Variable glucagon metabolic actions in diverse mouse models of obesity and type 2 diabetes

Abstract

Objective: The study aimed to investigate the effects of glucagon on metabolic pathways in mouse models of obesity, fatty liver disease, and type 2 diabetes (T2D) to determine the extent and variability of hepatic glucagon resistance in these conditions.

Methods: We investigated glucagon's effects in mouse models of fatty liver disease, obesity, and type 2 diabetes (T2D), including male BKS-db/db, high-fat diet-fed, and western diet-fed C57Bl/6 mice. Glucagon tolerance tests were performed using the selective glucagon receptor agonist acyl-glucagon (IUB288). Blood glucose, serum and liver metabolites include lipids and amino acids were measured. Additionally, liver protein expression related to glucagon signalling and a comprehensive liver metabolomics were performed.

Results: Western diet-fed mice displayed impaired glucagon response, with reduced blood glucose and PKA activation. In contrast, high-fat diet-fed and db/db mice maintained normal glucagon sensitivity, showing significant elevations in blood glucose and phospho-PKA motif protein expression. Acyl-glucagon treatment also lowered liver alanine and histidine levels in high-fat diet-fed mice, but not in western diet-fed mice. Additionally, some amino acids, such as methionine, were increased by acyl-glucagon only in chow diet control mice. Despite normal glucagon sensitivity in PKA signalling, db/db mice had a distinct metabolomic response, with acyl-glucagon significantly altering 90 metabolites in db/+ mice but only 42 in db/db mice, and classic glucagon-regulated metabolites, such as cyclic adenosine monophosphate (cAMP), being less responsive in db/db mice.

Conclusions: The study reveals that hepatic glucagon resistance in obesity and T2D is complex and not uniform across metabolic pathways, underscoring the complexity of glucagon action in these conditions.

Keywords: Acylcarnitine; Amino acids; Diabetes; Glucagon; Obesity.

Graphical abstract

Figure 1

Acyl-glucagon treatment acutely activates PKA signalling and affects glucose, amino acid and lipid metabolism of B6J male mice. B6J Male mice (7–8 weeks of age) were acutely injected with acyl-glucagon or vehicle via intraperitoneal injections. The mice were sacrificed either 15 min (n = 6) or 2 h post-injection (n = 5). (A): Western blot analysis of liver phospho-protein kinase A (pPKA) motif protein substrates and loading control vinculin (VCL) from mice treated acutely with acyl-glucagon or vehicle (15 min and 120 min). (B): Blood glucose levels in mice treated acutely with acyl-glucagon or vehicle (15 min). (C): Blood glucose levels in mice treated acutely with acyl-glucagon or vehicle (120 min). (D): Serum triglyceride levels in mice treated acutely with acyl-glucagon or vehicle (15 min and 120 min). (E): Liver glycogen levels in mice as in (B). (F): Liver free carnitine (C0) levels in mice treated acutely with acyl-glucagon or vehicle as in b. (G): Liver hexadecenoylcarnitine (C16:1) levels in mice as in (B). (H): Heatmap of liver amino acid levels in mice as in (B). (I): Liver citrulline (Cit), ornithine (Orn) and arginine (Arg) levels in mice as in (B). (J): Liver glutamine (Gln), alanine (Ala), glycine (Gly) and histidine (His) levels in mice as in (B). (K): Liver methionine (Met), Valine (Val) and Leucine/isoleucine (Leu/Ile) levels in mice as in (B). Data are presented as the mean ± s.e.m. Statistical analyses included two-tailed Student's t-test or two-way ANOVA with Sidak's post hoc tests. Significance thresholds were set as ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. Non-significant differences (P > 0.05) were labeled as ns.

