Opposing effects of chronic glucagon receptor agonism and antagonism on amino acids, hepatic gene expression, and alpha cells

Abstract

The pancreatic hormone, glucagon, is known to regulate hepatic glucose production, but recent studies suggest that its regulation of hepatic amino metabolism is equally important. Here, we show that chronic glucagon receptor activation with a long-acting glucagon analog increases amino acid catabolism and ureagenesis and causes alpha cell hypoplasia in female mice. Conversely, chronic glucagon receptor inhibition with a glucagon receptor antibody decreases amino acid catabolism and ureagenesis and causes alpha cell hyperplasia and beta cell loss. These effects were associated with the transcriptional regulation of hepatic genes related to amino acid uptake and catabolism and by the non-transcriptional modulation of the rate-limiting ureagenesis enzyme, carbamoyl phosphate synthetase-1. Our results support the importance of glucagon receptor signaling for amino acid homeostasis and pancreatic islet integrity in mice and provide knowledge regarding the long-term consequences of chronic glucagon receptor agonism and antagonism.

Keywords: Biological sciences; Endocrinology; Transcriptomics.

Graphical abstract

Figure 1

Chronic activation and inhibition of glucagon receptor signaling, respectively, enhances and reduces amino acid metabolism in female mice (A and B) Weekly body weight in female C57BL/6JRj mice treated (A) twice daily for four weeks with a long-acting glucagon analog (GCGA, NNC9204-0043, 1.5 nmol/kg body weight) or PBS +1% BSA (PBS) or (B) treated once weekly for four weeks with a glucagon receptor antibody (GCGR Ab, REGN1193, Regeneron, 10 mg/kg body weight) or control antibody (Ctl Ab, REGN1945, Regeneron, 10 mg/kg body weight). (C and D) Weekly blood glucose in mice treated with (C) GCGA or PBS or (D) GCGR Ab or Ctl Ab. (E and F) Total plasma amino acid levels in mice treated with (E) GCGA or PBS or (F) GCGR Ab or Ctl Ab. (G and H) Plasma urea levels in mice treated with (G) GCGA or PBS or (H) GCGR Ab or Ctl Ab. (I and J) Calculated urea index ([urea]/[amino acids]) for mice treated with (I) GCGA or PBS or (J) GCGR Ab or Ctl Ab. (K and L, NB! the y-axes differ) Fold change of individual amino acids at week four relative to baseline in mice treated with (K) GCGA or PBS or (L) GCGR Ab or Ctl Ab. Glycine was not measured in the samples, and aspartic acid was not detected in any of the samples. The fold changes in the treatment groups (GCGA and GCGR Ab) were statistically significant (p < 0.05) from week zero to week four for all amino acid measurements, except for glutamic acid in the GCGA-treated mice (p = 0.056). The corresponding absolute plasma concentrations and p-values are available in Table S1. All plasma samples were taken prior to injection. The mice were seven weeks old at the beginning of the study. Data in (A–D) and (K and L) are presented as mean ± SEM, n = 3–7. Data in (E–J) are presented as mean ± SD, n = 4–8. p-values (A) by unpaired t-tests of Δblood glucose concentrations, (C and D) by two-way ANOVA, and (E–J) by unpaired t-tests, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 2

