Hepatic energy state is regulated by glucagon receptor signaling in mice

The hepatic energy state, defined by adenine nucleotide levels, couples metabolic pathways with energy requirements. This coupling is fundamental in the adaptive response to many conditions and is impaired in metabolic disease. We have found that the hepatic energy state is substantially reduced following exercise, fasting, and exposure to other metabolic stressors in C57BL/6 mice. Glucagon receptor signaling was hypothesized to mediate this reduction because increased plasma levels of glucagon are characteristic of metabolic stress and because this hormone stimulates energy consumption linked to increased gluconeogenic flux through cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) and associated pathways. We developed what we believe to be a novel hyperglucagonemic-euglycemic clamp to isolate an increment in glucagon levels while maintaining fasting glucose and insulin. Metabolic stress and a physiological rise in glucagon lowered the hepatic energy state and amplified AMP-activated protein kinase signaling in control mice, but these changes were abolished in glucagon receptor–null mice and mice with liver-specific PEPCK-C deletion. 129X1/Sv mice, which do not mount a glucagon response to hypoglycemia, displayed an increased hepatic energy state compared with C57BL/6 mice in which glucagon was elevated. Taken together, these data demonstrate in vivo that the hepatic energy state is sensitive to glucagon receptor activation and requires PEPCK-C, thus providing new insights into liver metabolism.

The energy state in the cell is defined by adenine nucleotide levels and is critically coupled to nearly all metabolic processes (1). In the cell, the adenine nucleotides ATP, ADP, and AMP are tied directly or indirectly to all energetic pathways and allosterically control numerous regulatory enzymes (2–6). Changes in adenine nucleotides typically occur such that ATP and AMP deviate in reciprocal directions, while ADP remains constant (1). Such changes are the basis for using the ratio AMP/ATP (1, 7) or the equation for cellular energy charge ([ATP + (0.5 × ADP)] / [ATP + ADP + AMP]) (8, 9) as indices of the metabolic environment. Metabolic stress is thus characterized by a rise in AMP paired with a fall in ATP levels, reflecting a decrease in energy state. A fall in energy state is of considerable importance, in part due to the regulatory role of AMP/ATP ratios on AMPK activity (10). AMPK is a metabolic switch sensitive to high AMP/ATP ratios and functions to protect the energy state by inhibiting ATP-consuming processes while stimulating ATP-producing processes. (10).

In most tissues, the environment is controlled to maintain a high energy state (low AMP/ATP ratio). In skeletal muscle, for example, creatine kinase limits reductions in ATP during conditions such as exercise, when energy utilization is accelerated (11, 12). The liver, in contrast, lacks creatine kinase, and exercise has been shown to markedly decrease the hepatic energy state and increase the phosphorylation of AMPK (11, 13). The regulatory significance of deviations in the hepatic energy state is further illustrated by studies using 5-aminoimidazole-4-carboxamide-1-β-O-ribofuranoside (AICAR). AICAR is converted to the AMP analog AICA-riboside (ZMP) upon entering the cell and thus mimics an increase in cellular stress. In previous studies, AICAR has been shown to increase energy-producing pathways in the liver relative to energy-consuming pathways (14–16). The regulatory potential of the hepatic energy state is powerful, considering the key role of the liver in whole-body carbohydrate, lipid, and protein metabolism.

While the regulatory potential of an energy-depleted state in the liver is clear, the conditions that result in energy depletion and the mechanisms by which this state occurs are not well understood. Our goals, therefore, were to test the hypotheses that (a) reductions in the hepatic energy state are a physiological phenomenon not isolated to exhaustive exercise and (b) glucagon receptor activation mediates a lowered hepatic energy state sufficient to activate AMPK through a process requiring cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C). The foundation for this latter hypothesis is that a rise in plasma glucagon is characteristic of metabolic stress (17–24) and increased hormone levels are shown to amplify phosphorylation of AMPK in vitro and in perfused liver (25, 26). Studies in vivo have also shown that hepatic glucagon action is the primary determinant of gluconeogenesis via PEPCK-C and stimulation of coupled pathways, including fatty acid activation and ureagenesis, which are required to offset higher glucose use in the body during exercise (27–29).

