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. 2025 Feb 21;13(4):e70235. doi: 10.14814/phy2.70235

Female glucagon receptor knockout mice are prone to steatosis but resistant to weight gain when fed a MASH‐promoting GAN diet and a high‐fat diet

Katrine D Galsgaard 1,2, Emilie Elmelund 1, Jenna E Hunt 1,2, Mark M Smits 1,3, Trisha J Grevengoed 1, Christina Christoffersen 1,4, Nils J Færgeman 5, Jesper Havelund 5, Nicolai J Wewer Albrechtsen 6,7, Jens J Holst 1,2,✉
PMCID: PMC11845321  PMID: 39985139

Abstract

Glucagon is secreted from the pancreatic alpha cells and regulates not only hepatic glucose production, but also hepatic lipid and amino acid metabolism. Thus, glucagon provides a switch from hepatic glucose and lipid storage towards lipid and amino acid breakdown fueling glucose production during fasting. However, the effects of genetic deletion of the glucagon receptor on lipid metabolism are unclear. We therefore assessed parameters of lipid metabolism in fasted and non‐fasted male and female mice with permanent whole‐body deletion of the glucagon receptor (Gcgr −/− mice). To investigate whether Gcgr −/− mice tolerated a diet promoting metabolic dysfunction‐associated steatohepatitis (MASH) and steatosis, we fed female Gcgr −/− mice the Gubra Amylin Nonalcoholic steatohepatitis (GAN) diet and high‐fat diet (HFD), respectively. We found that non‐fasted Gcgr −/− mice fed standard chow showed hypercholesterolemia and increased liver fat (borderline significant in non‐fasted male Gcgr −/− mice, but significant in the remaining groups). In the fasted state these changes were insignificant due to fasting‐induced steatosis. When challenged with a GAN diet and HFD, female Gcgr −/− mice were prone to steatosis and dyslipidemia but resistant to weight gain. Taken together, our data highlight glucagon as an important physiological regulator of not just glucose, but also hepatic lipid metabolism.

Keywords: cholesterol, glucagon, high‐fat diet, steatosis, triglycerides

Female glucagon receptor knockout mice are prone to steatosis when fed a MASH‐promoting GAN diet and a high fat diet.

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1. INTRODUCTION

During conditions of decreasing blood glucose levels, glucagon is secreted from the pancreatic alpha cells. Upon reaching its primary target organ, the liver, glucagon increases not only hepatic glucose production (Jiang & Zhang, 2003; Ramnanan et al., 2011), but also amino acid metabolism (ureagenesis) (Fitzpatrick et al., 1977; Hamberg & Vilstrup, 1994), and lipid oxidation (Longuet et al., 2008; Pegorier et al., 1989; Roden et al., 2000; Witters & Trasko, 1979). Thus, glucagon increases plasma glucose concentrations (Jiang & Zhang, 2003; Ramnanan et al., 2011), while lowering concentrations of plasma amino acids (Boden et al., 1984; Fitzpatrick et al., 1977) and hepatic triglycerides (TG) (Jiang & Zhang, 2003; Roden et al., 2000; Witters & Trasko, 1979). Moreover, by decreasing very low‐density lipoprotein (VLDL) secretion from the liver (Longuet et al., 2008; Pegorier et al., 1989) glucagon reduces plasma TG and cholesterol concentrations (Amatuzio et al., 1962; Eaton, 1973; Guettet et al., 1988; Guettet, Rostaqui, Mathe, et al., 1991; Guettet, Rostaqui, Navarro, et al., 1991; Rudling & Angelin, 1993). These regulatory effects of glucagon are now often referred to as the liver‐alpha cell axis (Richter et al., 2022).

Subjects with metabolic dysfunction‐associated steatotic liver disease (MASLD) show increased plasma concentrations of glucagon and amino acids (Junker, 2017; Junker et al., 2016; Wewer Albrechtsen et al., 2018) suggesting that a disruption of the liver‐alpha cell axis has occurred. Supporting this, in both mice (Winther‐Sørensen et al., 2020) and humans (Heebøll et al., 2022; Suppli et al., 2020), steatosis has been linked to hepatic glucagon resistance which in turn affects both amino acid and lipid metabolism. The effect on amino acid metabolism shows up as a reduced glucagon‐induced decrease in plasma amino acid concentrations (Suppli et al., 2020) and a reduced increase in ureagenesis (Winther‐Sørensen et al., 2020), while the effect on lipid metabolism is evident as a reduced glucagon‐induced suppression of hepatic VLDL secretion (Heebøll et al., 2022). In the presence of a fatty liver, glucagon resistance may therefore create a vicious cycle of hyperglucagonemia, hyperaminoacidemia, hepatic lipid accumulation, and dyslipidemia (Galsgaard, 2020).

The glucagon receptor has been shown to be required for control of lipid metabolism during the adaptive metabolic response to fasting, as hepatic lipid oxidation failed to rise in response to fasting in mice with permanent deletion of the glucagon receptor (Gcgr −/− mice) causing increased hepatic TG secretion and increased plasma TG concentrations (Longuet et al., 2008). However, the effects of genetic deletion of the glucagon receptor on plasma and liver TG concentrations remain controversial (Conarello et al., 2007; Gelling et al., 2003; Parker et al., 2002; Sinclair et al., 2008). To investigate whether the inconsistent findings might be due to differences in feeding‐fasting‐regimes and perhaps the sex of the mice, we assessed lipid metabolism in male and female Gcgr −/− mice in the non‐fasted as well as the fasted state. We also investigated how female Gcgr −/− mice respond to a metabolic dysfunction‐associated steatohepatitis (MASH)‐promoting diet as well as a high‐fat diet (HFD; promoting steatosis).

This disentangling of glucagon's role in lipid metabolism is highly important considering the current development of glucagon‐based drugs to treat obesity and MASLD (Coskun et al., 2022; Day et al., 2009; Finan et al., 2015; Henderson et al., 2016; Urva et al., 2022). Our results suggest that permanent genetic deletion of the glucagon receptor may result in steatosis and hypercholesterolemia and thus highlight glucagon as an important physiological regulator of not just glucose, but also hepatic lipid metabolism.

2. METHODS

2.1. Mouse studies

Animal studies were conducted at the animal facilities at the Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, with permission from the Danish Animal Experiments Inspectorate, Ministry of Environment and Food of Denmark, permit 2018‐15‐0201‐01397. All studies were approved by the local ethical committee; Department of Experimental Medicine – University of Copenhagen. Glucagon receptor knockout (Gcgr −/−) mice (C57BL/6JGcgrtm1Mjch) and their wild‐type littermates (Gcgr +/+ mice) were bred in‐house with permission from Dr. Maureen J. Charron as described previously (Gelling et al., 2003). Female mice were housed in groups of four to eight and male mice in groups of two to six in individually ventilated cages. All mice followed a light cycle of 12 h (lights on 6 am to 6 pm) and were allowed a minimum of 1 week of acclimatization before being included in any experiment.

2.2. Characterization of glucagon receptor knockout mice in the non‐fasted and fasted state

Female and male Gcgr −/− and Gcgr +/+ mice, 14–18 weeks of age, had ad libitum access to chow (Autoclavable complete breeding vegetal diet for rats, mice and hamsters, SAFE® Complete Care Competence) or were fasted overnight (16 h). Blood glucose concentrations were measured after tail tip puncture using a handheld glucometer (Accu‐Chek® Mobile, catalog no. 05874149001; Roche Diagnostics, Mannheim, Germany), and the mice were weighed. Immediately after, blood was collected from the retrobulbar plexus using ethylenediaminetetraacetic acid (EDTA) coated capillary tubes (Micro Haematocrit Tubes, ref. no. 167313; Vitrex Medical A/S, Herlev, Denmark), subsequently stored on ice until spun (9000 g, 4°C, 10 min) and stored at −80°C. The mice were then euthanized by cervical dislocation and the liver, pancreas, and kidneys were excised, weighed, and snap‐frozen in liquid nitrogen. Finally, the tip of the ear was cut with a scissor washed with ethanol for genotyping.

In a separate experiment, female and male Gcgr −/− and Gcgr +/+ mice, 7–16 weeks of age, had ad libitum access to chow. Blood glucose concentrations were measured as described, and the mice were weighed and sedated using ketamine (catalog no. 511485, Ketaminol®, Intervet, Merck & CO., New Jersey, dose 100 mg/kg body weight) in combination with isotonic saline (0.9% NaCl) and xylazine (catalog no. 148999, Rompun® vet, Bayer AG, Leverkusen, Germany, dose 10 mg/kg body weight) given as an intraperitoneal injection. After sedation, blood was collected from the vena cava and stored on ice in EDTA coated Eppendorf tubes until spun (6500 g, 4°C, 10 min). Plasma was collected and stored at −80°C. Tissues were collected as described. To perform fast protein liquid chromatography (FPLC) analysis, aliquots of the plasma collected from each of the Gcgr −/− and Gcgr +/+ mice were pooled (according to the groups) in prechilled Eppendorf tubes and stored at −80°C until analysis as described in (Galsgaard et al., 2022).

2.2.1. Oral lipid tolerance test

Gcgr −/− (females n = 7 and males n = 8) and Gcgr +/+ (females n = 5 and males n = 9) mice, 10–14 weeks of age, were fasted overnight (16 h). The following morning, blood was collected from the retrobulbar plexus and blood glucose concentrations were measured using a handheld glucometer. Immediately after, the mice received 10 μL/g body weight of olive oil and blood was collected 30, 120, and 180 min after lipid administration. The mice were then killed by cervical dislocation and tissue for genotyping was collected as described.

2.2.2. Gubra amylin nonalcoholic steatohepatitis diet and high‐fat diet challenge

Female Gcgr −/− and Gcgr +/+ mice, 9–16 weeks of age, had ad libitum access to chow and body composition was assessed using a magnetic resonance scanner (MRI) scanner (Bruker LF90II MRI scanner). The mice were then fed the Gubra Amylin Nonalcoholic steatohepatitis (GAN) diet, a 40 kcal% fat, 20 kcal% fructose, and 2% cholesterol diet (GAN, D09100310i; Research Diets) for 5 weeks, and blood glucose, plasma, and tissue samples were measured and collected as described for un‐anesthetized mice. In a separate experiment, female Gcgr −/− and Gcgr +/+ mice, 7–16 weeks of age, were MRI scanned and then fed a high‐fat diet (HFD) diet, a 58 kcal% fat and sucrose, diet (D12331i; Research Diets) for 8 weeks. Following the 8 weeks the mice were again MRI scanned, blood glucose was measured, and plasma and tissue samples were collected as described for un‐anesthetized mice. Liver sections were stained with hematoxylin and eosin and steatosis was scored based on the following modified Kleiner (Kleiner et al., 2005) scoring system; steatosis (macro‐ and microvesicular) present in <5% of the hepatocytes = 0 point, 5%–33% = 1 point, 33%–66% = 2 point, and 66%–95% = 3 points.

