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<p>Glucagon receptor (GCGR) signaling is essential for glucose and lipid homeostasis, making it a potential therapeutic target for metabolic disorders. Zebrafish possess two GCGR co-orthologs, GCGRa and GCGRb, however, their distinct function remain unclear. In this study we employed CRISPR/Cas9 gene editing to generate GCGRa⁻/⁻, GCGRb⁻/⁻, and double-knockout (GCGR⁻/⁻) zebrafish to dissect isoform-specific functions. RNA-Seq analysis was performed to characterize transcriptomic alterations, while an overfeeding protocol was used to assess metabolic tolerance, and ligand-response assays in cell lines evaluated isoform activation dynamics. Transcriptomic analysis revealed that both isoforms regulate overlapping but distinct metabolic pathways. Functional enrichment analysis linked GCGRa to lipid and energy metabolism, cholesterol biosynthesis and glucose homeostasis, through key signaling cascades such as glucagon, PPARγ and PI3K-AKT. In contrast, GCGRb loss altered fatty acid β-oxidation, GPCR signaling, and oxidative phosphorylation networks, implicating roles in metabolism and cellular stress. The GCGR⁻/⁻ primarily impacted core metabolic networks including lipid, gluconeogenesis and energy metabolism, indicating complementary and overlapping functions of both receptors in maintaining hepatic metabolic homeostasis. Ligand-response assays revealed that GCGRb, but not GCGRa, is activated by both glucagon (GCGa) and glucagon like-peptide-1 (GLP1a), supporting the post-duplication receptor diversification theory. Notably, all knockouts exhibited impaired growth under high-nutrient conditions, confirming GCGR’s role in diet-responsive development. This study provides the first systematic functional comparison of zebrafish GCGR isoforms, establishing zebrafish as a valuable model for investigating glucagon-based metabolic regulation and therapeutic interventions.</p>

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Targeting glucagon signaling in metabolic disorders, functional insights from zebrafish receptor knockouts

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年6月22日

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  • Ashraf Al Madhoun,
  • Md Zubbair Malik,
  • Shaima Al-Beloushi,
  • Nermeen Abukhalaf,
  • Lavina Miranda,
  • Sindu Jacob,
  • Rasheeba Nizam,
  • Sumi John,
  • Preethi George,
  • Arshad Channanath,
  • Fatemah Bahman,
  • Meijiang Liao,
  • Rasheed Ahmad,
  • Wenbiao Chen,
  • Abdelhamid Bekri,
  • Pierre Drapeau,
  • Thangavel Alphonse Thanaraj,
  • Fahd Al-Mulla

摘要

Glucagon receptor (GCGR) signaling is essential for glucose and lipid homeostasis, making it a potential therapeutic target for metabolic disorders. Zebrafish possess two GCGR co-orthologs, GCGRa and GCGRb, however, their distinct function remain unclear. In this study we employed CRISPR/Cas9 gene editing to generate GCGRa⁻/⁻, GCGRb⁻/⁻, and double-knockout (GCGR⁻/⁻) zebrafish to dissect isoform-specific functions. RNA-Seq analysis was performed to characterize transcriptomic alterations, while an overfeeding protocol was used to assess metabolic tolerance, and ligand-response assays in cell lines evaluated isoform activation dynamics. Transcriptomic analysis revealed that both isoforms regulate overlapping but distinct metabolic pathways. Functional enrichment analysis linked GCGRa to lipid and energy metabolism, cholesterol biosynthesis and glucose homeostasis, through key signaling cascades such as glucagon, PPARγ and PI3K-AKT. In contrast, GCGRb loss altered fatty acid β-oxidation, GPCR signaling, and oxidative phosphorylation networks, implicating roles in metabolism and cellular stress. The GCGR⁻/⁻ primarily impacted core metabolic networks including lipid, gluconeogenesis and energy metabolism, indicating complementary and overlapping functions of both receptors in maintaining hepatic metabolic homeostasis. Ligand-response assays revealed that GCGRb, but not GCGRa, is activated by both glucagon (GCGa) and glucagon like-peptide-1 (GLP1a), supporting the post-duplication receptor diversification theory. Notably, all knockouts exhibited impaired growth under high-nutrient conditions, confirming GCGR’s role in diet-responsive development. This study provides the first systematic functional comparison of zebrafish GCGR isoforms, establishing zebrafish as a valuable model for investigating glucagon-based metabolic regulation and therapeutic interventions.