Hepatic glucagon action increases in response to accelerated metabolic demands and is associated with increased whole body substrate availability, including circulating lipids. The hypothesis that increases in hepatic glucagon action stimulate AMP-activated protein kinase (AMPK) signaling and peroxisome proliferator-activated receptor-α (PPARα) and fibroblast growth factor 21 (FGF21) expression in a manner modulated by fatty acids was tested in vivo. Wild-type (gcgr+/+) and glucagon receptor-null (gcgr−/−) littermate mice were studied using an 18-h fast, exercise, and hyperglucagonemic-euglycemic clamps plus or minus increased circulating lipids. Fasting and exercise in gcgr+/+, but not gcgr−/− mice, increased hepatic phosphorylated AMPKα at threonine 172 (p-AMPKThr172) and PPARα and FGF21 mRNA. Clamp results in gcgr+/+ mice demonstrate that hyperlipidemia does not independently impact or modify glucagon-stimulated increases in hepatic AMP/ATP, p-AMPKThr172, or PPARα and FGF21 mRNA. It blunted glucagon-stimulated acetyl-CoA carboxylase phosphorylation, a downstream target of AMPK, and accentuated PPARα and FGF21 expression. All effects were absent in gcgr−/− mice. These findings demonstrate that glucagon exerts a critical regulatory role in liver to stimulate pathways linked to lipid metabolism in vivo and shows for the first time that effects of glucagon on PPARα and FGF21 expression are amplified by a physiological increase in circulating lipids.
- adenosine 5′-monophosphate-activated protein kinase
- peroxisome proliferator-activated receptor-α
- fibroblast growth factor 21
hepatic glucagon receptor activation is a physiological stimulus that potently regulates whole body metabolism (19, 24, 42). During fasting and exercise, a rise in glucagon action acts in concert with lowered insulin and increased fatty acids to stimulate glucose-producing pathways in the liver that provide substrate to metabolically active tissues (39, 41–44). Exercise-induced increases in hepatic glucagon action are further linked to transcriptional changes that improve gluconeogenic efficiency and lower lipid content (21, 28).
The canonical pathway by which hepatic glucagon receptor activation exerts such effects is via cAMP/protein kinase A signaling (24). Recent studies, however, show that glucagon receptor activation has previously unforeseen fundamental regulatory effects in the liver. Our work in vivo showed that hepatic glucagon action causes a hepatic energy-depleted state characterized by an increased AMP-to-ATP ratio that is sufficient to activate AMP-activated protein kinase (AMPK) (6). Additional studies in vivo, in perfused liver, and in hepatocytes further support glucagon-mediated activation of AMPK (26, 29, 38). Recent work also extends glucagon's novel actions to include AMPK- and p38 mitogen-activated protein kinase-dependent activation of peroxisome proliferator-activated receptor-α (PPARα) (29). This finding is important because PPARα is a transcription factor critically required for many aspects of hepatic lipid metabolism as well as fibroblast growth factor 21 (FGF21), which regulates lipid metabolism (3, 40). Glucagon is also shown in vitro to induce hepatic expression of FGF21 (38). Increases in FGF21 are notable because administration of this endocrine factor positively impacts glucose and lipid metabolism in vivo (7, 13, 25, 51).
It is essential to understand glucagon receptor-mediated regulation of PPARα and subsequent effects on FGF21 because such targets may, in part, explain physiological adaptations linked to its action in the liver and in the whole body (i.e., fasting, exercise, etc.). Moreover, each component of this putative pathway (glucagon receptor, AMPK, PPARα, and FGF21) is dysregulated in metabolic disease (4, 5, 13, 16, 20, 24, 51) and is a current or proposed therapeutic target (13, 22, 24, 25, 51). It is therefore important to investigate the link between hepatic glucagon and PPARα/FGF21 under well-controlled in vivo conditions and to consider how such a pathway is influenced by other regulatory inputs. Here we test the hypotheses that 1) a rise in hepatic glucagon receptor activation previously shown to activate AMPK signaling causes increased PPARα signaling and 2) the effects of glucagon on AMPK and PPARα signaling are amplified by increased lipid availability. The latter hypothesis is based on evidence that fatty acids are the natural ligand for PPARα (15). This was assessed in vivo in wild-type (gcgr+/+) and littermate mice with genetic deletion of the glucagon receptor (gcgr−/−) using the following conditions: a fast, exhaustive treadmill exercise, and a hyperglucagonemic-euglycemic clamp with or without a simultaneous infusion of a lipid emulsion.