2.3. Biochemical analysis

Triglyceride and glycerol concentrations were quantified using Serum Triglyceride Determination Kit (catalog no. TR0100‐1KT; Sigma‐Aldrich), except when measuring plasma triglyceride concentrations from the HFD fed mice, plasma and liver TG concentrations in the non‐fasted and fasted male Gcgr −/− and Gcgr +/+ mice (shown in Figure S2), and when measuring plasma and liver triglyceride concentrations from the second group of HFD fed mice; here the Triglyceride Colorimetric Assay Kit (catalog no. 10010303; Cayman Chemical, MI, USA), the Triglyceride Quantification Colorimetric Kit (catalog no. MAK266‐1KT; Sigma‐Aldrich), and Triglyceride assay (catalog no. TR210, Randox) were used, respectively. Nonesterified fatty acid (NEFA) concentrations were measured with NEFA‐HR (R1 and R2) kit from Fujifilm Wako Chemicals and liver glycogen concentrations with EnzyChrom Glycogen Assay Kit (catalog no. E2GN‐100; BioAssay Systems, CA, USA). Plasma concentrations of glucagon were measured using a validated (Wewer Albrechtsen et al., 2016) low volume, two‐site enzyme immunoassay (catalog no. 10‐1281‐01; Mercodia, Upsala, Sweden). Fibroblast growth factor‐21 (FGF‐21) plasma concentrations were measured using mouse and rat FGF‐21 Elisa (catalog no. RD291108200R, BioVendor R&D, Czech Republic). Amino acid concentrations were measured using L‐Amino Acid Assay Kit (catalog no. ab65347; Abcam, USA). Total plasma Cholesterol concentrations were measured using a Total Cholesterol Assay (catalog no. CH200, Randox Laboratories Ltd), and the cholesterol content in plasma lipoproteins were measured as described in (Galsgaard et al., 2022).

2.4. Lipid extraction

Lipids from snap‐frozen liver tissue were extracted using a Triton™ X‐100 extraction buffer (3% Triton™ X‐100 (Catalog no. 10789704001; Sigma‐Aldrich) (25% solution in ethanol) in sodium acetate buffer (0.15 mol/L, pH 4.9)) as described in (Galsgaard et al., 2022).

2.5. Lipidomics

Lipids were extracted from liver samples (20 mg) from the mice feed the GAN diet and HFD using Folch extraction before transferring to HPLC vials as described previously (Hansen et al., 2024). A quality control (QC) sample was made by pooling 3 μL of each sample. Samples (0.5 μL) were injected using a Vanquish Horizon UPLC (Thermo Fisher Scientific) equipped with a Waters ACQUITY Premier CSH (2.1 × 100 mm, 1.7 μM) column operated at 55°C as described (Hansen et al., 2024). The flow from the UPLC was coupled to a TimsTOF Flex (Bruker) instrument for mass spectrometric analysis as described (Hansen et al., 2024). Compounds were annotated in Metaboscape (Bruker) using both an in‐built rule‐based annotation approach and using the LipidBlast MS2 library (Kind et al., 2013). Features were removed if their average signal were not >5× more abundant in the QC samples than blanks (water extraction). The signals were normalized to internal standards in the SPLASH mix before correction for signal drift using the statTarget R package (Luan et al., 2018), and signals were normalized using the QC samples. Peak intensities from each measured metabolite were scaled using Pareto scaling with MetabolAnalyze (version 1.3.1) and log2 transformed. Analysis of lipidomics data was done using the MetaboAnalyst 5.0 webtool (Pang et al., 2021). The lipodomics data will be made available.

2.6. Statistics

All statistical analyses were done in GraphPad Prism version 10.2.0 (La Jolla, California, USA). Groups were compared by unpaired t‐test, two‐way ANOVA, or mixed‐effects analysis as indicated in the figure legend. p < 0.05 was considered statistically significant.

3. RESULTS

3.1. Female glucagon receptor knockout mice show increased body weight

To determine the effects of permanent genetic deletion of the glucagon receptor on parameters of lipid metabolism in the non‐fasted and fasted state, we investigated non‐fasted and overnight fasted (16 h) female and male Gcgr −/− and Gcgr +/+ mice.

Overnight fasting decreased blood glucose concentrations (p < 0.0001), body weight (p < 0.0001), and liver weights (p < 0.0001) in both female Gcgr −/− and Gcgr +/+ mice.

Blood glucose concentrations were lower in female Gcgr −/− compared to Gcgr +/+ mice both in the non‐fasted and fasted state (p = 0.0002 and p < 0.0001, respectively) (Figure 1a). The body weight was increased in female Gcgr −/− compared to Gcgr +/+ mice both in the non‐fasted and fasted state (p = 0.001 and p = 0.008, respectively) (Figure 1b). The glucagon receptor has been shown to be expressed in three organs; the liver, pancreas, and kidney (Bomholt et al., 2021). We therefore assessed the weight of these organs and found the weight of all three organs to be increased in female Gcgr −/− mice compared to Gcgr +/+ mice both in the non‐fasted and fasted state (p < 0.0001) (Figure 1c–e). The increased pancreas weight is most likely due hyperaminoacidemia mediated alpha‐cell hyperplasia (Dean et al., 2017; Gelling et al., 2003). Similar results were obtained in male mice (Figure S1).

FIGURE 1.

FIGURE 1

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Glucagon receptor knockout female mice show increased body weight. (a) Blood glucose, (b) body weights, (c) liver weights, (d) pancreas weights, and (e) kidney weights in non‐fasted (squares) and overnight fasted (16 h) (circles) female wild‐type littermates (Gcgr +/+, open symbols) and glucagon receptor knockout mice (Gcgr −/−, closed symbols). Mice 14–18 weeks of age, n = 8–22. p value by unpaired t‐test. (f) Body weights of female Gcgr −/− and Gcgr +/+ mice, the numbers in black indicate the number of Gcgr +/+ and the numbers in magenta indicate the number of Gcgr −/− mice. p value by mixed‐effects analysis. Data shown as mean ± SD.

To further investigate the difference observed in body weight, we weighed a separate cohort of female Gcgr −/− and Gcgr +/+ mice once weekly and found the body weights to be increased at all ages ranging from 7 to 24 weeks (Figure 1f).

To investigate the details behind the increased body weight of Gcgr −/− mice, we assessed the body composition using MR scans in a group of female mice in the non‐fasted state (following the scan these mice were fed either a GAN diet or HFD). The Gcgr −/− mice showed increased body weight (p = 0.006) (Figure 2a), and both the absolute fat mass (Figure 2b) and lean mass (Figure 2c) were higher in Gcgr −/− mice compared to Gcgr +/+ mice (p = 0.002 and p = 0.003, respectively).

FIGURE 2.

FIGURE 2

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Female glucagon receptor knockout mice show an increased absolute lean and fat mass. (a) Body weights, (b) absolute fat mass, and (c) absolute lean mass in female wild‐type littermates (Gcgr +/+, open symbols) and glucagon receptor knockout mice (Gcgr −/−, closed symbols). Data shown as mean ± SD, n = 26–28, mice 7–18 weeks of age. p value by unpaired t‐test.

3.2. Fasting‐induced steatosis masks the increase in liver triglycerides observed in non‐fasted female glucagon receptor knockout mice

3.2.1. Plasma parameters

Overnight fasting increased plasma nonesterified fatty acid (NEFA) (p < 0.0005) and liver TG concentrations (p < 0.0003) while decreasing liver glycogen concentrations (p < 0.0001) in both female Gcgr −/− and Gcgr +/+ mice.

In both the non‐fasted and fasted state, plasma TG concentrations were numerically higher in Gcgr −/− than in Gcgr +/+ mice (p = 0.053 and p = 0.096, respectively) (Figure 3a). Plasma glycerol concentrations were similar in female Gcgr −/− and Gcgr +/+ mice both in the non‐fasted and fasted state (p > 0.1) (Figure 3b). When compared to female Gcgr +/+ mice, plasma NEFA concentrations were numerically higher in female Gcgr −/− mice in the non‐fasted state (p = 0.051), but not in the fasted state (p = 0.2) (Figure 3c). Fibroblast growth factor 21 (FGF‐21) regulates lipid metabolism by increasing lipolysis in adipocytes and in the liver (Inagaki et al., 2007). We therefore assessed plasma concentrations of FGF‐21 and found that in the non‐fasted state plasma FGF‐21 concentrations were increased in Gcgr −/− mice (p = 0.005), but in the fasted state plasma FGF‐21 concentrations were similar to those of Gcgr +/+ mice (p = 0.4) (Figure 3d). In female Gcgr +/+ mice, fasting increased FGF‐21 concentrations (p = 0.008), whereas in Gcgr −/− mice fasting, if anything, lowered FGF‐21 concentrations (p = 0.051).

FIGURE 3.

FIGURE 3

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Non‐fasted female glucagon receptor knockout mice show increased liver triglyceride concentrations. (a) Plasma triglyceride, (b) glycerol, (c) nonesterified fatty acid (NEFA), (d) fibroblast growth factor 21 (FGF‐21), (e) liver triglyceride, and (f) liver glycogen (five of the measurements were under the detection limit and shown as 0 μg/mg) concentrations in non‐fasted (squares) and overnight fasted (16 h) (circles) female wild‐type littermates (Gcgr +/+, open symbols) and glucagon receptor knockout mice (Gcgr −/−, closed symbols). Data shown as mean ± SD, n = 7–12, mice 14–18 weeks of age. p value by unpaired t‐test.

