Glucagon-induced extracellular cAMP regulates hepatic lipid metabolism

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   Figure 1

   Glucagon differently stimulates gluconeogenesis and fatty acid oxidation pathway. (A) Effect of short-term and long-term fasting on hepatic G6Pase luciferase reporter activity as well as Aox luciferase reporter activity in mouse liver. Significance was determined by Student’s t test (*P < 0.05 compared with 12 h fasted mice, n = 8). (B) Effect of short-term and long-term fasting on serum amounts of glucagon, cAMP, triacylglycerol and ketones. Significance was determined by one-way ANOVA (*P < 0.05 compared with ad libitum mice; ^#P < 0.05 compared with 12 h fasted mice, n = 8). (C) Effect of short-term and long-term fasting on mRNA amounts for gluconeogenic genes including Pepck, G6Pase and Pgc1a and fatty acid oxidation genes including Cpt1a, Aox and Mcad in mouse liver. Significance was determined by one-way ANOVA (*P < 0.05, **P < 0.01 compared with ad libitum mice; ^#P < 0.05 compared with 12 h fasted mice, n = 8).
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   Figure 2

   Extracellular cAMP stimulates hepatic fatty acid oxidation. (A) Effect of glucagon (20 nmol/L) stimulation on extracellular cAMP levels at different time point in primary hepatocytes. Significance was determined by one-way ANOVA (*P < 0.05, **P < 0.01 compared with respective controls, n = 6). (B) Effect of IBMX (0.1 mmol/L) exposure on glucagon-induced extracellular cAMP levels at different time point in primary hepatocytes. Primary hepatocytes were pretreated with IBMX for 1 h and followed by glucagon stimulation for indicated times. Significance was determined by Student’s t test (*P < 0.05 compared between indicated groups, n = 6). (C) Effect of IBMX (0.1 mmol/L) exposure on glucagon-induced Cpt1a and Aox mRNA amounts at indicated time in primary hepatocytes. Significance was determined by Student’s t test (*P < 0.05 compared between indicated groups, n = 6). (D) Effect of Ad-AC6 on glucagon-induced extracellular cAMP levels. Primary hepatocytes were infected with Ad-GFP or Ad-AC6 for 24 h and followed by glucagon stimulation for indicated times. Significance was determined by Student’s t test (*P < 0.05 compared between indicated groups, n = 6). (E) Effect of Ad-AC6 on glucagon-induced Cpt1a and Aox mRNA amounts at indicated time in primary hepatocytes. Significance was determined by Student’s t test (*P < 0.05 compared between indicated groups, n = 6). (F) Effect of probenecid (500 μmol/L) exposure on glucagon-induced extracellular cAMP levels at different time point in primary hepatocytes. Primary hepatocytes were pretreated with probenecid for 1 h and followed by glucagon stimulation for indicated times. Significance was determined by Student’s t test (*P < 0.05 compared between indicated groups, n = 6). (G) Effect of probenecid (500 μmol/L) exposure on glucagon-induced Cpt1a and Aox mRNA amounts at indicated time in primary hepatocytes. Significance was determined by Student’s t test (*P < 0.05 compared between indicated groups, n = 6). (H) Effect of Ad-MRP4i on glucagon-induced extracellular cAMP levels. Primary hepatocytes were infected with Ad-USi or Ad-MRP4i for 48 h and followed by glucagon stimulation for indicated times. Significance was determined by Student’s t test (*P < 0.05 compared between indicated groups, n = 6). (I) Effect of Ad-MRP4i on glucagon-induced Cpt1a and Aox mRNA amounts at indicated time in primary hepatocytes. Significance was determined by Student’s t test (*P < 0.05 compared between indicated groups, n = 6). (J) Effect of cAMP (10, 50, or 100 μmol/L) exposure on glucagon-induced Cpt1a, Aox and Mcad mRNA amounts in primary hepatocytes. Primary hepatocytes were pretreated with indicated amount of cAMP for 1 h and followed by control or glucagon stimulation for 24 h. Significance was determined by one-way ANOVA (*P < 0.05 compared with the control only; ^#P < 0.05 compared with glucaon only, n = 6).
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   Figure 3