Chronic activation or inhibition of glucagon receptor signaling changes hepatic expression of amino acid metabolism and ureagenesis genes in opposite directions and regulates CPS-1 activity in female mice (A) Workflow of RNA sequencing of liver biopsies from female C57BL/6JRj mice treated twice daily for eight weeks with a long-acting glucagon analog (GCGA, NNC9204-0043, 1.5 nmol/kg body weight) or PBS +1% BSA (PBS) or treated once weekly for eight weeks with a glucagon receptor antibody (GCGR Ab, REGN1193, Regeneron, 10 mg/kg body weight) or control antibody (Ctl Ab, REGN1945, Regeneron, 10 mg/kg body weight) (all mice seven weeks of age at the beginning of the study). (B and C) Principal component analysis (PCA) of RNA sequencing samples from (B) GCGA- or PBS-treated mice or (C) GCGR Ab- or Ctl Ab-treated mice. (D–F) Log2foldchanges of selected genes associated with (D) urea cycle, (E) amino acid transport, and (F) amino acid metabolism in the livers of mice treated for eight weeks with GCGA or GCGR Ab compared to their respective control groups (PBS or Ctl Ab). Only genes that were differentially expressed by FDR<0.05 are shown, thus when no bars are shown for GCGA (orange) or GCGR Ab (green) it means that the gene was not significantly differentially expressed when compared to the respective control groups (PBS or Ctl Ab). A detailed table of differentially expressed genes of interest is available in Table S2. (G) Venn diagram showing the number of significantly down-regulated genes related to amino acid processes in GCGR Ab mice (green) and male Gcgr^−/− mice (purple) from (Winther-Sorensen et al., 2020). The genes were selected using the Gene Ontology Biological Pathways (GOBP) umbrella terms “Cellular amino acid metabolic process,” “Amino acid transport,” “Amino acid homeostasis,” and “Response to amino acid,” and all related child terms. A table of genes included in this Venn Diagram is available in Table S3. (H, I, and L) Enzymatic activity of carbamoyl phosphate synthetase-1 (CPS-1) in liver biopsies from female mice treated for eight weeks with (H) GCGA or PBS or (I) GCGR Ab or Ctl Ab; or in (L) Gcgr^−/− and Gcgr^+/+ mice (10–11 weeks of age, females are circles, and males are squares). (J and K) RNA sequencing data (DESeq2 normalized counts) of expression of Cps-1 in female mice treated with (J) GCGA or PBS or (K) GCGR Ab or Ctl Ab. Data in (D-F) presented as mean ± SEM and (H–L) presented as mean ± SD, n = 4–8. FDR<0.05 was applied to all RNA sequencing analyses to correct for multiple testing, p-values in (H and I) by unpaired t-tests, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 3

Chronic activation and inactivation of glucagon receptor signaling, respectively, reduce and increase pancreatic glucagon content in female mice (A and B) Pancreas weights from female C57BL/6JRj mice treated (A) twice daily for eight weeks with a long-acting glucagon analog (GCGA, NNC9204-0043, 1.5 nmol/kg body weight) or PBS +1% BSA (PBS) or (B) treated once weekly for eight weeks with a glucagon receptor antibody (GCGR Ab, REGN1193, Regeneron, 10 mg/kg body weight) or control antibody (Ctl Ab, REGN1945, Regeneron, 10 mg/kg, body weight). (C and D, NB! the y axes differ) Pancreatic glucagon and (E and F) insulin content in mice treated for eight weeks with (C and E) GCGA or PBS or (D and F) GCGR Ab or Ctl Ab. (G–J) Immunohistochemical staining for glucagon-positive cells in the pancreases of mice treated with (G) GCGA, (H) PBS, (I) GCGR Ab, or (J) Ctl Ab. (K and L) Immunohistochemical staining for insulin-positive cells in pancreases of mice treated with (K) GCGR Ab or (L) Ctl Ab. Black arrows indicate (G–J) glucagon- or (K and L) insulin-positive cells. Scale bar: 50 μm. See also Figure S2. (M and O, NB! the y axes differ) Plasma glucagon levels in mice treated for four weeks with (M) GCGA or PBS or (O) GCGR Ab or Ctl Ab. (N and P) Plasma insulin levels in mice treated for four weeks with (N) GCGA or PBS or (P) GCGR Ab or Ctl Ab. All blood samples were taken prior to injection. The mice were seven weeks of age at the beginning of the study. Data presented as mean ± SD, n = 4–8. p-values by unpaired t-tests, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.