3.2.2. Liver parameters

In the non‐fasted state, liver TG concentrations were increased in female Gcgr −/− mice (p = 0.02), whereas in the fasted state liver TG concentrations were similar in Gcgr −/− and Gcgr +/+ mice (p = 0.7) but increased compared to the non‐fasted state (Figure 3e), suggesting that fasting induced‐steatosis masks the steatosis‐promoting effect of permanent genetic deletion of the glucagon receptor. Liver glycogen concentrations were increased in Gcgr −/− mice compared to female Gcgr +/+ mice in the non‐fasted (p = 0.03) and fasted state (p = 0.007) (Figure 3f).

In male mice similar results were obtained with the exception of plasma TG concentrations being increased in non‐fasted male Gcgr +/+ compared to Gcgr −/− mice (p = 0.03), and the increased in liver TG concentrations being numerically higher in male Gcgr −/− compared to Gcgr +/+ mice (p = 0.0579) (Figure S2).

3.3. Glucagon receptor knockout mice show hypercholesterolemia

To further investigate the lipid metabolism in Gcgr −/− mice, we investigated another cohort of non‐fasted anesthetized Gcgr −/− and Gcgr +/+ female and male mice. Blood glucose concentrations in Gcgr −/− mice were, as expected, decreased compared to Gcgr +/+ mice (p = 0.0001) (Figure 4a). Body weights were similar in Gcgr −/− and Gcgr +/+ mice (females; p = 0.1, males; p = 0.9) (Figure 4b), possibly because of the small number of mice investigated. Liver and kidney weights were again increased in Gcgr −/− compared to Gcgr +/+ mice (both p < 0.0001) (Figure 4c,d). No difference in plasma TG, glycerol, or NEFA concentrations were found between the genotypes (p > 0.6, p > 0.9, and p > 0.9, respectively) (Figure 4e–g). Liver TG and glycogen concentrations were increased in Gcgr −/− compared to Gcgr +/+ mice (p = 0.0006 and p = 0.001, respectively) (Figure 4h,i). When liver TG concentrations were stratified by sex, both female and male Gcgr −/− mice had increased liver TG compared to Gcgr +/+ mice of the same sex (females; p = 0.006, males; p = 0.0003). Finally, we found plasma concentrations of low‐density lipoprotein (LDL) and high‐density lipoprotein (HDL) to be increased in Gcgr −/− when compared to Gcgr +/+ mice (Figure 4j,k).

FIGURE 4.

FIGURE 4

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Non‐fasted glucagon receptor knockout mice show hypercholesterolemia. (a) Blood glucose, (b) body weights, (c) liver weights, (d) kidney weights, (e) plasma triglyceride, (f) glycerol, (g) nonesterified fatty acid (NEFA), (h) liver triglyceride (one mouse, shown in gray, had an abnormally high liver triglyceride concentration (>30 μmol/g tissue) and was excluded), and (i) liver glycogen concentrations in wild‐type littermates (Gcgr +/+, black squares) and glucagon receptor knockout mice (Gcgr −/−, purple circles). Values obtained from female mice are shown in closed symbols and values obtained from male mice are shown in open symbols. Plasma cholesterol profiles from female (j) and male (k) Gcgr −/− and Gcgr +/+ mice. Very‐low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL). Data shown as mean ± SD, n = 4–15, mice 7–16 weeks of age. p value by unpaired t‐test.

3.4. Glucagon receptor knockout mice show lipid intolerance

To investigate postprandial lipid metabolism, we challenged a mixed cohort of male and female Gcgr −/− and Gcgr +/+ mice with an oral lipid tolerance test (olive oil, 10 μL/g body weight via oral gavage; OLTT). The mice were overnight fasted, and fasting blood glucose concentrations (0 min time‐point of the OLTT) were decreased in Gcgr −/− mice compared to Gcgr +/+ mice (p < 0.0001), while fasting TG and glycerol concentrations were numerically increased in Gcgr −/− mice (p = 0.09 and p = 0.09), whereas plasma NEFA concentrations were similar in Gcgr −/− and Gcgr +/+ mice (p = 0.5) (Figure 5).

FIGURE 5.

FIGURE 5

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Glucagon receptor knockout mice show lipid intolerance. (a) Blood glucose, (b) plasma triglyceride (c) glycerol, (d) non‐esterified fatty acid (NEFA) concentrations in glucagon receptor knockout mice (Gcgr −/−, purplee circles) and their wild‐type littermates (Gcgr +/+, black circles) during a lipid tolerance test (olive oil, 10 μL/g body weight). Data shown as mean ± SEM, n = 9–16, mice 10–14 weeks of age. p value by (a) two‐way ANOVA or (b–d) mixed‐effects analysis.

During the OLTT the Gcgr −/− mice had lower blood glucose concentrations than Gcgr +/+ mice (genotype effect p < 0.0001) (Figure 5a). After lipid administration plasma TG concentrations were increased in Gcgr −/− compared to mice Gcgr +/+ (genotype effect p > 0.03) (Figure 5b). Glycerol (genotype effect p > 0.2) (Figure 5c) and NEFA (genotype effect p > 0.09) (Figure 5d) concentrations were similar during the OLTT. Female and male mice are shown individually Figure S3.

3.5. Female glucagon receptor knockout mice show dyslipidemia and increased liver fat when challenged with GAN diet and HFD

To investigate whether the disturbances observed in lipid metabolism in Gcgr −/− mice would be augmented when challenged with a diet high in cholesterol and fat, and to investigated if mice lacking glucagon receptor signaling were able to tolerate such a diet, we challenged female Gcgr −/− and Gcgr +/+ mice with a GAN diet (the Gubra Amylin Nonalcoholic steatohepatitis (GAN) diet consisting of 40 kcal% fat, 20 kcal% fructose, and 2% cholesterol) for 5 weeks and in separate experiments female Gcgr −/− and Gcgr +/+ mice were challenged with a HFD (58 kcal% fat and sucrose) for 8 weeks.

Before the 5 weeks of GAN diet feeding, the female Gcgr −/− mice had a higher body weight than Gcgr +/+ mice (p = 0.03; these data are included in Figure 2a), however after 5 weeks of GAN diet feeding the Gcgr −/− mice had lost weight while the Gcgr +/+ mice had gained weight (−1.2 ± 0.8 vs. 3.2 ± 0.9 g) (Figure 6a). Blood glucose concentrations were lower in the Gcgr −/− mice before (data not shown) and after the diet (Figure 6b) (both p < 0.0001). After the 5 weeks of diet, plasma TG concentrations were increased in the Gcgr −/− compared to Gcgr +/+ mice (p = 0.04) (Figure 6c) (also after excluding the highest value measured in the Gcgr −/− mice (p < 0.0001)). Plasma NEFA (p = 0.055) (Figure 6d) and total cholesterol (p < 0.0001) (Figure 6e) concentrations were also increased in the Gcgr −/− compared to Gcgr +/+ mice. After the diet, the liver weights of the Gcgr −/− mice were increased compared to Gcgr +/+ mice (p < 0.0001) (Figure 6f). H&E staining's of the livers revealed that Gcgr −/− mice had a higher degree of hepatic lipid accumulation compared to Gcgr +/+ mice (representative sections shown in Figure 6g,h and steatosis score in Figure 6i). When quantifying the liver TG content enzymatically, the histology results were not confirmed as Gcgr −/− mice showed decreased amounts of extractable liver TGs compared to Gcgr +/+ mice (p < 0.0001) (Figure 6j). Consistent with our previous data, glycogen concentrations were increased after the GAN diet in the livers of Gcgr −/− mice (p = 0.001) (Figure 6k). Kidney weights were again increased in Gcgr −/− mice compared to Gcgr +/+ mice after the GAN diet (p = 0.01) (Figure 6l).

FIGURE 6.

FIGURE 6

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Female glucagon receptor knockout mice show dyslipidemia and increased liver fat when challenged with a GAN diet. (a) ΔBody weights, (b) blood glucose, (c) plasma triglyceride, (d) non‐esterified free fatty acid (NEFA), (e) cholesterol, (f) liver weights, (g) representive H&E staining of wild‐type littermates (Gcgr +/+) and (h) glucagon receptor knockout mice (Gcgr −/−) livers, (i) steatosis score, (j) liver triglyceride concentrations, (k) liver glycogen, and (l) kidney weights in female Gcgr +/+ (open symbols) and Gcgr −/− (closed symbols) mice after 5 weeks of Gubra Amylin Nonalcoholic steatohepatitis diet feeding. Data shown as mean ± SD, n = 10–12, mice 22–29 weeks of age. p value by unpaired t‐test.

The results observed after the GAN diet were replicated in female Gcgr −/− and Gcgr +/+ mice challenged with a HFD for 8 weeks (Figure 7). Again, when quantifying the liver TG content enzymatically, the histology results were not confirmed as Gcgr −/− mice showed decreased amounts of extractable liver TGs compared to Gcgr +/+ mice (p < 0.002) (Figure 7j). During the 8 weeks of HFD the Gcgr −/− gained significantly less fat mass compared to Gcgr +/+ (p = 0.0005) and tended to also gain less lean mass (p = 0.0517) (Figure S4).

FIGURE 7.

FIGURE 7

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Female glucagon receptor knockout mice show dyslipidemia and increased liver fat when challenged with a high‐fat diet. (a) ΔBody weights, (b) blood glucose, (c) plasma triglyceride, (d) nonesterified free fatty acid (NEFA), (e) cholesterol, (f) liver weights, (g) representive H&E staining of wild‐type littermates (Gcgr +/+) and (h) glucagon receptor knockout mice (Gcgr −/−) livers, (i) steatosis score, (j) liver triglyceride concentrations, (k) liver glycogen concentrations, and (l) kidney weights in female Gcgr +/+ (open symbols) and Gcgr −/− (closed symbols) mice 8 weeks after high‐fat diet feeding. Data shown as mean ± SD, n = 12–18, mice 16–25 weeks of age. p value by unpaired t‐test.

We repeated the HFD study, and fed a small cohort of female and male Gcgr −/− and Gcgr +/+ mice the same HFD for 8 weeks. The results were replicated except the change in body weight, plasma TG, plasma NEFA concentrations, liver TG, and steatosis score not being significantly different between Gcgr −/− and Gcgr +/+ mice. Additionally, we noticed that male Gcgr −/− mice showed hypercholesterolemia, increased liver weight, and steatosis to a lesser degree than female Gcgr −/− mice; however, our study was not powered to detect significant sex differences (Figure S5).