   Extracellular cAMP activates AMPK. (A) Immunoblot showing effects of 24 h cAMP (10, 50, 100 or 200 μmol/L) or 2 h AICAR (500 μmol/L) treatment on AMPK phosphorylation in primary hepatocytes. Immunoblot intensities for pAMPK/AMPK were quantitated. Significance was determined by one-way ANOVA (*P < 0.05 compared with control, n = 4). (B) Immunoblot showing effects of 24 h glucagon (20 nmol/L) treatment on AMPK phosphorylation in primary hepatocytes. Immunoblot intensities for pAMPK/AMPK were quantitated. Significance was determined by Student’s t test (*P < 0.05 compared with control, n = 4). (C) Immunoblot showing effects of probenecid (500 μmol/L) or Ad-MRP4i treatment on AMPK phosphorylation in primary hepatocytes. Primary hepatocytes were pretreated with probenecid for 1 h and followed by glucagon stimulation for 24 h or primary hepatocytes were infected with Ad-USi or Ad-MRP4i for 48 h and followed by glucagon stimulation for 24 h. Immunoblot intensities for pAMPK/AMPK were quantitated. Significance was determined by one-way ANOVA (*P < 0.05 compared with respective controls, n = 4). (D) Immunoblot showing effects of AMPCP (0.1 mmol/L), NBTI (10 μmol/L) or DPSPX (10 nmol/L) on cAMP (100 μmol/L)-induced AMPK phosphorylation in primary hepatocytes. Primary hepatocytes were pretreated with indicated reagents for 1 h and followed by glucagon stimulation for 24 h. Immunoblot intensities for pAMPK/AMPK were quantitated. Significance was determined by one-way ANOVA (*P < 0.05 compared between indicated groups, n = 4). (E) Effect of 4 h AICAR (500 μmol/L) exposure on glucagon-induced Cpt1a and Aox mRNA amounts in primary hepatocytes. Significance was determined by Student’s t test (*P < 0.05 compared between indicated groups, n = 4). (F) Effect of compound C (50 μmol/L) exposure on cAMP (100 μmol/L)-induced Cpt1a and Aox mRNA amounts in primary hepatocytes. Primary hepatocytes were pretreated with compound C for 1 h and followed by cAMP or glucagon stimulation for 24 h. Significance was determined by one-way ANOVA (*P < 0.05 compared with respective controls, n = 4). (G) Effect of AMPCP (0.1 mmol/L), NBTI (10 μmol/L) or DPSPX (10 nmol/L) exposure on cAMP (100 μmol/L) or glucagon (20 nmol/L)-induced Cpt1a and Aox mRNA amounts in primary hepatocytes. Primary hepatocytes were pretreated with indicated reagents for 1 h and followed by cAMP or glucagon stimulation for 24 h. Significance was determined by one-way ANOVA (*P < 0.05 compared between indicated groups, n = 4).
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   Figure 4

