Remodeling of hepatic metabolism and hyperaminoacidemia in mice deficient in proglucagon-derived peptides

Abstract

Glucagon is believed to be one of the most important peptides for upregulating blood glucose levels. However, homozygous glucagon-green fluorescent protein (gfp) knock-in mice (Gcg(gfp/gfp): GCGKO) are normoglycemic despite the absence of proglucagon-derived peptides, including glucagon. To characterize metabolism in the GCGKO mice, we analyzed gene expression and metabolome in the liver. The expression of genes encoding rate-limiting enzymes for gluconeogenesis was only marginally altered. On the other hand, genes encoding enzymes involved in conversion of amino acids to metabolites available for the tricarboxylic acid cycle and/or gluconeogenesis showed lower expression in the GCGKO liver. The expression of genes involved in the metabolism of fatty acids and nicotinamide was also altered. Concentrations of the metabolites in the GCGKO liver were altered in manners concordant with alteration in the gene expression patterns, and the plasma concentrations of amino acids were elevated in the GCGKO mice. The insulin concentration in serum and phosphorylation of Akt protein kinase in liver were reduced in GCGKO mice. These results indicated that proglucagon-derived peptides should play important roles in regulating various metabolic pathways, especially that of amino acids. Serum insulin concentration is lowered to compensate the impacts of absent proglucagon-derived peptide on glucose metabolism. On the other hand, impacts on other metabolic pathways are only partially compensated by reduced insulin action.

FIG. 1.

Expression levels of genes encoding enzymes involved in gluconeogenesis, concentrations of intermediate metabolites for gluconeogenesis, and genes differentially expressed in control and GCGKO livers. Control liver (□); GCGKO liver (■). A–C: Expression levels of PYCS, PEPCK, and G6PC mRNA in the liver. The mice were either fed ad libitum (A) or starved for 5 (B) or 24 h (C). The relative expression levels controlled by the expression levels of β-actin mRNA are presented. The data are means ± SEM (n = 4–6). D: Relative concentrations of pyruvate, phosphoenolpyruvate, and glucose-6-phosphate in control and GCGKO liver. The data are means ± SEM (n = 3). E: The relative expression levels controlled by the expression levels of β-actin mRNA are presented. The data are means ± SEM (n = 14–16). *P < 0.05; **P < 0.01.

FIG. 2.

Expression level of genes encoding enzymes involved in amino acid metabolism and schematic representation of metabolic pathways. A–C: Expression levels of mRNA encoding enzymes involved in amino acid metabolism in control (□) and GCGKO (■) liver. The mice were either fed ad libitum (A) or starved for 5 (B) or 24 h (C). The data are means ± SEM (n = 4–6). *P < 0.05; **P < 0.01. D: Schematic representation of metabolic pathways. The enzymes and metabolites involved in glycolysis, gluconeogenesis, TCA cycle, and urea cycle are summarized. The enzymes analyzed in Fig. 3A–C are shown in bold letters and those analyzed in Fig. 1 are shown in halftone bold letters. Among the metabolites depicted, aspartate and glutamate are marked with asterisks because they appear twice in this scheme. It should be noted that most of the enzymes analyzed in Fig. 3A–C are involved in the conversion of amino acids to metabolites required for gluconeogenesis via the TCA cycle.

FIG. 3.

Relative concentrations of amino acids in liver and plasma. A–C: Relative concentrations of amino acids in control (□) and GCGKO (■) livers. The data are means ± SEM (n = 3). *P < 0.05; **P < 0.01. The relative concentrations of the 20 amino acids, urea cycle components, and cystathionine are depicted in A, B, and C, respectively. D and E: Plasma concentrations of amino acids and urea. The data are means ± SEM (n = 6). *P < 0.05; **P < 0.0001. The concentrations of the 20 amino acids and other variables are depicted in D and E, respectively.

FIG. 4.

Expression levels of genes encoding proteins involved in fatty acid metabolism and serum lipid concentrations. A–C: Expression levels of FGF21, ACAC, and FABP5 mRNA in control (□) and GCGKO (■) liver. The mice were either fed ad libitum (A) or starved for 5 (B) or 24 h (C). The data are means ± SEM (n = 4–6). *P < 0.05; **P < 0.01. D: Serum concentrations of triglyceride, HDL, LDL, and total cholesterol. The data are means ± SEM (n = 9). *P < 0.05; **P < 0.01.

FIG. 5.

Gene expression levels and enzymatic activities of NNMT and concentrations of metabolites involved in nicotinamide metabolism. A: Expression levels of NNMT mRNA. The data are means ± SEM (n = 4–6). **P < 0.01. B: NNMT activities in the liver. The data are means ± SEM (n = 3). *P < 0.05. C: Relative concentrations of metabolites involved in nicotinamide metabolism. The data are means ± SEM (n = 3). *P < 0.05; **P < 0.01.

FIG. 6.

Pyruvate tolerance and glucagon challenge tests. A: For the pyruvate tolerance test, sodium pyruvate was administered at 1.5 g/kg to 4-month-old male mice of the indicated genotypes. The data are means ± SEM (n = 6). *P < 0.05. B: For the glucagon challenge test, glucagon was administered at 30 mg/kg to 4-month-old male mice of the indicated genotypes. The data are means ± SEM (n = 6). *P < 0.05.

FIG. 7.

Indirect calorimetry, food intake, and locomotor activities. A: Respiratory exchange ratios of control and GCGKO mice. The respiratory exchange ratios during ad libitum feeding or starvation for 24 h are shown. B: Oxygen consumptions of control and GCGKO mice. C: Food intake in control and GCGKO mice. D: Locomotor activities in control and GCGKO mice. The locomotor activity was monitored by sensor beam lines and horizontal movements in counts (×10⁻²) per day are shown. The data are means ± SEM (n = 6) for all variables (A–D).

FIG. 8.

Serum insulin concentrations and immunoblot analyses of AKT in the liver under ad libitum feeding. A: Serum insulin concentrations in the control and GCGKO mice fed ad libitum. The data are presented as means ± SEM (n = 6). ***P < 0.0001. B: Phosphorylation of Akt. Representative images of immunoblots for phospho-Akt (Thr473), total Akt, and β-actin are shown together with quantitative data. The relative intensities of the phospho-kinase bands were normalized by the band intensities of either β-actin or total Akt and are presented as means ± SEM. The intensities in the control livers were set as 1 (n = 4). **P < 0.01.