3.6. Lipidomics analysis reveals significant difference in the lipidome of GAN diet and HFD fed Gcgr−/− and Gcgr+/+ female mice

To investigate the discrepancy between the histological manifestation of hepatic lipid droplets and the enzymatic quantification of liver TG following the GAN diet and HFD in detail we conducted a mass‐spectromety based lipidomics analysis of liver biopsies from the Gcgr −/− and Gcgr +/+ female mice fed a GAN diet and HFD. When comparing all four groups (Gcgr −/− GAN diet, Gcgr +/+ GAN diet, Gcgr −/− HFD, and Gcgr +/+ HFD) the diets completely separated the four groups into two, thus revealing that the mice fed the GAN diet have a different lipidome that the mice fed the HFD (Figure S6a). Not surprisingly sterols were significantly upregulated in the livers of mice receiving the GAN diet (Figure S6b). Only six lipid species showed a significant effect due to genotype alone and four lipid species showed a significant interaction effect between genotype and diet (Figure S6c).

When comparing the Gcgr −/− and Gcgr +/+ fed the GAN diet, the mice clustered together based on genotype (Figure 8a). The Gcgr −/− showed enrichment of specific diacylglycerols, TG, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine species whereas sphingomyelin and cardiolipin species among others were less abundant (Figure 8b). In the Gcgr −/− 87 and 57 lipid species were found to be significantly down‐ and up‐regulated, respectively (Figure 8c), the 20 most down‐ and up‐regulated species are shown in Table S1.

FIGURE 8.

FIGURE 8

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Lipidomics analysis reveals significant difference in the lipidome of female glucagon receptor knockout mice challenged with a GAN diet. (a) Principal component analysis plot, (b) heat map, and (c) volcano plot (fold change treshold 2.0, p value treshold 0.05 false discovery rate corrected, direction of comparison KO_GAN_F/ WT_GAN_F) showing differencences in the lipidome of female glucagon receptor knockout mice (KO_GAN_F) and wild‐type littermates (WT_GAN_F) after 5 weeks of Gubra Amylin Nonalcoholic (GAN) steatohepatitis diet feeding.

When comparing the Gcgr −/− and Gcgr +/+ fed the HFD mice, the mice again clustered together based on genotype (Figure 9a). The Gcgr −/− mice showed enrichment of specific TG and sphingomyelin species among others whereas the Gcgr +/+ showed an enrichment of diacylglycerol species among others (Figure 9b). In the Gcgr −/− 56 and 41 lipid species were found to be significantly down‐ and up‐regulated, respectively (Figure 9c), the 20 most down‐ and up‐regulated species are shown in Table S2.

FIGURE 9.

FIGURE 9

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Lipidomics analysis reveals significant difference in the lipidome of female glucagon receptor knockout mice challenged with high‐fat diet. (a) Principal component analysis plot, (b) heat map, and (c) volcano plot (fold change treshold 2.0, p value treshold 0.05 false discovery rate corrected, direction of comparison KO_HFD_F/ WT_HFD_F) showing differencences in the lipodome of female glucagon receptor knockout mice (KO_HFD_F) and wild‐type littermates (WT_HFD_F) after 8 weeks of high‐fat diet feeding.

3.7. Female glucagon receptor knockout mice show a decreased urea index

To investigate how Gcgr −/− mice adapt protein metabolism in response to fasting we assessed plasma amino acid and urea concentrations in non‐fasted and fasted Gcgr −/− and Gcgr +/+ mice. Plasma amino acid concentrations were increased in non‐fasted (p < 0.0001) and fasted (p < 0.0001) female Gcgr −/− compared to Gcgr +/+ mice (Figure 10a). In female Gcgr +/+ mice, plasma amino acid concentrations did not differ between the non‐fasted and fasted state (p = 0.8), whereas in Gcgr −/− mice plasma amino acid concentrations decreased upon fasting (p = 0.005). Plasma urea concentrations were increased in non‐fasted (p = 0.02) and fasted (0.07) Gcgr −/− female mice compared to Gcgr +/+ mice (Figure 10b). In neither genotype did plasma urea concentrations change upon fasting (p > 0.2). The plasma urea index ([urea]/[amino acids]) was decreased in non‐fasted (p < 0.0001) and fasted (p < 0.0001) Gcgr −/− female mice compared to Gcgr +/+ mice (Figure 10c). In Gcgr +/+ mice, the urea index did not differ between the non‐fasted and fasted state (p = 0.7), whereas in Gcgr −/− mice the urea index increased upon fasting (p = 0.01).

FIGURE 10.

FIGURE 10

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Female glucagon receptor knockout mice show a decreased urea index. (a) Plasma amino acid, (b) urea concentrations, and (c) urea index in non‐fasted (squares) and overnight fasted (16 h) (circles) female wild‐type littermates (Gcgr +/+, open symbols) and glucagon receptor knockout mice (Gcgr −/−, closed symbols). Data shown as mean ± SD, n = 8–12, mice 14–18 weeks of age. p value by unpaired t‐test.

Similar results were obtained in male mice (Figure S7), with the exception of plasma urea concentrations being similar in non‐fasted (p = 0.2) and fasted (0.9) Gcgr −/− and Gcgr +/+ male mice, and that in Gcgr −/− mice plasma urea concentrations increased upon fasting (p = 0.04).

4. DISCUSSION

We here show that female Gcgr −/− mice challenged with a GAN diet (promoting MASH) and a HFD (promoting hepatic steatosis) were resistant to weight gain but prone to steatosis and dyslipidemia. When fed a chow diet, non‐fasted Gcgr −/− mice showed hypercholesterolemia (increased LDL and HDL plasma concentrations) and increased liver fat. The latter was masked by fasting‐induced steatosis. The increase in liver TG concentrations was borderline significant (p = 0.0579) in non‐fasted male Gcgr −/− mice (Figure S2), but significant (p < 0.02) in the remaining groups. Additionally, fasted Gcgr −/− mice, fed a standard chow diet, show impaired lipid tolerance. In response to overnight (16 h) fasting, Gcgr −/− mice, fed a standard chow diet, were able to decrease hepatic glycogen content and did not become hypoglycemic. Upon fasting, plasma amino acid concentrations decreased in Gcgr −/− mice while the urea‐index increased (suggesting increased substrate induced ureagenesis and thus increased amino acid catabolism), whereas these parameters remained unchanged in Gcgr +/+ mice. Taken together, our data suggest that in Gcgr −/− mice lipid metabolism is impaired and the impairment becomes augmented when the mice are challenge with a GAN diet and HFD, thus supporting glucagon as an important physiological regulator of not just glucose and amino acid, but also lipid metabolism.

During fasting, lipolysis in adipose tissue is increased, resulting in increased flux of lipids to the liver causing fasting‐induced steatosis. The TGs consequently stored in the liver are used for energy, and glucagon may be required for this adaptive response to fasting in mice (Longuet et al., 2008). A study showed that Gcgr −/− mice (both sexes) fasted for 16 h failed to increase hepatic lipid oxidation and showed an increase in hepatic lipid secretion, resulting in increased plasma TG concentrations (Longuet et al., 2008). In contrast, fasted and non‐fasted TG plasma concentrations were found to be similar in Gcgr −/− and Gcgr +/+ male mice, while fed TG plasma concentrations were 60% decreased in female Gcgr −/− mice (Gelling et al., 2003). Similar plasma TG and slightly elevated plasma cholesterol concentrations were found in male and female Gcgr −/− mice following a 14–16 h fast (Parker et al., 2002). This is consistent with the clinical finding that treatment with glucagon receptor antagonists increased plasma LDL, HDL, and TG concentrations in individuals with type 2 diabetes (Kazda et al., 2017). We also found non‐fasted Gcgr −/− mice, both males and females, to have increased LDL and HDL plasma concentrations. Similarly, plasma LDL was increased in non‐fasted Gcgr −/− mice (Gelling et al., 2003). In addition, Gcgr −/− mice showed impaired lipid tolerance during an OLTT following an overnight fast, as also shown previously (Galsgaard et al., 2022). We found a tendency to plasma TG concentrations to be numerically higher in Gcgr −/− mice, however this was dependent on the sex and fasting state of the mice. Male Gcgr −/− mice fed a HFD have been shown to be prone to steatosis (Longuet et al., 2008), and show a greater hepatic lipid accumulation, despite similar food intake and body weight, when fed a methionine and choline deficient diet (Sinclair et al., 2008). We likewise found that female Gcgr −/− mice were prone to steatosis and dyslipidemia in response a GAN diet and HFD. In contrast, resistance to HFD‐induced steatosis has been reported in male Gcgr −/− mice (Conarello et al., 2007). We did observe that male Gcgr −/− mice were not as prone to steatosis when fed a HFD as female Gcgr −/− mice were, however, our study was not powered to test for sex differences. Taken together, these studies do not give a final and conclusive answer to how parameters of lipid metabolism are changed when glucagon receptor signaling is inhibited, however most data, including the data presented in the current paper, points to a change in hepatic lipid metabolism that may be reflected in elevated plasma cholesterol and perhaps TG concentrations. Also, by quantifying the liver TG content in Gcgr −/− mice fed a HFD and GAN diet enzymatically, we were unable to explain the histology results. This prompted us to perform lipidomics on liver biopsies from these mice and here we found significant differences in the lipidome based on genotype, suggesting that permanent deletion of the glucagon receptor has significant effects on hepatic lipid metabolism.

Gcgr −/− mice have been reported to have similar plasma insulin levels and similar pancreatic insulin content when compared to control mice (Gelling et al., 2009; Parker et al., 2002). This is consistent with Gcgr −/− mice showing improved glycemic control and increased insulin sensitivity (Gelling et al., 2009; Parker et al., 2002). Increased plasma levels of glucagon‐like peptide 1 (GLP‐1) and FGF‐21 were found to contribute to the improved glycemic control in Gcgr −/− mice and to work in a complementary way to prevent postprandial hyperglycemia in mice lacking insulin and glucagon action (Omar et al., 2014). Increased hepatic insulin sensitivity might contribute to the observed hepatic lipid and glycogen accumulation in Gcgr −/− mice. This is supported by one of our previous studies in which mice treated with a glucagon receptor antibody showed an upregulation of hepatic genes involved in insulin response (Galsgaard et al., 2022). Insulin is also a powerful regulator of adipocyte lipolysis; however, basal lipolysis was similar in white adipose tissue of Gcgr −/− and control mice while epinephrine stimulated white adipose tissue lipolysis was increased in Gcgr −/− mice when compared to control mice (Gelling et al., 2009). This might explain the observed tendency to increased free fatty acid plasma concentrations in Gcgr −/− mice.