Figure 2

Actions of acyl-glucagon on glucose and lipid metabolism in mouse models of obesity and fatty liver. Male B6J mice (7–8 weeks old) were fed either a chow or Western diet, or a chow or high-fat diet, for 6 weeks before receiving an acute intraperitoneal injection of acyl-glucagon or vehicle (n = 5 per group). The mice were sacrificed 2 h post-injection. Male db/db and db/+ mice (9–10 weeks old) were similarly injected with acyl-glucagon or vehicle and sacrificed 2 h post-injection (n = 7 per group). (A): Blood glucose levels in chow-fed mice (as control for Western diet) were measured after acute treatment with acyl-glucagon or vehicle. (B): Blood glucose levels in high-fat diet-fed mice treated acutely with acyl-glucagon or vehicle. (C): Blood glucose levels in db/db mice treated with acyl-glucagon or vehicle. (D): Liver glycogen levels in mice fed either a chow diet or a western diet, following acute treatment with acyl-glucagon or vehicle. (E): Liver glycogen levels in mice fed either a chow diet or a high-fat diet, following acute treatment with acyl-glucagon or vehicle. (F): Liver glycogen levels in db/db or db/+ mice treated acutely with acyl-glucagon or vehicle. (G): Western blot quantification of liver phospho-PKA motif protein expression in mice as in (D). (H): Western blot quantification of liver phospho-PKA motif protein expression in mice as in (E). (I): Western blot quantification of liver phospho-PKA motif protein expression in mice as in (F). (J): Serum triglyceride levels in mice as in (D). (K): Serum triglyceride levels in mice as in (E). (L): Serum triglyceride levels in mice as in (F). Data are presented as mean ± s.e.m. Statistical analysis was performed using two-way ANOVA with Sidak's post hoc tests. For comparisons between vehicle and acyl-glucagon, significance levels are indicated as ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001. For comparisons between chow and Western/high-fat diet, thresholds are #P < 0.05, ##P < 0.01, ###P < 0.001 and ####P < 0.0001. Non-significant differences (P > 0.05) are marked as ns.

Figure 3

Actions of acyl-glucagon on liver acylcarnitines in mice fed a western or high-fat diet. (A): Heatmap of liver acylcarnitine species in mice fed either a chow diet or a western diet, following acute treatment with acyl-glucagon or vehicle (n = 5 per group). CD: chow diet; WD: western diet. (B): Liver free carnitine (C0) levels in mice as in (A). (C): Liver hexanoylcarnitine (C6) levels in mice as in (A). (D): Liver hexadecenoylcarnitine (C16:1) levels in mice as in (A). (E): Liver octadecenoylcarnitine (C18:1) levels in mice as in (A). (F): Heatmap of liver acylcarnitine species in mice fed either a chow diet or a high-fat diet, following acute treatment with acyl-glucagon or vehicle. CD: chow diet; HFD: high-fat diet. (G): Liver C0 levels in mice as in (F). (H): Liver C6 levels in mice as in (F). (I): Liver C16:1 levels in mice as in (F). (J): Liver C18:1 levels in mice as in (F). Data are presented as mean ± s.e.m. Statistical analysis was performed using two-way ANOVA with Sidak's post hoc tests. For comparisons between vehicle and acyl-glucagon, significance levels are indicated as ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001. For comparisons between chow and western/high-fat diet, thresholds are #P < 0.05, ##P < 0.01, ###P < 0.001 and ####P < 0.0001. Non-significant differences (P > 0.05) are marked as ns.

Figure 4

Actions of acyl-glucagon on liver amino acids in mice fed a western or high-fat diet. (A): Heatmap of liver amino acid levels in mice fed either a chow diet or a Western diet, following acute treatment with acyl-glucagon or vehicle. (B): Liver glutamine (Gln) levels in mice as in (A). (C): Liver alanine (Ala) levels in mice as in (A). (D): Liver histidine (His) levels in mice as in (A). (E): Liver methionine (Met) levels in mice as in (A). (F): Heatmap of liver amino acid levels in mice fed either a chow diet or a high-fat diet, following acute treatment with acyl-glucagon or vehicle. (G): Liver glutamine (Gln) levels in mice as in (F). (H): Liver alanine (Ala) levels in mice as in (F). (I): Liver histidine (His) levels in mice as in (F). (J): Liver methionine (Met) levels in mice as in (F). Data are presented as mean ± s.e.m. Statistical analysis was performed using two-way ANOVA with Sidak's post hoc tests. For comparisons between vehicle and acyl-glucagon, significance levels are indicated as ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001. For comparisons between chow and Western/high-fat diet, thresholds are #P < 0.05, ##P < 0.01, ###P < 0.001 and ####P < 0.0001. Non-significant differences (P > 0.05) are marked as ns.

