Glucagon Receptor Deficiency Causes Early-Onset Hepatic Steatosis

Abstract

In mice, glucagon regulates lipid metabolism by activating receptors in the liver; however, its role in human lipid metabolism is incompletely understood. Here we describe three normal-weight individuals from a consanguineous family with early-onset hepatic steatosis and/or cirrhosis. Using exome sequencing, we found they were homozygous for two missense variants in the glucagon receptor gene (GCGR). In cells, the double GCGR mutation reduced cell membrane expression and signaling, resulting in an almost complete loss of function. Carriers of pathogenic GCGR mutations had substantially elevated circulating glucagon and amino acid levels and increased adiposity. Introducing the double GCGR mutation into human induced pluripotent stem cell-derived hepatocytes using CRISPR/Cas9 caused increased lipid accumulation. Our results provide an explanation for increased liver fat seen in clinical trials of GCGR antagonists and reduced liver fat in people with obesity and steatotic liver disease treated with GCGR agonists.

Article highlights: In this study, we investigated a consanguineous family in whom normal-weight individuals had hepatic steatosis and cirrhosis. Using whole-exome sequencing we found two rare homozygous variants in the glucagon receptor (GCGR) gene that cosegregated with the phenotype. In cells, the GCGR mutations result in a loss of function and increased lipid accumulation. These results highlight the potential risks associated with GCGR antagonists and the benefits of GCGR agonists, currently in clinical trials.

Graphical abstract

Figure 1

A homozygous double mutation in the gene encoding the glucagon receptor identified in a consanguineous family with early-onset hepatic steatosis. A: Pedigree of the family with GCGR deficiency: proband (case patient 1) indicated by an arrow, black filled symbols indicate homozygous variant carriers, heterozygotes shown in gray, empty symbols denote unknown genotypes, squares denote males, circles denote females, double lines indicate consanguineous marriages. B: Percutaneous liver biopsy specimen from a 27-year-old man homozygous for GCGR^R225H/V368M stained using hematoxylin-eosin; black arrow, macrovesicular steatosis; red arrow, ballooned hepatocytes; green arrow, chronic inflammatory infiltrate (original magnification ×10). C: White arrow indicates a 15-mm nodule in the head of the pancreas in GCGR^R225H/V368M mutation carrier (case patient 2). D: Explanted liver of GCGR^R225H/V368M mutation carrier aged 50 (case patient 2) demonstrating macronodular cirrhosis; stain picrosirius red, original magnification ×5.

Figure 2

Metabolic phenotype of GCGR variant carriers. A: MRI of the proband’s mother depicting a 7-mm nodule in the tail of the pancreas (arrow). B: Glucagon immunostaining of the pancreas obtained from a GCGR mutation carrier (left) and healthy control individual (right). Brown, glucagon; blue, nuclei. Scale bar = 100 µm. C: Plasma total GLP-1 concentrations measured before and during an oral 75-g glucose load in case patient 1 (black squares), his mother (black circles), and matched control individuals (open circles); n = 6; mean ± SD shown. D: MRI measurement of abdominal fat distribution at the level of the L3/L4 intervertebral disc analyzed using Osirix to differentiate subcutaneous (green) and visceral (red) fat in case patient 1 (left) and his mother (right). Plasma insulin (E), glucose (F), and FGF21 (G) concentrations were measured after the oral administration of 75 g glucose (time 0) in case patient 1 (black, closed squares), his mother (black, closed circles), and eight control individuals matched for age, sex, and BMI (gray, open circles).

Figure 3

Double mutation in the glucagon receptor causes a loss of function in cells. Functional studies of WT and mutant forms of GCGR. A: Cell surface expression in the ebBRET assay expressed as a percentage of WT in each experiment; n = 5, mean ± SEM shown. B: GCGR-mediated cAMP production in response to glucagon stimulation in HEK293 cells, expressed as percentage of WT; n = 4, mean ± SEM. C: GCGR-mediated cAMP production in iPSC-derived hepatocytes, expressed as AUC; n = 5, mean ± SEM.

Figure 4

Generation of homozygous knockin human-iPSC (hiPSC) line GCGR^R225H/V368M using CRISPR/Cas9. A: CRISPR/Cas9 strategy targeting exons 8 and 12 to generate the mutant model. White boxes noncoding exons; blue boxes coding exons; purple residues Protospacer adjacent motif (PAM) sequence; blue residues, target sequence; green residues depict single-stranded oligodeoxynucleotides (ssODN); red residues, mutated residues. B: Chromatograms, which show sequencing analysis to confirm the presence of the two targeted mutations. RNP, ribonucleoprotein.

Figure 5

iPSC differentiation to iPSC-derived hepatocytes. A: Summary of four-stage differentiation protocol from human iPSCs to definitive endoderm, hepatic progenitor, and hepatocytes over a 21-day period, using a combination of growth factors and small molecules indicated in blue. B: Bright-field microscopy using EVOS cell imaging system, depicting typical hepatocyte morphology with characteristic polyhedral appearance; GCGR WT (left), GCGR^R225H/V368M (right); scale bar = 100 µm. C: Expression of canonical hepatocyte markers (albumin, HNF4A1, SERPINA1) in GCGR WT and R225H/V368M hepatocytes relative to the housekeeping gene HMBS; n = 6–7 experiments; mean ± SEM.

Figure 6

Increased lipid accumulation in GCGR R225H/V368M human iPSC-derived hepatocytes. A: Lipid accumulation in GCGR^R225H/V368M human iPSC-derived hepatocytes (bottom row) compared with WT GCGR (top row) using confocal microscopy; BODIPY staining of neutral lipid (green), Hoechst 33342 staining of nuclei (blue); scale bar = 50 µm. B: Lipid accumulation measured as lipid surface area divided by number of nuclei in WT GCGR (white) and GCGR^R225H/V368M (gray) iPSC-derived hepatocytes treated with increasing concentrations of oleic acid. Data were acquired using confocal microscopy and quantified using Harmony High-Content Imaging and Analysis Software; n = 7, mean ± SEM; ns, not significant; two-tailed Student t test.