It is evident from studies in Gcgr −/− mice that additional compensatory mechanisms exist to prevent hypoglycemia. These were investigated in (Gelling et al., 2009), suggesting that Gcgr −/− mice may compensate for loss of glucagon receptor signaling in part by increased basal cAMP signaling in the liver as well as an increased responsiveness to epinephrine in the liver and white adipose tissue as both basal and epinephrine stimulated cAMP levels in liver membranes from Gcgr −/− mice were increased and epinephrine stimulated (but not basal) white adipose tissue lipolysis was increased in knockout mice when compared to control.

Glucagon administration reduce food intake through reduced meal size and increased satiety (Bagger et al., 2015; Geary et al., 1992, 1993; Holloway & Stevenson, 1964; Langhans et al., 1982; Le Sauter & Geary, 1991; Martin & Novin, 1977; Penick & Hinkle Jr, 1961; Schulman et al., 1957; Stunkard et al., 1955; Weick & Ritter, 1986) and glucagon receptor agonism may result in body weight loss (Hinds et al., 2021; Pedersen et al., 2018). Conversely, treatment with certain inhibitors of glucagon receptor signaling have been reported to increase body weight (Engel et al., 2011; Guzman et al., 2017; Kazda et al., 2017). We found Gcgr −/− mice fed a standard chow diet to have increased body weight compared to Gcgr +/+ mice, and this was associated with an increase in both lean and fat mass. In contrast to our findings, Gcgr −/− mice have been shown to have increased wholebody lean mass and decreased fat mass (Gelling et al., 2003; Hinds et al., 2021), and body weight have been found to be similar in whole body Gcgr −/− and wild‐type mice given a standard chow diet (Gelling et al., 2003). Mice carrying a naturally occurring deleterious glucagon receptor mutation also showed unaltered food intake and body weight with reduced adiposity (Lin et al., 2020). Mice with a partially inactivating mutation in the glucagon receptor gene showed increased body weight from 9 to 11 months of age (Xu et al., 2024). When fed a GAN diet and HFD, we found female Gcgr −/− mice to not gain weight whereas the Gcgr +/+ mice gained weight as expected. This is consistent with a study showing that Gcgr −/− mice fed a HFD for 8 weeks did not gain weight while the wild‐type mice did (Longuet et al., 2008). Furthermore, wild‐type mice given a HFD for 12 weeks gained more weight than Gcgr −/− mice (Conarello et al., 2007). This was most likely due to the Gcgr −/− mice consuming less HFD than the wild‐type mice (Conarello et al., 2007). In both studies the Gcgr −/− mice had a reduced fat mass compared to wild‐type mice after the HFD feeding. However, in one of the studies (Longuet et al., 2008) the Gcgr −/− mice developed steatosis to a higher degree than the wild‐type mice, while in the other study the Gcgr −/− mice developed less steatosis compared to the wild‐type mice (Conarello et al., 2007).

The liver is a highly sexually dimorphic organ. In response to a short‐term fast, female mice use amino acids as substrates to increase lipid storage in the liver, whereas males simply slow down anabolic pathways (Della Torre et al., 2018). FGF‐21 regulates transcriptional and metabolic responses in the liver, and reduced liver TG concentrations, in a sex‐dependent manner (Chaffin et al., 2022). In diet‐induced obese male mice FGF‐21 decreases hepatic triglyceride levels (in a weight‐loss independent manner) and decreases body weight via loss of fat mass, neither of which were observed in diet‐induced obese female mice (Chaffin et al., 2022). FGF‐21 increased caloric intake and improved glucose tolerance in both male and female mice (Chaffin et al., 2022). FGF‐21 drives the reduction in liver TGs partly through adiponectin, whereas FGF‐21 did not increase adiponectin levels in female mice (Chaffin et al., 2022). FGF‐21 works via the brain to reduce adipose tissue, and it was suggested that FGF‐21 treatment (e.g., high FGF‐21 levels) promotes a “FGF‐21‐resistant” state in white adipose tissue of female mice thereby preserving lipid storage (Chaffin et al., 2022). These sex‐differences may to some degree explain the differences we observed in this study. We previously performed RNA sequencing of livers from Gcgr −/− and Gcgr +/+ male mice fasted for 4 h and found FGF‐21 expression to be upregulated (Winther‐Sørensen et al., 2020), this is in line with the increased FGF‐21 plasma concentrations observed in both male and female Gcgr −/− mice. FGF‐21 has been reported to act centrally to induce sympathetic nerve activity, energy expenditure, and weight loss (Owen et al., 2014). Hypoglycemia also increases sympathetic nerve activity. Thus, it is possible that sympathetic nerve activity is increased in Gcgr −/− mice. This would potentially affect lipid metabolism and might explain the resistance to weight gain when Gcgr −/− mice were put on the GAN diet and HFD. However, an increased sympathetic nerve activity would contrast to the finding that the chow‐fed Gcgr −/− mice had increased body weight compared to Gcgr +/+ mice and that Gcgr −/− mice had similar basal epinephrine levels compared to control mice (Gelling et al., 2009).

During fasting amino acids are released from the muscles, and catabolized and converted to glucose thus increasing ureagenesis (Li et al., 2016; Schimke, 1962); however, urea is rapidly excreted in the urine, a process that may be stimulated by glucagon (Ahloulay et al., 1995). Amino acid concentrations decreased during fasting in Gcgr −/− mice but remained unchanged in Gcgr +/+ mice. The rapid urea excretion most likely explains the similar urea plasma concentrations in non‐fasted and fasted Gcgr +/+ mice. The decreased urea index in Gcgr −/− mice compared to Gcgr +/+ mice may reflect an impaired amino acid catabolism (decreased ureagenesis). The increase in the urea index upon fasting in Gcgr −/− mice may be caused by an increased urea plasma concentration due to impaired renal excretion (perhaps a consequence of glucagon receptor deletion as glucagon increase renal urea excretion (Ahloulay et al., 1992; Ahloulay et al., 1995) and the glucagon receptor is express in the kidney (Bomholt et al., 2021)), combined with a decreased amino acid plasma concentration due to lack of muscle derived plasma amino acids. In line with this urine urea in Gcgr −/− mice were reduced, however the daily excretion of urea in the urine and blood nitrogen levels were similar to wild‐type mice (Wang et al., 2024).

Glucagon‐based drugs are currently being investigated as therapeutic tools in the treatment of obesity and MASLD. In obese or overweight individuals with type 2 diabetes, a GLP‐1/glucagon co‐agonist decreased hepatic fat content (Robertson et al., 2020), and in mice with diet‐induced MASH the co‐agonist reduced body weight and liver TG concentrations (Trevaskis et al., 2020). A long‐acting glucagon analog decreased plasma and hepatic TG concentrations and decreased body weight in diet induced obese mice; the effect was mainly on fat mass and not lean mass (Lee et al., 2020). Glucagon analog treatment moreover resulted in a greater weight loss than GLP‐1 agonists (Lee et al., 2019). However a concern for loss of lean mass has been raised due to glucagon‐induced hypoaminoacidemia, this might be prevented by protein supplementation which can defend lean mass at a glucagon dose of that is sub‐anorectic and does not reduce fat mass, but still reduce steatosis and glycemia in obese mice (Hope et al., 2022; Lopes et al., 2024).

Our data show that female mice with genetic deletion of the glucagon receptor are prone to steatosis and dyslipidemia when fed a GAN diet and HFD. Thus, our data highlight glucagon as an important physiological regulator of not just glucose, but also hepatic lipid metabolism.

AUTHOR CONTRIBUTIONS

Conceptualization; K.D.G., N.J.W.A., and J.J.H. Data curation; K.D.G., E.E., J.E.H., M.M.S., N.J.F., and J.H. Formal analysis; K.D.G., N.J.F., J.H., N.J.W.A., and J.J.H. Funding acquisition; K.D.G., N.J.W.A., and J.J.H. Project administration/supervision; N.J.W.A. and J.J.H. Writing—original draft; K.D.G. Writing—review and editing; E.E., J.E.H., M.M.S., T.J.G., C.C., N.J.F., J.H., N.J.W.A., and J.J.H. All authors approved the final version of the manuscript.

FUNDING INFORMATION

The project is supported by grants from the A.P. Møller Foundation; NNF Tandem Programme (NNF Application Number: 31526); NNF Project Support in Endocrinology and Metabolism–Nordic Region (NNF Application Number: 34250). Katrine D. Galsgaard is supported by the BRIDGE‐Translational Excellence Programme (bridge.ku.dk) at Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, funded by the Novo Nordisk Foundation. Grant agreement no. NNF20SA0064340. Associate Prof. Nicolai J. Wewer Albrechtsen is supported by NNF Excellence Emerging Investigator Grant – Endocrinology and Metabolism (Application No. NNF19OC0055001), EFSD Future Leader Award (NNF21SA0072746) and DFF Sapere Aude (1052‐00003B). Prof. Jens Juul Holst is supported by the Novo Nordisk Foundation (NNF) Center for Basic Metabolic Research University of Copenhagen (NNF Application Number: 13563). Novo Nordisk Foundation Center for Basic Metabolic Research is an independent Research Center, based at the University of Copenhagen, Denmark and partially funded by an unconditional donation from the Novo Nordisk Foundation (Grant number NNF18CC0034900 and NNF23SA0084103). Emilie Elmelund is supported by the Novo Scholarship Program (2022). N.J.F. received funding from Independent Research Fund Denmark (grant no. 2032‐00062B). Lipidomics work was performed within the Integra Infrastructure supported by the Novo Nordisk Foundation (NNF20OC0061575).

CONFLICT OF INTEREST STATEMENT

None.

PRIOR PRESENTATION

Some of the data were presented at American Diabetes Association's 82nd Scientific Session, June 3rd–7th, New Orleans, LA, USA.

ETHICS STATEMENT

Animal studies were conducted at the animal facilities at the Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, with permission from the Danish Animal Experiments Inspectorate, Ministry of Environment and Food of Denmark, permit 2018‐15‐0201‐01397. All studies were approved by the local ethical committee; Department of Experimental Medicine – University of Copenhagen.

Supporting information

Appendix S1.