All animal procedures were approved by the Vanderbilt University Animal Care and Use Committee. Experiments used equal numbers of male and female gcgr+/+ and gcgr−/− mice at 12 wk of age on a C57BL/6J background. gcgr−/− mice were previously shown to exhibit reduced fasting blood glucose, lowered plasma insulin, supraphysiological glucagon levels, and have normal sensitivity to catecholamines (19). Mice were housed in a temperature-controlled environment on a 12:12-h light-dark cycle and fed a standard chow diet (5% fat by kcal).
Overnight (18-h) fast.
gcgr+/+ and gcgr−/− mice were either killed at ∼6:00 A.M. after an 18-h overnight fast or in the fed state.
gcgr+/+ and gcgr−/− mice were acclimated to treadmill exercise 2 days before study by performing 10 min of exercise at 10 m/min. On the day of study after a 5-h morning fast, mice either ran until exhaustion or were placed on the nonmoving treadmill belt for 60 min as a control. The exercise protocol was a speed of 10 m/min for 3 min followed by 20 m/min until exhaustion. Exhaustion was defined by mice remaining on a shock grid placed at the end of the treadmill (1.5 mA, 200-ms pulses, 4 Hz) for 5 s.
Surgical techniques used for the hyperglucagonemic-euglycemic experiments have been previously described (2, 8). Briefly, carotid artery and jugular vein catheters were implanted for sampling and infusing, respectively, 5 days before study. Only mice returning to within 10% of presurgical body weight were studied. These techniques permit serial arterial blood sampling, which prevents the associated rise in catecholamines due to cut-tail blood sampling (2).
The in vivo clamp technique used to simultaneously infuse glucagon and/or lipid in 5-h-fasted, 12-wk-old mice has been described (6). The advantage of this protocol is that an increase in blood glucose due to an increment in glucagon is controlled using phloridzin and a variable glucose infusion. The 5-h-fast period also ensures that the mouse is in a postabsorptive state without the marked hepatic glycogen depletion characteristic of an overnight fast (2). At time (t) = −60 min, phloridzin (80 μg·kg−1·min−1) was infused to prevent renal reuptake of glucose. Saline-washed erythrocytes were infused (5 μl/min) to prevent a fall in hematocrit. Blood glucose (3 μl) was measured every 5 min, and a variable glucose infusion rate (GIR) was infused to maintain euglycemia (∼8.5 mM). At t = 0, glucagon (10 ng·kg−1·min−1; Glucagen), lipid (5 U·kg−1·h−1; Intralipid), or glucagon + lipid was infused in gcgr+/+ and gcgr−/− mice from t = 0 to 120 min. Vehicle was infused as needed to account for differences in infusion volume. Blood glucose (3 μl) was measured every 5 min during t = 0–30 min and every 10 min thereafter (t = 30–120 min). GIR was adjusted to maintain euglycemia. Blood samples for plasma insulin (50 μl) and glucagon (50 μl) were taken at t = −15, −5, 100, and 120 min. Samples for plasma catecholamines (100 μl) and nonesterified fatty acid (NEFA, 25 μl) were taken at t = −15 and 110 min. Urine glucose was measured at t = 120 min using urinalysis strips (Diastix; Bayer, Elkhart, IN). All blood samples were collected in 0.6-ml tubes containing EDTA, traysolol, and glutathione and stored at −20°C.