   AMPK phosphorylation promotes PPARα activation. (A) Conserved AMPK consensus site on PPARα. (B) Immunoblot showing effects of 2 h AICAR (500 μmol/L) treatment on PPARα phosphorylation in primary hepatocytes. Immunoblot intensities for pPPARα/PPARα were quantitated. Significance was determined by Student’s t test (*P < 0.05 compared with control, n = 4). (C) Immunoblot showing effect of mutations in the AMPK phosphorylation site (S452A) on PPARα phosphorylation by AMPK CA in HEK293T cells. HEK293T cells were transfected with indicated plasmid for 24 h. Immunoblot intensities for pPPARα/PPARα were quantitated. Significance was determined by one-way ANOVA (*P < 0.05 compared with control in PPARα WT, n = 4). (D) Immunoblot showing effect of 2 h AICAR (500 μmol/L) or 24 h cAMP (100 μmol/L) treatment on PPARα or PPARα S452A phosphorylation by AMPK in HepG2 cells. HepG2 cells were transfected with indicated plasmid for 48 h before treatment. Immunoblot intensities for pPPARα/PPARα were quantitated. Significance was determined by one-way ANOVA (*P < 0.05, compared between indicated groups, n = 4). (E) Effect of wild-type or S452A mutant PPARα on Aox-luc reporter activity. Primary hepatocytes were infected with Ad-Aox-luc, Ad-RSV-β-gal together with Ad-PPARα, Ad-PPARα S452A and Ad-AMPK CA adenovirus for 24 h. Significance was determined by one-way ANOVA (*P < 0.05, compared between indicated groups, n = 6). Effect of AMPK CA expression was shown. (F) Effect of wild-type or S452A mutant PPARα on cAMP (100 μmol/L)-induced or AICAR (500 μmol/L)-induced Cpt1a and Aox mRNA amounts in primary hepatocytes. Primary hepatocytes were infected with Ad-GFP, Ad-PPARα or Ad-PPARα S452A for 24 h. Cells were pretreated with cAMP or AICAR for 1 h and followed by glucagon stimulation for 24 h. Significance was determined by two-way ANOVA (*P < 0.05, GFP vs PPARα WT, n = 6).
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   Figure 5

   Extracellular cAMP maintains lipid homeostasis under fasted stage. (A) Effect of cAMP (20 mg/kg) injection on hepatic and serum triacylglycerol, NEFA, serum ketones, blood glucose and insulin levels in 24 h fasted mice. Significance was determined by Student’s t test (*P < 0.05 compared with control, n = 8). (B) Representative H&E and Oil Red O staining of liver from cAMP injected mice (scale bar, 50 μm). (C) Effect of cAMP (20 mg/kg) injection on hepatic fatty acid oxidation gene expression. Significance was determined by Student’s t test (*P < 0.05, **P < 0.01 compared with control, n = 8). (D) Effect of Ad-MRP4i injection on hepatic and serum triacylglycerol, NEFA, serum ketones, blood glucose and insulin levels in 24 h fasted mice. Significance was determined by Student’s t test (*P < 0.05 compared with USi, n = 8). (E) Representative H&E and Oil Red O staining of liver from Ad-MRP4i injected mice (scale bar, 50 μm). (F) Effect of Ad-MRP4i injection on hepatic fatty acid oxidation gene expression. Significance was determined by Student’s t test (*P < 0.05, **P < 0.01 compared with USi, n = 8).
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   Figure 6

   cAMP administration attenuates hepatic steatosis caused by HFD. (A) Hepatic and serum cAMP levels in RD or HFD-fed mice. Significance was determined by Student’s t test (*P < 0.05, **P < 0.01 compared with RD-fed mice, n = 8). (B) Hepatic Mrp4 mRNA (left) and protein (right) levels in mice fed on RD or HFD. Immunoblot intensities for MRP4/αtubulin were quantitated. Significance was determined by Student’s t test (*P < 0.05 compared with RD-fed mice, n = 8). (C) Effect of cAMP (20 mg/kg) injection on hepatic and serum triacylglycerol, NEFA and serum ketones levels in HFD-fed mice. Significance was determined by Student’s t test (*P < 0.05 compared with control, n = 8). (D) Representative H&E and Oil Red O staining of liver sections from HFD-fed mice injected with cAMP (scale bar, 50 μm). (E) Effect of cAMP (20 mg/kg) injection on hepatic fatty acid oxidation gene expression. Significance was determined by Student’s t test (*P < 0.05, **P < 0.01 compared with control, n = 8). (F) Effect of cAMP (20 mg/kg) injection on blood glucose and insulin levels in HFD-fed mice. Significance was determined by Student’s t test (*P < 0.05 compared with control, n = 8). (G) Effect of cAMP (20 mg/kg) injection on GTT and ITT in HFD-fed mice. Significance was determined by one-way ANOVA (*P < 0.05 compared with control mice, n = 8). (H) Schematic of proposed mechanism.

• © 2017 Society for Endocrinology