PHY2-13-e70235-s001.docx (3.5MB, docx)

ACKNOWLEDGMENTS

We thank Maureen J. Charron, Departments of Biochemistry, Obstetrics and Gynecology and Women's Health, and Medicine, Albert Einstein College of Medicine, New York, for providing glucagon receptor knockout mice. We thank laboratory technician Christine Rasmussen for assistance at the Department of Clinical Biochemistry, Rigshospitalet. We thank the Rodent Metabolic Phenotyping platform at Center for Metabolic Research for providing MRI scanner access and assistance. We thank Biomedical Laboratory Technologist Heidi Paulsen and Biomedical Laboratory Technologist Bente Stærgaard for performing the histological staining's and Associate Professor Jens Christian Brings Jacobsen and Associate professor Cathrine Ørskov for assistance with the histological analysis. The graphical abstract was created with BioRender.com.

Galsgaard, K. D. , Elmelund, E. , Hunt, J. E. , Smits, M. M. , Grevengoed, T. J. , Christoffersen, C. , Færgeman, N. J. , Havelund, J. , Wewer Albrechtsen, N. J. , & Holst, J. J. (2025). Female glucagon receptor knockout mice are prone to steatosis but resistant to weight gain when fed a MASH‐promoting GAN diet and a high‐fat diet. Physiological Reports, 13, e70235. 10.14814/phy2.70235

DATA AVAILABILITY STATEMENT

All data reported in this paper will be shared by the lead contact upon request. All lipidomics data is available at Figshare (https://doi.org/10.6084/m9.figshare.27924528.v1).