All mice were killed, and livers were quickly removed in situ. Tissues were snap-frozen in liquid nitrogen using precooled aluminum pliers and stored at −80°C. All tissues were crushed into powder before analyses. Liver adenine nucleotides were measured as previously described (6). Liver glycogen was measured enzymatically after ethanol precipitation (12). Liver triglycerides, cholesterol esters, and phospholipids were measured by thin-layer chromatography in the Vanderbilt Mouse Metabolic Phenotypic Center (MMPC) Lipid Core. Total AMPKα, phosphorylated AMPKα at threamine 172 (p-AMPKThr172), total acetyl-CoA carboxylase (ACC), and phosphorylated ACC at serine 79 (p-ACCSer79) were determined in the liver using previously described methods (6). Plasma glucagon (33), insulin (33), epinephrine (31), and norepinephrine (31) were determined by the Vanderbilt MMPC Hormone Assay and Analytical Resources Core. mRNA for PPARα and FGF21 were isolated using a commercial kit (Qiagen RNAeasy) and converted to cDNA using standard methods. Real-time RT-PCR was used to measure mRNA levels of PPARα and FGF21 in the liver. mRNA was detected using TaqMan Assay-on-Demand primers.
Calculations and statistics.
Total glucose requirement was assessed by area under the curve (calculated using the trapezoidal method) of infused glucose during t = 0–180 min. mRNA levels in the liver were quantified in duplicate using the ΔΔCT method normalized to glyceraldehydes-3-phosphate dehydrogenase. Statistical comparisons were made using one-way ANOVA followed by the Fisher least-significant difference test for post hoc comparisons. Data are presented as means ± SE. Statistical significance was defined as P < 0.05.
The glucagon receptor is required for fasting- and treadmill exercise-induced increases in hepatic AMPKThr172 phosphorylation as well as PPARα and FGF21 expression.
Fasting in gcgr+/+ mice induced increases in hepatic p-AMPKThr172/AMPKα as well as PPARα and FGF21 mRNA levels. These responses were attenuated by 95, 65, and 51%, respectively, in gcgr−/− littermates (Fig. 1, A–C). Endurance exercise in gcgr+/+ mice also increased hepatic p-AMPKThr172/AMPKα, PPARα mRNA, and FGF21 mRNA, but this effect was abolished in gcgr−/− mice despite comparable treadmill run times (Fig. 1, E and F; 54 ± 3 and 59 ± 4 min in gcgr+/+ and gcgr−/− mice, respectively).
AMPK signaling and PPARα/FGF21 expression in the liver are stimulated by a physiological increment in glucagon in a manner that is enhanced by increased lipid.
Hyperglucagonemic-euglycemic clamps were performed in gcgr+/+ and gcgr−/− littermate mice with and without infusion of lipid to isolate the interaction of increased glucagon and lipid on AMPK signaling and hepatic expression of PPARα and FGF21 mRNA. There were no differences in body weight or body composition between gcgr+/+ and gcgr−/− mice. The approximately fourfold rise in arterial glucagon during both clamps in gcgr+/+ mice was comparable to the response to exercise in the mouse (17) and is consistent with previous experiments (6) (Fig. 2A). Basal glucagon levels were supraphysiological (>8,000 ng/l) in gcgr−/− mice consistent with prior data (6, 19), and the infusion of glucagon had no measurable effect. Basal insulin and NEFA were slightly lower in gcgr−/− mice compared with gcgr+/+ littermates, but differences were normalized by the hyperglucagonemic-euglycemic clamp (Fig. 2, B and C). Insulin and NEFA were elevated to a similar extent during the clamp in all mice infused with lipid (Fig. 2, B and C). The lipid-induced increase in clamp insulin may be due to changes in insulin clearance or compensatory secretion due to lipid-induced changes in insulin sensitivity (35, 37). Arterial glucose was lower in gcgr−/− mice compared with gcgr+/+ mice, but this difference was alleviated by the clamp (Fig. 3, A and B). The variable GIR needed to achieve comparable glycemia between genotypes and/or treatment is shown in Fig. 3, C and D. The total glucose infusion required to clamp arterial glucose in glucagon-infused gcgr+/+ mice with and without lipid was markedly reduced, indicating potent effects on hepatic glucose production (Fig. 3E). The total glucose requirement in lipid-infused gcgr+/+ mice was also reduced with and without glucagons, further indicating effects on hepatic substrate flux (Fig. 3E). There was no effect of glucagon on the glucose infusion required to clamp glucose in gcgr−/− mice, but lipid did reduce the glucose requirement (Fig. 3F).
