that mediates the activity of glucagon. Disruption of GCGR results in many metabolic alterations, including increased glucose tolerance, decreased adiposity, hypoglycemia, and pancreatic α-cell hyperplasia. To better understand the global transcriptomic changes resulting from GCGR deficiency, we performed whole-organism RNA sequencing analysis in wild type and gcgr-deficient zebrafish. We found that the expression of 1645 genes changes more than two-fold among mutants. Most of these genes are related to metabolism of carbohydrates, lipids, and amino acids. Genes related to fatty acid β-oxidation, amino acid catabolism, and ureagenesis are often downregulated. Among gcrgr-deficient zebrafish, we experimentally confirmed increases in lipid accumulation in the liver and whole-body glucose uptake, as well as a modest decrease in total amino acid content. These results provide new information about the global metabolic network that GCGR signaling regulates in addition to a better understanding of the receptor’s physiological functions.

Integrated Metabolomics and Lipidomics Analysis Reveal Remodeling of Lipid Metabolism and Amino Acid Metabolism in Glucagon Receptor–Deficient Zebrafish

“…In order to study the changes in GCGR-deficient animal, as well as for screening chemical modifiers to mitigate these side effects, we generated a GCGR knockout zebrafish model (Li et al, 2015 ). Our studies suggested that gcgr −/− mutant zebrafish displayed phenotypes similar to Gcgr -deficient mice, which had lower free glucose content, higher glucagon content, α-cell hyperplasia, and accentuation of fat in the liver (Li et al, 2015 ; Kang et al, 2020 ). We further analyzed the regulated metabolic network in transcriptional level by RNA-seq and found that many genes related to metabolism of carbohydrates, lipids, and amino acids were dysregulated in gcgr −/− mutant zebrafish (Kang et al, 2020 ).…”

“…Among all of these changed lipids in glycerophospholipid metabolism, LPC (16:0) (−2.10) and LPC (P-18:0) (−1.91) were altered the most ( Supplementary Table 1 ). To further investigate how GCGR affects lipid metabolism in multiple levels, we also carried out the integrated analysis of metabolomics data with our previous transcriptomics data (Kang et al, 2020 ). We found 29 significantly altered transcripts ( Supplementary Table 3 ) were associated with glycerophospholipid metabolism.…”

“…Based on the different metabolites, we performed KEGG analysis by using R language especially the R package “clusterProfiler” (Yu et al, 2012 ). Further, integrative analyses of metabolomics and lipidomics with our previous transcriptomics data (Kang et al, 2020 ) were conducted through display of metabolites associated with genes on the pathways. On the basis of pathways analysis by different metabolites, the transcription level of relative genes in these pathways was selected for analysis.…”

“…Nevertheless, Gcgr −/− mice exhibited enhanced susceptibility to hepatosteatosis following exposure to the high-fat diet (Longuet et al, 2008 ). Similarly, knocking out GCGR and treatment with GCGR antagonist (LY2409021) induced liver fat accumulation in zebrafish and patients with type 2 diabetes (T2D), respectively (Guzman et al, 2017 ; Kang et al, 2020 ). Moreover, the plasma triglycerides and free fatty acids were increased in Gcgr −/− mice after 16 h fasting (Longuet et al, 2008 ).…”

“…Although the serum metabolome has been studied in Gcgr −/− mice (Yang et al, 2011 ; Dean et al, 2017 ), the whole-animal metabolomics changes are still unclear. In this study, we performed global metabolomics and lipidomics profiling using the whole organism of GCGR knockout zebrafish, which were further analyzed with our previous global transcriptomics data (Kang et al, 2020 ). We aimed to reveal the comprehensive metabolic alterations in gcgr −/− zebrafish.…”