REFERENCES

  1. Ahloulay, M. , Bouby, N. , Machet, F. , Kubrusly, M. , Coutaud, C. , & Bankir, L. (1992). Effects of glucagon on glomerular filtration rate and urea and water excretion. The American Journal of Physiology, 263(1 Pt 2), F24–F36. [DOI] [PubMed] [Google Scholar]
  2. Ahloulay, M. , Déchaux, M. , Laborde, K. , & Bankir, L. (1995). Influence of glucagon on GFR and on urea and electrolyte excretion: Direct and indirect effects. American Journal of Physiology, 269(2 Pt 2), F225–F235. [DOI] [PubMed] [Google Scholar]
  3. Amatuzio, D. S. , Grande, F. , & Wada, S. (1962). Effect of glucagon on the serum lipids in essential hyperlipemia and in hypercholesterolemia. Metabolism, 11, 1240–1249. [PubMed] [Google Scholar]
  4. Bagger, J. I. , Holst, J. J. , Hartmann, B. , Andersen, B. , Knop, F. K. , & Vilsbøll, T. (2015). Effect of oxyntomodulin, glucagon, GLP‐1, and combined glucagon +GLP‐1 infusion on food intake, appetite, and resting energy expenditure. The Journal of Clinical Endocrinology and Metabolism, 100(12), 4541–4552. [DOI] [PubMed] [Google Scholar]
  5. Boden, G. , Rezvani, I. , & Owen, O. E. (1984). Effects of glucagon on plasma amino acids. Journal of Clinical Investigation, 73(3), 785–793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bomholt, A. B. , Johansen, C. D. , Kjeldsen, S. A. , Galsgaard, K. D. , Christensen, J. B. , Winther‐Sørensen, M. , Serizawa, R. , Hornum, M. , Porrini, E. , Pedersen, J. , Ørskov, C. , Gluud, L. L. , Sørensen, C. M. , Holst, J. J. , Albrechtsen, R. , & Wewer Albrechtsen, N. J. (2021). Evaluation of commercially available glucagon receptor antibodies and glucagon receptor expression. bioRxiv . 10.1101/2021.12.21.473442 [DOI] [PMC free article] [PubMed]
  7. Chaffin, A. T. , Larson, K. R. , Huang, K. P. , Wu, C. T. , Godoroja, N. , Fang, Y. , Jayakrishnan, D. , Soto Sauza, K. A. , Sims, L. C. , Mohajerani, N. , Goodson, M. L. , & Ryan, K. K. (2022). FGF21 controls hepatic lipid metabolism via sex‐dependent interorgan crosstalk. Journal of Clinical Investigation. Insight, 7(19), e155848. 10.1172/jci.insight.155848 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Conarello, S. L. , Jiang, G. , Mu, J. , Li, Z. , Woods, J. , Zycband, E. , Ronan, J. , Liu, F. , Roy, R. S. , Zhu, L. , Charron, M. J. , & Zhang, B. B. (2007). Glucagon receptor knockout mice are resistant to diet‐induced obesity and streptozotocin‐mediated beta cell loss and hyperglycaemia. Diabetologia, 50(1), 142–150. [DOI] [PubMed] [Google Scholar]
  9. Coskun, T. , Urva, S. , Roell, W. C. , Qu, H. , Loghin, C. , Moyers, J. S. , O'Farrell, L. S. , Briere, D. A. , Sloop, K. W. , Thomas, M. K. , Pirro, V. , Wainscott, D. B. , Willard, F. S. , Abernathy, M. , Morford, L. R. , du, Y. , Benson, C. , Gimeno, R. E. , Haupt, A. , & Milicevic, Z. (2022). LY3437943, a novel triple glucagon, GIP, and GLP‐1 receptor agonist for glycemic control and weight loss: From discovery to clinical proof of concept. Cell Metabolism, 34, 1234–1247.e9. [DOI] [PubMed] [Google Scholar]
  10. Day, J. W. , Ottaway, N. , Patterson, J. T. , Gelfanov, V. , Smiley, D. , Gidda, J. , Findeisen, H. , Bruemmer, D. , Drucker, D. J. , Chaudhary, N. , Holland, J. , Hembree, J. , Abplanalp, W. , Grant, E. , Ruehl, J. , Wilson, H. , Kirchner, H. , Lockie, S. H. , Hofmann, S. , … Tschöp, M. H. (2009). A new glucagon and GLP‐1 co‐agonist eliminates obesity in rodents. Nature Chemical Biology, 5(10), 749–757. [DOI] [PubMed] [Google Scholar]
  11. Dean, E. D. , Li, M. , Prasad, N. , Wisniewski, S. N. , Von Deylen, A. , Spaeth, J. , Maddison, L. , Botros, A. , Sedgeman, L. R. , Bozadjieva, N. , Ilkayeva, O. , Coldren, A. , Poffenberger, G. , Shostak, A. , Semich, M. C. , Aamodt, K. I. , Phillips, N. , Yan, H. , Bernal‐Mizrachi, E. , … Powers, A. C. (2017). Interrupted glucagon signaling reveals hepatic alpha‐cell axis and role for L‐glutamine in alpha‐cell proliferation. Cell Metabolism, 25(6), 1362–1373.e5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Della Torre, S. , Mitro, N. , Meda, C. , Lolli, F. , Pedretti, S. , Barcella, M. , Ottobrini, L. , Metzger, D. , Caruso, D. , & Maggi, A. (2018). Short‐term fasting reveals amino acid metabolism as a major sex‐discriminating factor in the liver. Cell Metabolism, 28(2), 256–267.e5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Eaton, R. P. (1973). Hypolipemic action of glucagon in experimental endogenous lipemia in the rat. Journal of Lipid Research, 14(3), 312–318. [PubMed] [Google Scholar]
  14. Engel, S. S. , Xu, L. , Andryuk, P. J. , Davies, M. J. , Amatruda, J. , Kaufman, K. , & Goldstein, B. J. (2011). Efficacy and tolerability of MK‐0893, a glucagon receptor antagonist (GRA), in patients with type 2 diabetes (T2DM). Diabetes, 60, A85. [Google Scholar]
  15. Finan, B. , Yang, B. , Ottaway, N. , Smiley, D. L. , Ma, T. , Clemmensen, C. , Chabenne, J. , Zhang, L. , Habegger, K. M. , Fischer, K. , Campbell, J. E. , Sandoval, D. , Seeley, R. J. , Bleicher, K. , Uhles, S. , Riboulet, W. , Funk, J. , Hertel, C. , Belli, S. , … Tschöp, M. H. (2015). A rationally designed monomeric peptide triagonist corrects obesity and diabetes in rodents. Nature Medicine, 21(1), 27–36. [DOI] [PubMed] [Google Scholar]
  16. Fitzpatrick, G. F. , Meguid, M. M. , Gitilitz, P. H. , & Brennan, M. F. (1977). Glucagon infusion in normal man: Effects on 3‐methylhistidine excretion and plasma amino acids. Metabolism, 26(5), 477–485. [DOI] [PubMed] [Google Scholar]
  17. Galsgaard, K. D. (2020). The vicious circle of hepatic glucagon resistance in non‐alcoholic fatty liver disease. Journal of Clinical Medicine, 9(12), 4049. 10.3390/jcm9124049 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Galsgaard, K. D. , Elmelund, E. , Johansen, C. D. , Bomholt, A. B. , Kizilkaya, H. S. , Ceutz, F. , Hunt, J. E. , Kissow, H. , Winther‐Sørensen, M. , Sørensen, C. M. , Kruse, T. , Lau, J. F. , Rosenkilde, M. M. , Ørskov, C. , Christoffersen, C. , Holst, J. J. , & Wewer Albrechtsen, N. J. (2022). Glucagon receptor antagonism impairs and glucagon receptor agonism enhances triglycerides metabolism in mice. Molecular Metabolism, 66, 101639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Geary, N. , Kissileff, H. R. , Pi‐Sunyer, F. X. , & Hinton, V. (1992). Individual, but not simultaneous, glucagon and cholecystokinin infusions inhibit feeding in men. American Journal of Physiology, 262(6 Pt 2), R975–R980. [DOI] [PubMed] [Google Scholar]
  20. Geary, N. , Le Sauter, J. , & Noh, U. (1993). Glucagon acts in the liver to control spontaneous meal size in rats. American Journal of Physiology, 264(1 Pt 2), R116–R122. [DOI] [PubMed] [Google Scholar]
  21. Gelling, R. W. , du, X. Q. , Dichmann, D. S. , Rømer, J. , Huang, H. , Cui, L. , Obici, S. , Tang, B. , Holst, J. J. , Fledelius, C. , Johansen, P. B. , Rossetti, L. , Jelicks, L. A. , Serup, P. , Nishimura, E. , & Charron, M. J. (2003). Lower blood glucose, hyperglucagonemia, and pancreatic alpha cell hyperplasia in glucagon receptor knockout mice. Proceedings of the National Academy of Sciences of the United States of America, 100(3), 1438–1443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gelling, R. W. , Vuguin, P. M. , du, X. Q. , Cui, L. , Rømer, J. , Pederson, R. A. , Leiser, M. , Sørensen, H. , Holst, J. J. , Fledelius, C. , Johansen, P. B. , Fleischer, N. , McIntosh, C. H. S. , Nishimura, E. , & Charron, M. J. (2009). Pancreatic beta‐cell overexpression of the glucagon receptor gene results in enhanced beta‐cell function and mass. American Journal of Physiology. Endocrinology and Metabolism, 297(3), E695–E707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Guettet, C. , Mathe, D. , Riottot, M. , & Lutton, C. (1988). Effects of chronic glucagon administration on cholesterol and bile acid metabolism. Biochimica et Biophysica Acta, 963(2), 215–223. [DOI] [PubMed] [Google Scholar]
  24. Guettet, C. , Rostaqui, N. , Mathe, D. , Lecuyer, B. , Navarro, N. , & Jacotot, B. (1991). Effect of chronic glucagon administration on lipoprotein composition in normally fed, fasted and cholesterol‐fed rats. Lipids, 26(6), 451–458. [DOI] [PubMed] [Google Scholar]
  25. Guettet, C. , Rostaqui, N. , Navarro, N. , Lecuyer, B. , & Mathe, D. (1991). Effect of chronic glucagon administration on the metabolism of triacylglycerol‐rich lipoproteins in rats fed a high sucrose diet. The Journal of Nutrition, 121(1), 24–30. [DOI] [PubMed] [Google Scholar]
  26. Guzman, C. B. , Zhang, X. M. , Liu, R. , Regev, A. , Shankar, S. , Garhyan, P. , Pillai, S. G. , Kazda, C. , Chalasani, N. , & Hardy, T. A. (2017). Treatment with LY2409021, a glucagon receptor antagonist, increases liver fat in patients with type 2 diabetes. Diabetes, Obesity & Metabolism, 19(11), 1521–1528. [DOI] [PubMed] [Google Scholar]
  27. Hamberg, O. , & Vilstrup, H. (1994). Regulation of urea synthesis by glucose and glucagon in normal man. Clinical Nutrition, 13(3), 183–191. [DOI] [PubMed] [Google Scholar]
  28. Hansen, S. S. K. , Krautz, R. , Rago, D. , Havelund, J. , Stigliani, A. , Færgeman, N. J. , Prézelin, A. , Rivière, J. , Couturier‐Tarrade, A. , Akimov, V. , Blagoev, B. , Elfving, B. , Neess, D. , Vogel, U. , Khodosevich, K. , Hougaard, K. S. , & Sandelin, A. (2024). Pulmonary maternal immune activation does not cross the placenta but leads to fetal metabolic adaptation. Nature Communications, 15(1), 4711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Heebøll, S. , Risikesan, J. , Ringgaard, S. , Kumarathas, I. , Sandahl, T. D. , Grønbæk, H. , Søndergaard, E. , & Nielsen, S. (2022). Impaired glucagon‐mediated suppression of VLDL‐triglyceride secretion in individuals with metabolic dysfunction‐associated fatty liver disease (MAFLD). Diabetes, 71(11), 2402–2411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Henderson, S. J. , Konkar, A. , Hornigold, D. C. , Trevaskis, J. L. , Jackson, R. , Fritsch Fredin, M. , Jansson‐Löfmark, R. , Naylor, J. , Rossi, A. , Bednarek, M. A. , Bhagroo, N. , Salari, H. , Will, S. , Oldham, S. , Hansen, G. , Feigh, M. , Klein, T. , Grimsby, J. , Maguire, S. , … Coghlan, M. P. (2016). Robust anti‐obesity and metabolic effects of a dual GLP‐1/glucagon receptor peptide agonist in rodents and non‐human primates. Diabetes, Obesity & Metabolism, 18(12), 1176–1190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Hinds, C. E. , Owen, B. M. , Hope, D. C. D. , Pickford, P. , Jones, B. , Tan, T. M. , Minnion, J. S. , & Bloom, S. R. (2021). A glucagon analogue decreases body weight in mice via signalling in the liver. Scientific Reports, 11(1), 22577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Holloway, S. A. , & Stevenson, J. A. (1964). Effect of glucagon on food intake and weight gain in the young rat. Canadian Journal of Physiology and Pharmacology, 42, 867–869. [DOI] [PubMed] [Google Scholar]
  33. Hope, D. C. D. , Hinds, C. E. , Lopes, T. , Vincent, M. L. , Shrewsbury, J. V. , Yu, A. T. C. , Davies, I. , Scott, R. , Jones, B. , Murphy, K. G. , Minnion, J. S. , Sardini, A. , Carling, D. , Lutz, T. A. , Bloom, S. R. , Tan, T. M. M. , & Owen, B. M. (2022). Hypoaminoacidemia underpins glucagon‐mediated energy expenditure and weight loss. Cell Reports Medicine, 3(11), 100810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Inagaki, T. , Dutchak, P. , Zhao, G. , Ding, X. , Gautron, L. , Parameswara, V. , Li, Y. , Goetz, R. , Mohammadi, M. , Esser, V. , Elmquist, J. K. , Gerard, R. D. , Burgess, S. C. , Hammer, R. E. , Mangelsdorf, D. J. , & Kliewer, S. A. (2007). Endocrine regulation of the fasting response by PPARalpha‐mediated induction of fibroblast growth factor 21. Cell Metabolism, 5(6), 415–425. [DOI] [PubMed] [Google Scholar]
  35. Jiang, G. , & Zhang, B. B. (2003). Glucagon and regulation of glucose metabolism. American Journal of Physiology. Endocrinology and Metabolism, 284(4), E671–E678. [DOI] [PubMed] [Google Scholar]
  36. Junker, A. E. (2017). The role of incretin hormones and glucagon in patients with liver disease. Danish Medical Journal, 64(5), B5363. [PubMed] [Google Scholar]
  37. Junker, A. E. , Gluud, L. , Holst, J. J. , Knop, F. K. , & Vilsbøll, T. (2016). Diabetic and nondiabetic patients with nonalcoholic fatty liver disease have an impaired incretin effect and fasting hyperglucagonaemia. Journal of Internal Medicine, 279(5), 485–493. [DOI] [PubMed] [Google Scholar]
