Glucagon acutely regulates hepatic amino acid catabolism and the effect may be disturbed by steatosis

Abstract

Objective: Glucagon is well known to regulate blood glucose but may be equally important for amino acid metabolism. Plasma levels of amino acids are regulated by glucagon-dependent mechanism(s), while amino acids stimulate glucagon secretion from alpha cells, completing the recently described liver-alpha cell axis. The mechanisms underlying the cycle and the possible impact of hepatic steatosis are unclear.

Methods: We assessed amino acid clearance in vivo in mice treated with a glucagon receptor antagonist (GRA), transgenic mice with 95% reduction in alpha cells, and mice with hepatic steatosis. In addition, we evaluated urea formation in primary hepatocytes from ob/ob mice and humans, and we studied acute metabolic effects of glucagon in perfused rat livers. We also performed RNA sequencing on livers from glucagon receptor knock-out mice and mice with hepatic steatosis. Finally, we measured individual plasma amino acids and glucagon in healthy controls and in two independent cohorts of patients with biopsy-verified non-alcoholic fatty liver disease (NAFLD).

Results: Amino acid clearance was reduced in mice treated with GRA and mice lacking endogenous glucagon (loss of alpha cells) concomitantly with reduced production of urea. Glucagon administration markedly changed the secretion of rat liver metabolites and within minutes increased urea formation in mice, in perfused rat liver, and in primary human hepatocytes. Transcriptomic analyses revealed that three genes responsible for amino acid catabolism (Cps1, Slc7a2, and Slc38a2) were downregulated both in mice with hepatic steatosis and in mice with deletion of the glucagon receptor. Cultured ob/ob hepatocytes produced less urea upon stimulation with mixed amino acids, and amino acid clearance was lower in mice with hepatic steatosis. Glucagon-induced ureagenesis was impaired in perfused rat livers with hepatic steatosis. Patients with NAFLD had hyperglucagonemia and increased levels of glucagonotropic amino acids, including alanine in particular. Both glucagon and alanine levels were reduced after diet-induced reduction in Homeostatic Model Assessment for Insulin Resistance (HOMA-IR, a marker of hepatic steatosis).

Conclusions: Glucagon regulates amino acid metabolism both non-transcriptionally and transcriptionally. Hepatic steatosis may impair glucagon-dependent enhancement of amino acid catabolism.

Keywords: Amino acids; Glucagon; Liver-alpha cell axis; Non-alcoholic fatty liver disease.

Graphical abstract

Figure 1

Pharmacological inhibition of glucagon receptor signaling alters amino acid metabolism and ureagenesis in mice. (A) Female C57BL/6JRj mice received a glucagon receptor antagonist (GRA, 100 mg/kg) or vehicle three hours prior to intraperitoneal administration of amino acids or saline. (B) Baseline plasma levels of total l-amino acids after treatment with GRA or vehicle (n = 23–24). (C) Plasma levels of total l-amino acids upon administration of saline (open circles) or amino acids (Vamin, 3.5 μmol/g body weight, closed squares) in mice treated with GRA (red symbols and lines) or vehicle (blue symbols and lines). Data are shown as incremental area under the curve (iAUC) in the right panel (n = 9–14). (D) Baseline plasma levels of urea after three hours of treatment with GRA or vehicle (n = 23–24). (E) Plasma levels of urea (baseline corrected) upon administration of saline or amino acids in mice treated with GRA or vehicle. Data are shown as net area under the curve (nAUC, positive and negative peaks were included) in the right panel (n = 9–14). Levels of (F) plasma glucagon, (G) plasma insulin, and (H) blood glucose in response to administration of amino acids or saline in mice treated with GRA or vehicle (n = 23–24 except for insulin: n = 15–17). (B + D) Error bars indicate SD, and data are analyzed by unpaired t-tests. (C, E, F, G, H) Data are shown as mean ± SEM. AUCs are analyzed by one-way ANOVA and time-resolved data by multiple t-tests on delta values and corrected for multiple testing using the Holm-Sidak algorithm; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗∗P < 0.0001.

Figure 2

Glucagon acutely increases amino acid turnover in mice with a 95% reduction in pancreatic glucagon content. (A) Representative histological pictures of endocrine islets stained for glucagon in female transgenic (TgN(GCG.DTR)) mice treated with vehicle (left panel) or diphtheria toxin (DT, 10 ng/g, right panel). Images are shown at ×200 magnification. The scale bar indicates 50 μm. (B) Extracted pancreatic glucagon content in a separate group of transgenic (TG) mice treated with vehicle or DT. Error bars indicate SD (n = 6–8). (C) Plasma levels of glucagon in response to saline (open circles), amino acids (Vamin, 3.5 μmol/g body weight, closed squares), or amino acids + glucagon (200 ng/g body weight, triangles) in mice treated with DT (red symbols) or vehicle (blue symbols and lines). (D) Plasma levels of total l-amino acids (left panel) and incremental area under the curve (iAUC, right panel), and (E) plasma levels of urea (baseline corrected, left panel) and netAUC (nAUC, positive and negative peaks were included, right panel) in response to saline, amino acids, or amino acids + glucagon in TG mice treated with vehicle or DT. (F) Plasma insulin and (G) blood glucose levels in response to saline, amino acids, or amino acids + glucagon in TG mice treated with vehicle or DT. n = 9–15. Data in (C–G) are shown as mean ± SEM and AUCs were analyzed by one-way ANOVA corrected for multiple testing using the Holm-Sidak algorithm. ∗P < 0.05 and ∗∗P < 0.01. See also Fig. S2.