Plasma catecholamines were measured to validate that mice were unstressed during the clamp experiments. Arterial epinephrine levels were <15 pg/ml at t = 0 min in all groups and did not increase during the clamp. Arterial norepinephrine levels in vehicle-infused gcgr+/+, glucagon-infused gcgr+/+, lipid-infused gcgr+/+, and glucagon + lipid-infused gcgr+/+ mice were 83 ± 6, 77 ± 10, 70 ± 6, and 79 ± 5 pg/ml, respectively, at t = 0 min and were unchanged at t = 120 min. Arterial norepinephrine levels in the equivalent groups of gcgr−/− mice were 95 ± 6, 82 ± 8, 74 ± 8, and 83 ± 7 pg/ml, respectively, at t = 0 min and were also unchanged at t = 120 min. The low catecholamine levels reflect the absence of stress in our mouse model.
In addition to GIR, the physiological impact of glucagon and/or lipid was assessed based on liver glycogen, triglycerides, cholesterol esters, and phospholipid content. As expected, glucagon reduced hepatic glycogen in vehicle-infused gcgr+/+ mice (Fig. 4A). Infusion of lipid increased hepatic glycogen in gcgr+/+ mice, but this accumulation was prevented by coinfusion of glucagon (Fig. 4B). Neither glucagon nor lipid altered hepatic glycogen in gcgr−/− mice (Fig. 4B). Similar to the effects on glycogen, glucagon also reduced hepatic triglyceride in vehicle-infused gcgr+/+ mice (Fig. 4B). Infusion of lipid increased hepatic triglyceride content in gcgr+/+ mice, and this effect was attenuated by glucagon (Fig. 4B). Glucagon did not affect hepatic triglycerides in gcgr−/− littermates, whereas lipid modestly increased hepatic triglyceride (Fig. 4B). Neither glucagon nor lipid had any effect on cholesterol esters or phospholipids in either genotype (Fig. 4, C and D).
The clamp-mediated rise in arterial glucagon in both vehicle- and lipid-infused gcgr+/+ mice lowered ATP and increased AMP levels by similar magnitudes in the liver (Fig. 5). These increases in hepatic AMP/ATP indicate a comparable reduction in energy state (Fig. 5). Lipid did not independently alter hepatic energy state in gcgr+/+ mice (Fig. 5). There were no glucagon- or lipid-induced changes in hepatic energy state in gcgr−/− littermates (Fig. 5). The glucagon-induced transition to a hepatic energy-depleted state in both groups of gcgr+/+ mice corresponded to similar increases in p-AMPKThr172/AMPKα (Fig. 6A). Infusion of lipid in gcgr+/+ mice had no independent impact on p-AMPKThr172/AMPKα (Fig. 6A). Glucagon and/or lipid did not modulate p-AMPKThr172/AMPKα in gcgr−/− mice (Fig. 6A). Glucagon-induced increases in hepatic p-AMPKThr172/AMPKα were further linked to increased p-ACCSer79/ACC in gcgr+/+ mice. This association was uncoupled in mice receiving lipid (Fig. 6B). Lipid did not independently alter p-ACCSer79/ACC in gcgr+/+ mice but attenuated the glucagon-induced increase (Fig. 6B). Glucagon stimulation of p-ACCSer79/ACC was also abolished in all gcgr−/− mice (Fig. 6B).
The glucagon-induced increase in hepatic AMPK signaling in gcgr+/+ mice was associated with elevated hepatic PPARα and FGF21 mRNA (Fig. 7, A and B). Lipid had no independent effect in gcgr+/+ mice to induce hepatic expression of PPARα and FGF21 mRNA but markedly potentiated the effects of glucagon (Fig. 7, A and B). Effects of glucagon plus or minus lipid on hepatic PPARα and FGF21 mRNA expression were absent in gcgr−/− mice (Fig. 7, C and D).