Figure 5

Liver metabolome analysis of acyl-glucagon action in db/db mice. The male db/db and db/+ mice (9–10 weeks of age) were acutely injected with acyl-glucagon or vehicle via intraperitoneal injections. The mice were sacrificed 30 min post-injection (n = 4–5) for liver metabolome analysis. (A): Western blot quantification of liver phospho-PKA motif protein expression in db/db or db/+ mice treated acutely with acyl-glucagon or vehicle. (B): Principal component analysis (PCA) of the liver metabolome in mice as in a. (C): Volcano plots of metabolites (from mice as in a) were generated by plotting unadjusted p-values (-log10) against fold change (log2) between treatments. Significant changes (p < 0.05 and absolute fold change >1.5) are highlighted with colored circles, where color indicates fold change and circle size represents p-value. Labeled metabolites are highlighted. (D): Liver cyclic adenosine monophosphate (cAMP) levels in mice as in (A). (E): Liver Ureidosuccinic acid levels in mice as in (A). (F): Liver N-Acetylglucosamine 6-phosphate levels in mice as in (A). (G): Liver 5-Hydroxyindoleacetylglycine levels in mice as in (A). (H): Liver Carnitine levels in mice as in (A). (I): Liver Sorbitol-6-phosphate levels in mice as in (A). (J): Liver Hypotaurine levels in mice as in (A). (K): Liver Taurine levels in mice as in (A). (L): Liver Histidine levels in mice as in (A). (M): Liver Citrulline levels in mice as in (A). (N): Liver Glutamine levels in mice as in (A). (O): Liver Glutamic acid levels in mice as in (A). Data are presented as mean ± s.e.m. Statistical analysis was performed using two-way ANOVA with Sidak's post hoc tests. For comparisons between vehicle and acyl-glucagon, significance levels are indicated as ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001. For comparisons between db/db and db/+ mice, thresholds are #P < 0.05, ##P < 0.01, ###P < 0.001 and ####P < 0.0001. Non-significant differences (P > 0.05) are marked as ns.

Figure 6

Metabolome pathway analysis of db/db mice. (A–B): Pathway analysis (via MetaboAnalyst 6.0, SMPDB database) of significantly altered by acyl-glucagon (30 min) in db/db and db/+ mice. (C): liver 5′-Phosphoribosyl-N-formylglycinamide in db/db and db/+ mice treated acutely with acyl-glucagon or vehicle (30 min). (D): Liver Uric acid levels in mice as in (C). (E): Liver Inosine levels in mice as in (C). (F): Liver Deoxyinosine levels in mice as in (C). (G): Liver Xanthosine levels in mice as in (C). (H): Liver Hypoxanthine levels in mice as in (C). (I): Liver 5-L-Glutamyl-taurine levels in mice as in (C). (J): Liver 3-Sulfinoalanine levels in mice as in (C). (K): Liver Phosphoenolpyruvic acid levels in mice as in (C). (L): Liver Uridine diphosphate-N-acetylglucosamine levels in mice as in (C). (M): Liver N-Formimino-l-glutamate levels in mice as in (C). (N): Liver 1-Pyrroline-5-carboxylic acid levels in mice as in (C). (O): Liver Glutamic gamma-semialdehyde levels in mice as in (C). (P): Liver Formyl-isoglutamine levels in mice as in (C). (Q): Liver N-Acetylglutamic acid levels in mice as in (C). (R): Liver N-Acetylglutamine levels in mice as in (C). Data are presented as mean ± s.e.m. Statistical analysis was performed using two-way ANOVA with Sidak's post hoc tests. For comparisons between vehicle and acyl-glucagon, significance levels are indicated as ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001. For comparisons between db/db and db/+ mice, thresholds are #P < 0.05, ##P < 0.01, ###P < 0.001 and ####P < 0.0001. Non-significant differences (P > 0.05) are marked as ns.

Figure 7

Summary of acyl-glucagon action across all the obesity and diabetes models in this study. TG, triglyceride; TC, cholesterol; ALT, alanine aminotransferase.