Figure 3

Glucagon acutely impacts the release of metabolites from perfused rat livers and potentiates amino acid-induced urea production in perfused rat livers and primary human hepatocytes. (A) Experimental design of the perfused rat liver using Krebs-Henseleit buffer (KHB). n = 4 male rats. (B) Principal component analysis based on both positive and negative mode LC-MS metabolomics of perfusate collected from perfused rat livers during baseline 2 (grey) and glucagon stimulation (10 nM, green) (n = 4). (C) Formation of urea (normalized to liver weight) in perfusate during baseline (gray), amino acids (Vamin, 1 mM, yellow), glucagon (10 nM, green), and amino acids + glucagon (blue) stimulations. Data are shown as mean ± SEM. Statistical analyses were performed on mean output: ^##P < 0.01 compared to amino acid stimulation, ∗∗P < 0.001 compared to baseline. (D) Concentration of urea in media of primary human hepatocytes incubated with glucagon (200 nM, green), amino acids (Vamin, 175 mM, yellow), or both glucagon and amino acids (blue) at indicated time periods. Error bars indicate SDs of two technical replicates. See also Fig. S3 and S4.

Figure 4

Expression of amino acid metabolism genes is downregulated in glucagon receptor knockout mice and mice with hepatic steatosis. (A) Work flow of RNA sequencing of liver biopsies from global glucagon receptor knock-out mice (Gcgr^−/−) and wild-type (WT) littermates. (B) Gene clustering of the 50 most differentially expressed genes between Gcgr^−/− (pink) and WT (blue). Scale represents normalized counts using the variance stabilizing transformation (VST). (C) Gene ontology (GO) biological processes enriched for up- and downregulated genes in Gcgr^−/− mice compared to WT littermates. Only genes downregulated by more than 2 (log2(fold-change)) are included. FDR<0.1 was applied to correct for multiple testing. Gcgr^−/− mice: n = 4 male mice, 10 weeks of age; WT littermates: n = 5 male mice, 10 weeks of age. (D) Design of the study with female C57BL/6JRj mice (start weight: 21.7 ± 0.1 g) fed high-fat diet and 15% fructose water (HFD + FW) for 8–10 weeks with representative histological pictures of a liver section from a mouse fed HFD + FW (top) and control diet (bottom). Scale bar = 50 μm. (E) Gene expression of the same 50 genes shown in (B) in mice fed high-fat diet and 15% fructose water (HFD + FW) or control diet and water. Scale represents normalized counts using the VST. HFD + FW: n = 3 female mice; control diet: n = 3 female mice. (F) Venn diagram analysis showing the number of genes significantly (FDR<0.1) regulated in Gcgr^−/− mice (blue) and mice fed HFD + FW (red) and overlapping genes. See also Tables S3 and S4.

Figure 5

Increased liver fat impairs the liver-alpha cell axis in mice. (A) Design of the study with female C57BL/6JRj mice fed high-fat diet and 15% fructose water (HFD + FW) for 8–10 weeks. Plasma levels of (B) glucagon, (C) amino acids, (D) urea (baseline subtracted), (E) insulin, and (F) blood glucose levels in response to amino acids (Vamin, 3.5 μmol/g body weight) in mice fed HFD + FW (red) or control diet and water (blue) (n = 9–12). Data are shown as mean ± SEM and analyzed by multiple t-tests corrected for multiple testing using the Holm-Sidak algorithm ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. (G) Urea in the culture media of primary hepatocytes from male ob/ob and control mice upon stimulation with mixed amino acids (Vamin, 175 mM) or vehicle (n = 3 mice). Data are shown as mean ± SEM and data are analyzed by AUC using one-way ANOVA. ∗P < 0.05. (H) Formation of urea (normalized to liver weight) in perfusate during baseline (grey), amino acids (Vamin, 10 mM, yellow), glucagon (10 nM, green), and amino acids + glucagon (blue) stimulations in male Zucker lean (black, n = 5) and Zucker fatty rats (red, n = 5). Data are shown as mean ± SEM. See also Fig. S5 and S6.

Figure 6

Increased liver fat distorts the liver-alpha cell axis in humans. (A) Overview of the three cohorts included in the study. Discovery cohort: patients with biopsy-verified steatosis (light blue, n = 12) and non-alcoholic steatohepatitis (NASH, dark blue, n = 13), and controls (grey, n = 10). Validation cohort: patients with biopsy-verified steatosis (light blue, n = 18), NASH (dark blue, n = 17), and healthy controls (grey, n = 9). Weight loss study cohort: obese individuals were subjected to eight weeks of calorie restriction (800 kcal/day, n = 52) and thereafter a 52-week weight maintenance program (n = 39). (B) Fasting plasma levels of individual amino acids in individuals in the discovery cohort. Amino acids are ranked according to mean plasma levels in the control group. Bars indicate median and interquartile range. ∗P < 0.05 between control and both steatosis and NASH, †P < 0.05 between control and steatosis, #P < 0.05 between control and NASH. (C) Glucagon-alanine index (the product of fasting levels of plasma glucagon (pmol/L) and alanine (μmol/L)) in the discovery cohort and (D) validation cohort. Bars indicate median and interquartile range. The data were analyzed by the Kruskal–Wallis test and compared to the healthy control group using Dunn's post hoc test to correct for multiple testing. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. (E) Glucagon-alanine index in individuals before weight loss (week −8), after weight loss (week 0), and after 1-year weight loss maintenance (week 52). Statistical significance was tested by paired t-tests corrected for multiple testing using the Holm-Sidak algorithm. ∗P < 0.05. See also Figure S7.