The current studies were performed to investigate acute glucagon-mediated physiological regulation of hepatic AMPK and PPARα and FGF21 mRNA expression in vivo. Studies were performed in wild-type and littermate mice with genetic deletion of the glucagon receptor. Fasting, exercise, and hyperglucagonemic-euglycemic clamps were used to study physiological actions of increased arterial glucagon. Increased availability of lipids was also studied during the hyperglucagonemic-euglycemic clamps because this is an important component of the response to metabolic stresses and has marked effects on the liver (41), including ligand activation of PPARα (15). Coinfusion of lipid with glucagon during the glucose clamp was designed to recreate the parallel increases seen in physiological conditions [e.g., fasting (32) and exercise (18, 27, 42, 43, 46)].
Fasting was initially used as a physiological stressor because this condition has been previously shown to increase hepatic glucagon action (32), AMPK activity (26, 29), and expression of PPARα and FGF21 (23, 30). Exercise was also used because this condition increases hepatic glucagon action and AMPK activity (10, 11, 41–44, 46). Exercise is of further value in these studies because it increases hepatic glucagon action without elevated blood glucose and insulin (41). The current results in gcgr+/+ mice are consistent with evidence that fasting increases hepatic p-AMPKThr172/AMPKα (47), PPARα mRNA (23, 30), and FGF21 mRNA (23, 30). The current data using exhaustive treadmill exercise also recapitulate increases in hepatic p-AMPKThr172/AMPKα (10) and show for the first time elevated hepatic expression of PPARα and FGF21 mRNA. These data show for the first time the mechanistic link between fasting and exercise-stimulated increases in hepatic p-AMPKThr172/AMPKα, PPARα mRNA, and FGF21 mRNA are attenuated in response to fasting and exercise in gcgr−/− mice.
It is noteworthy that the current results using fasting and exercise in gcgr+/+ and gcgr−/− mice to assess the role of glucagon to stimulate changes in PPARα and, in turn, FGF21 are not in full agreement with recent studies by Longuet et al. (29). Longuet et al. (29) concluded that glucagon injection increases hepatic PPARα mRNA, but not hepatic FGF21 mRNA. Glucagon injection as was performed by Longuet et al. (29) results in hyperglycemia and hyperinsulinemia, which blocks glucagon action. The hyperglucagonemic-euglycemic clamps were performed to circumvent these issues, since this technique permits an isolated physiological increase in glucagon. This is not possible using injections/infusion of glucagon due to stimulation of hepatic glucose production or pancreatic clamps because such techniques result in hyperglycemia, which counteracts the effects of glucagon. This clamp protocol also permits the investigator to normalize blood glucose and insulin between genotypes using techniques that are, in contrast to other methods such as collecting blood from the cut tail, stress-free (validated by low catecholamine levels) (2). The current results in gcgr+/+ and gcgr−/− mice using the hyperglucagonemic-euglycemic clamp support our hypothesis that a physiological link exists between hepatic glucagon action, AMPK signaling, and expression of PPARα and FGF21. Results in gcgr+/+ mice finding a marked increase in hepatic AMP/ATP, increased phosphorylation of AMPK and its downstream target ACC, as well as decreased hepatic glycogen content are in agreement with prior work (6), and the current data extend this pathway to include increased expression of hepatic PPARα and FGF21 mRNA and potent reductions in triglycerides. These latter findings are consistent with previous work showing that glucagon reduces liver triglyceride (29, 45).
The current finding in gcgr+/+ and gcgr−/− mice that lipid infusion did not affect hepatic energy state or p-AMPKThr172/AMPKα was unexpected considering that fatty acids are a critical source of energy in the liver and activate AMPK signaling in vitro (36, 50). This result in gcgr+/+ mice indicates that hepatic energy depletion is a normal physiological response to glucagons stimulation of liver metabolism and that fatty acids neither augment nor protect against the rise in hepatic AMP/ATP. This is also consistent with the present finding that physiological hyperlipidemia did not independently induce changes in p-AMPKThr172/AMPKα. Infusion of lipids did surprisingly suppress the glucagon-induced increase in p-ACCSer79/ACC. p-ACCSer79 was assessed here as a downstream marker of AMPK activity. Prior work in skeletal muscle, however, shows that p-ACCSer79 is regulated by additional kinases (14, 48), and studies in liver show that lipid-induced increases in protein phosphatase 2A activity modify ACC enzyme activity (1, 49). These mechanisms may explain reduced p-ACCSer79/ACC in the context of the hyperglucagonemic-euglycemic clamp with lipid. An additional possibility is higher insulin levels during the clamp performed with lipid-modified p-ACCSer79/ACC via an unknown mechanism. Nonetheless, this component of AMPK signaling is not proposed to modulate PPARα signaling, and the functional effect of glucagon to reduce hepatic triglycerides is intact in lipid-infused gcgr+/+ mice. This effect is particularly robust, since it attenuated the ∼35% increase in hepatic triglyceride associated with infusion of lipid. This is likely due to stimulation of hepatic fat oxidation and not to triglyceride secretion, since glucagon has been shown to inhibit this latter pathway (29). The acute effect of glucagon to reduce hepatic triglycerides also appears to be specific, since there were no changes in phospholipids or cholesterol esters in the present data.