  38. Kazda, C. M. , Frias, J. , Foga, I. , Cui, X. , Guzman, C. B. , Garhyan, P. , Heilmann, C. , Yang, J. A. , & Hardy, T. A. (2017). Treatment with the glucagon receptor antagonist LY2409021 increases ambulatory blood pressure in patients with type 2 diabetes. Diabetes, Obesity & Metabolism, 19(8), 1071–1077. [DOI] [PubMed] [Google Scholar]
  39. Kind, T. , Liu, K. H. , Lee, D. Y. , DeFelice, B. , Meissen, J. K. , & Fiehn, O. (2013). LipidBlast in silico tandem mass spectrometry database for lipid identification. Nature Methods, 10(8), 755–758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Kleiner, D. E. , Brunt, E. M. , Van Natta, M. , Behling, C. , Contos, M. J. , Cummings, O. W. , Ferrell, L. D. , Liu, Y. C. , Torbenson, M. S. , Unalp‐Arida, A. , Yeh, M. , McCullough, A. J. , Sanyal, A. J. , & Nonalcoholic Steatohepatitis Clinical Research Network . (2005). Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology, 41(6), 1313–1321. [DOI] [PubMed] [Google Scholar]
  41. Langhans, W. , Zeiger, U. , Scharrer, E. , & Geary, N. (1982). Stimulation of feeding in rats by intraperitoneal injection of antibodies to glucagon. Science, 218(4575), 894–896. [DOI] [PubMed] [Google Scholar]
  42. Le Sauter, J. , & Geary, N. (1991). Hepatic portal glucagon infusion decreases spontaneous meal size in rats. American Journal of Physiology, 261(1 Pt 2), R154–R161. [DOI] [PubMed] [Google Scholar]
  43. Lee, J. S. , Kim, J. K. , Kim, J. , Park, E. , Lee, S. H. , Bae, S. , & Choi, I. Y. (2019). 109‐LB: Potent weight loss mechanism by a novel long‐acting glucagon analog, HM15136, in animal models. Diabetes, 68(Supplement 1), 109.30352879 [Google Scholar]
  44. Lee, S. , Lee, J. , Choi, J. , Park, E. , Lee, J. , Lee, S. , Bae, S. , Kim, D. , & Choi, I. (2020). Beneficial effects of a novel long‐acting glucagon analog, HM15136, on obesity and obesity‐related metabolic disorders in animal models. Diabetes, 69(Supplement 1), 1925. [Google Scholar]
  45. Li, L. , Zhang, P. , Bao, Z. , Wang, T. , Liu, S. , & Huang, F. (2016). PGC‐1alpha promotes ureagenesis in mouse periportal hepatocytes through SIRT3 and SIRT5 in response to glucagon. Scientific Reports, 6, 24156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Lin, G. , Liu, Q. , Dai, A. , Cai, X. , Zhou, Q. , Wang, X. , Chen, Y. , Ye, C. , Li, J. , Yang, D. , & Wang, M. W. (2020). Characterization of a naturally occurring mutation V368M in the human glucagon receptor and its association with metabolic disorders. Biochemical Journal, 477(13), 2581–2594. [DOI] [PubMed] [Google Scholar]
  47. Longuet, C. , Sinclair, E. M. , Maida, A. , Baggio, L. L. , Maziarz, M. , Charron, M. J. , & Drucker, D. J. (2008). The glucagon receptor is required for the adaptive metabolic response to fasting. Cell Metabolism, 8(5), 359–371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Lopes, T. , Hope, D. C. D. , Ramos‐Pittol, J. M. , Curtis, A. , Shrewsbury, J. V. , Davies, I. , Zhou, Z. , Sardini, A. , Minnion, J. S. , Dormann, D. , Bewick, G. A. , Murphy, K. G. , Carling, D. , Bloom, S. R. , Tan, T. M. M. , & Owen, B. M. (2024). Dietary protein defends lean mass and maintains the metabolic benefits of glucagon receptor agonism in mice. Molecular Metabolism, 89, 102024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Luan, H. , Ji, F. , Chen, Y. , & Cai, Z. (2018). statTarget: A streamlined tool for signal drift correction and interpretations of quantitative mass spectrometry‐based omics data. Analytica Chimica Acta, 1036, 66–72. [DOI] [PubMed] [Google Scholar]
  50. Martin, J. R. , & Novin, D. (1977). Decreased feeding in rats following hepatic‐portal infusion of glucagon. Physiology & Behavior, 19(4), 461–466. [DOI] [PubMed] [Google Scholar]
  51. Omar, B. A. , Andersen, B. , Hald, J. , Raun, K. , Nishimura, E. , & Ahrén, B. (2014). Fibroblast growth factor 21 (FGF21) and glucagon‐like peptide 1 contribute to diabetes resistance in glucagon receptor‐deficient mice. Diabetes, 63(1), 101–110. [DOI] [PubMed] [Google Scholar]
  52. Owen, B. M. , Ding, X. , Morgan, D. A. , Coate, K. C. , Bookout, A. L. , Rahmouni, K. , Kliewer, S. A. , & Mangelsdorf, D. J. (2014). FGF21 acts centrally to induce sympathetic nerve activity, energy expenditure, and weight loss. Cell Metabolism, 20(4), 670–677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Pang, Z. , Chong, J. , Zhou, G. , de Lima Morais, D. A. , Chang, L. , Barrette, M. , Gauthier, C. , Jacques, P. É. , Li, S. , & Xia, J. (2021). MetaboAnalyst 5.0: Narrowing the gap between raw spectra and functional insights. Nucleic Acids Research, 49(W1), W388–w396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Parker, J. C. , Andrews, K. M. , Allen, M. R. , Stock, J. L. , & McNeish, J. D. (2002). Glycemic control in mice with targeted disruption of the glucagon receptor gene. Biochemical and Biophysical Research Communications, 290(2), 839–843. [DOI] [PubMed] [Google Scholar]
  55. Pedersen, C. , Porsgaard, T. , Thomsen, M. , Rosenkilde, M. M. , & Roed, N. K. (2018). Sustained effect of glucagon on body weight and blood glucose: Assessed by continuous glucose monitoring in diabetic rats. PLoS One, 13(3), e0194468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Pegorier, J. P. , Garcia‐Garcia, M. V. , Prip‐Buus, C. , Duée, P. H. , Kohl, C. , & Girard, J. (1989). Induction of ketogenesis and fatty acid oxidation by glucagon and cyclic AMP in cultured hepatocytes from rabbit fetuses. Evidence for a decreased sensitivity of carnitine palmitoyltransferase I to malonyl‐CoA inhibition after glucagon or cyclic AMP treatment. The Biochemical Journal, 264(1), 93–100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Penick, S. B. , & Hinkle, L. E., Jr. (1961). Depression of food intake induced in healthy subjects by glucagon. New England Journal of Medicine, 264, 893–897. [DOI] [PubMed] [Google Scholar]
  58. Ramnanan, C. J. , Edgerton, D. S. , Kraft, G. , & Cherrington, A. D. (2011). Physiologic action of glucagon on liver glucose metabolism. Diabetes, Obesity & Metabolism, 13(Suppl 1), 118–125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Richter, M. M. , Galsgaard, K. D. , Elmelund, E. , Knop, F. K. , Suppli, M. P. , Holst, J. J. , Winther‐Sørensen, M. , Kjeldsen, S. A. S. , & Wewer Albrechtsen, N. J. (2022). The liver‐alpha‐cell axis in health and in disease. Diabetes Care, 71(9), 1852–1861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Robertson, D. , Hansen, L. , Ambery, P. , Esterline, R. L. , Jermutus, L. , Chang, Y. T. , Petrone, M. , Johansson, E. , Johansson, L. , Sjöberg, F. B. , & Parker, V. (2020). 354‐OR: Cotadutide (medi0382), a dual receptor agonist with glucagon‐like Peptide‐1 and glucagon activity, modulates hepatic glycogen and fat content. Diabetes, 69(Supplement 1), 354. [Google Scholar]
  61. Roden, M. , Anderwald, C. , Fürnsinn, C. , Waldhäusl, W. , & Lohninger, A. (2000). Effects of short‐term leptin exposure on triglyceride deposition in rat liver. Hepatology, 32(5), 1045–1049. [DOI] [PubMed] [Google Scholar]
  62. Rudling, M. , & Angelin, B. (1993). Stimulation of rat hepatic low density lipoprotein receptors by glucagon. Evidence of a novel regulatory mechanism in vivo. Journal of Clinical Investigation, 91(6), 2796–2805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Schimke, R. T. (1962). Differential effects of fasting and protein‐free diets on levels of urea cycle enzymes in rat liver. Journal of Biological Chemistry, 237, 1921–1924. [PubMed] [Google Scholar]
  64. Schulman, J. L. , Carleton, J. L. , Whitney, G. , & Whitehorn, J. C. (1957). Effect of glucagon on food intake and body weight in man. Journal of Applied Physiology, 11(3), 419–421. [DOI] [PubMed] [Google Scholar]
  65. Sinclair, E. M. , Yusta, B. , Streutker, C. , Baggio, L. L. , Koehler, J. , Charron, M. J. , & Drucker, D. J. (2008). Glucagon receptor signaling is essential for control of murine hepatocyte survival. Gastroenterology, 135(6), 2096–2106. [DOI] [PubMed] [Google Scholar]
  66. Stunkard, A. J. , Van Itallie, T. B. , & Reis, B. B. (1955). The mechanism of satiety: Effect of glucagon on gastric hunger contractions in man. Proceedings of the Society for Experimental Biology and Medicine, 89(2), 258–261. [DOI] [PubMed] [Google Scholar]
  67. Suppli, M. P. , Bagger, J. I. , Lund, A. , Demant, M. , van Hall, G. , Strandberg, C. , Kønig, M. J. , Rigbolt, K. , Langhoff, J. L. , Wewer Albrechtsen, N. J. , Holst, J. J. , Vilsbøll, T. , & Knop, F. K. (2020). Glucagon resistance at the level of amino acid turnover in obese subjects with hepatic steatosis. Diabetes, 69(6), 1090–1099. [DOI] [PubMed] [Google Scholar]
  68. Trevaskis, J. L. , Norlin, J. , Miranda, D. A. , Bates, J. G. , Zois, N. E. , Veidal, S. , Feigh, M. , & Latta, M. (2020). 1813‐P: Coadministration of a GLP‐1/glucagon receptor agonist with an FXR agonist and ACC inhibitor reverses nonalcoholic Steatohepatitis (NASH) in diet‐induced and biopsy‐confirmed mice. Diabetes, 69(Supplement 1), 1813. [Google Scholar]
  69. Urva, S. , Coskun, T. , Loh, M. T. , Du, Y. , Thomas, M. K. , Gurbuz, S. , Haupt, A. , Benson, C. T. , Hernandez‐Illas, M. , D'Alessio, D. A. , & Milicevic, Z. (2022). LY3437943, a novel triple GIP, GLP‐1, and glucagon receptor agonist in people with type 2 diabetes: A phase 1b, multicentre, double‐blind, placebo‐controlled, randomised, multiple‐ascending dose trial. The Lancet, 400(10366), 1869–1881. [DOI] [PubMed] [Google Scholar]
  70. Wang, M.‐Y. , Zhang, Z. , Zhao, S. , Onodera, T. , Sun, X. N. , Zhu, Q. , Li, C. , Li, N. , Chen, S. , Paredes, M. , Gautron, L. , Charron, M. J. , Marciano, D. K. , Gordillo, R. , Drucker, D. J. , & Scherer, P. E. (2024). Downregulation of the kidney glucagon receptor, essential for renal function and systemic homeostasis, contributes to chronic kidney disease. Cell Metabolism, 36(3), 575–597.e7. 10.1016/j.cmet.2023.12.024 [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Weick, B. G. , & Ritter, S. (1986). Dose‐related suppression of feeding by intraportal glucagon infusion in the rat. American Journal of Physiology, 250(4 Pt 2), R676–R681. [DOI] [PubMed] [Google Scholar]
  72. Wewer Albrechtsen, N. J. , Junker, A. E. , Christensen, M. , Hædersdal, S. , Wibrand, F. , Lund, A. M. , Galsgaard, K. D. , Holst, J. J. , Knop, F. K. , & Vilsbøll, T. (2018). Hyperglucagonemia correlates with plasma levels of non‐branched‐chain amino acids in patients with liver disease independent of type 2 diabetes. American Journal of Physiology. Gastrointestinal and Liver Physiology, 314(1), G91–G96. [DOI] [PubMed] [Google Scholar]
  73. Wewer Albrechtsen, N. J. , Kuhre, R. E. , Windeløv, J. A. , Ørgaard, A. , Deacon, C. F. , Kissow, H. , Hartmann, B. , & Holst, J. J. (2016). Dynamics of glucagon secretion in mice and rats revealed using a validated sandwich ELISA for small sample volumes. American Journal of Physiology. Endocrinology and Metabolism, 311(2), E302–E309. [DOI] [PubMed] [Google Scholar]
  74. Winther‐Sørensen, M. , Galsgaard, K. D. , Santos, A. , Trammell, S. A. J. , Sulek, K. , Kuhre, R. E. , Pedersen, J. , Andersen, D. B. , Hassing, A. S. , Dall, M. , Treebak, J. T. , Gillum, M. P. , Torekov, S. S. , Windeløv, J. A. , Hunt, J. E. , Kjeldsen, S. A. S. , Jepsen, S. L. , Vasilopoulou, C. G. , Knop, F. K. , … Wewer Albrechtsen, N. J. (2020). Glucagon acutely regulates hepatic amino acid catabolism and the effect may be disturbed by steatosis. Molecular Metabolism, 42, 101080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Witters, L. A. , & Trasko, C. S. (1979). Regulation of hepatic free fatty acid metabolism by glucagon and insulin. American Journal of Physiology, 237(1), E23–E29. [DOI] [PubMed] [Google Scholar]
  76. Xu, Y. , Liu, Q. , Chen, C. W. , Wang, Q. , du, T. , Yu, R. , Zhou, Q. , Yang, D. , & Wang, M. W. (2024). Absence of PNET formation and normal longevity in a mouse model of Mahvash disease. Heliyon, 10(15), e35362. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Appendix S1.

PHY2-13-e70235-s001.docx (3.5MB, docx)

Data Availability Statement

All data reported in this paper will be shared by the lead contact upon request. All lipidomics data is available at Figshare (https://doi.org/10.6084/m9.figshare.27924528.v1).

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