The finding that hyperlipidemia does not independently stimulate hepatic PPARα and FGF21 mRNA, but potentiates glucagon-mediated expression, also highlights a complex regulatory interaction in vivo. This finding was surprising considering that fatty acids are the natural ligand of PPARα (15). PPARα and FGF21 mRNA levels were assessed here as indexes of lipid-induced ligand-mediated activation based on evidence that PPARα is autoregulated (9) and that it is required for expression of FGF21 (3, 23). It is possible that disparities between ligand activation and expression of PPARα exist in the liver, but the fact that FGF21 expression is induced suggests that activation occurred. It is also unknown if such changes in hepatic expression of FGF21 impact circulating levels of the hormone. Plasma FGF21 levels were below levels of detection in the clamp experiments, but this was not surprising considering the short duration of the protocol. Nevertheless, these observations suggest that glucagon-mediated stimulation of AMPK is potentially critical to facilitate lipid-induced PPARα signaling. This notion is supported by in vitro findings that glucagon requires AMPK to stimulate PPARα expression (29).
The fact that the glucagon + lipid-induced changes in hepatic PPARα and FGF21 mRNA occurred despite increased arterial insulin is also noteworthy. This is because elevation in insulin levels may have prevented an even greater increase in PPARα and FGF21 mRNA expression. This notion is based on previous work in hepatocytes showing that insulin and glucagon have opposing effects on FGF21 expression (38). This point is important because a fall in the ratio of insulin to glucagon and increase in circulating fatty acids are characteristic of physiological conditions such as exercise. Fat oxidation is increased during exercise, and repeated bouts of exercise have been shown to improve oxidative pathways in the liver in a glucagon-dependent manner (28). We speculate that glucagon stimulation of PPARα and FGF21 expression may be an underlying mechanism responsible, at least in part, for such changes in the liver. An analogous pathway linking repeated AMPK activation to PPARδ and increased oxidative capacity has been proposed for skeletal muscle (34).
Taken together, the present results confirm the hypothesis that hepatic expression of PPARα and FGF21 is stimulated by hepatic glucagon receptor activation in a manner that is further augmented by fatty acids. Overall, these studies support the view that glucagon receptor signaling is important to stimulate acute and long-term physiological adaptations in both hepatic glucose and lipid metabolism. The present findings further suggest the possibility that antagonism of the glucagon receptor in metabolic disease may have unintended negative consequences related to the blunting of oxidative pathways controlled by AMPK, PPARα, and/or FGF21. In summary, these studies highlight the role of glucagon in regulation of physiological processes in the liver and how such changes interact with other physiological signals (i.e., lipids). These findings are important in understanding adaptations to exercise, fasting, or other conditions in which glucagon is elevated and lends insight into disruption of glucagon action as a potential treatment for metabolic disease.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-50277 and DK-59637 to D. H. Wasserman. E. D. Berglund was supported by an National Institutes of Health T32 Molecular Endocrinology Training Program Grant (DK-07563 awarded to R. O'Brien).
No conflicts of interest are declared by the authors.
We thank the Vanderbilt University Mouse Metabolic Phenotypic Center Analytical Core and Metabolic Pathophysiology Core. We also thak Dr. Joyce Repa for helpful discussion regarding the manuscript.
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