These hypotheses were tested in conscious mice in response to metabolic stress associated with elevated glucagon; metabolic stress in mice with genetic deletion of the glucagon receptor; hypoglycemia in 129X1/Sv mice, in whom the glucagon response to a fall in glucose is blunted; metabolic stress in which glucagon-stimulated flux through PEPCK-C is inhibited by liver-specific deletion of the enzyme; and a physiological increment in glucagon levels. Experiments in the last situation were carried out by developing a hyperglucagonemic-euglycemic clamp technique which, for what we believe is the first time, makes it possible for an increment in glucagon levels to be examined while maintaining euglycemia and fasting insulin. Collectively, our studies show that reductions in hepatic energy state due to metabolic stress are provoked by glucagon receptor activation and PEPCK-C.

Metabolic stress induces dramatic changes in hepatic adenine nucleotide levels

Liver adenine nucleotides were assessed in C57BL/6 mice following metabolic stressors in which glucagon has been shown to be elevated (fasting, exercise, hypoglycemia, diabetes, and high-fat diet; refs. 17–24). In response to an 18-hour fast, hepatic ATP, ADP, and AMP levels were decreased, unchanged, and increased, respectively, resulting in a nearly 6-fold increase in AMP/ATP ratios compared with fed mice and mice fasted for 5 hours (Figure 1A). Changes in adenine nucleotides induced by an 18-hour fast corresponded to a fall in hepatic energy charge from 0.69 ± 0.02 and 0.69 ± 0.03 in fed and 5-hour-fasted mice, respectively, to 0.46 ± 0.05 (P < 0.05). In response to maximal treadmill exercise, hepatic ATP, ADP, and AMP levels were also decreased, unchanged, and increased, respectively, compared with sedentary mice (Figure 1B). Exercise lowered hepatic energy charge from 0.69 ± 0.03 in sedentary mice to 0.33 ± 0.02 (P < 0.05), consistent with previous findings (11).

Figure 1

Metabolic stress reduces hepatic energy state in C57BL/6 mice. Hepatic adenine nucleotides measured by HPLC in mice following (A) a 5- or 18-hour fast (n = 6–8/group), (B) maximal treadmill exercise (starting at 10 m/min, adding 4 m/min every 3 minutes, after a 5-hour fast; n = 6–8/group), (C) insulin-induced hypoglycemic clamp (n = 6–8/group), and (D) with STZ-induced diabetes (1 s.c. injection of 150 mg/kg; n = 6/group) or fed a high-fat diet (HFD; 60% fat by kcal for 9 weeks) and after fasting for 5 hours (n = 4/group). Hepatic AMP/ATP ratios are also shown for each condition. TAN content is the sum total of ATP, ADP, and AMP. All mice were 12 weeks of age and on a C57BL/6 background except in the experiments shown in C, in which 129X1/Sv mice were also studied. Mice were compared with respective controls (fed, C57BL/6, and 5-hour-fasted chow-fed mice). Data are presented as mean ± SEM. *P < 0.05 compared with controls. ^†P < 0.05 compared with 5-hour-fasted mice.

Insulin-induced hypoglycemia also led to marked changes in hepatic AMP and ATP concentrations, while ADP did not change in C57BL/6 mice (Figure 1C). Hypoglycemia-induced changes in adenine nucleotide corresponded to a significant increase in AMP/ATP ratios (Figure 1C). In 129X1/Sv mice, a strain previously shown to lack a glucagon response to insulin-induced hypoglycemia (30), hepatic adenine nucleotides remained relatively unchanged (Figure 1C) despite hypoglycemia, such that the energy charge was 0.53 ± 0.02. This was in contrast to C57BL/6 mice, in which glucagon levels were increased and hepatic energy charge was diminished to 0.30 ± 0.02 (P < 0.05 vs. 129X1/Sv). Hepatic adenine nucleotides were similar in C57BL/6 and 129X1/Sv mice fasted for 5 hours, corresponding to basal energy charges of 0.71 ± 0.03 and 0.70 ± 0.04, respectively.

In addition to these acute physiological stresses, streptozotocin-induced (STZ-induced) diabetes and a high-fat diet led to decreased ATP and ADP as well as increased AMP levels in the liver (Figure 1D), corresponding to approximately 17- and 23-fold increases, respectively, in hepatic AMP/ATP ratios compared with control mice (Figure 1D). The hepatic energy charge was reduced from 0.70 ± 0.02 in healthy control mice to 0.22 ± 0.01 and 0.18 ± 0.01 in STZ-induced diabetes and high-fat fed mice, respectively (P < 0.05 in both conditions). Total adenine nucleotide (TAN) content in the liver (Figure 1) and energy state in the gastrocnemius (Supplemental Table 1; supplemental material available online with this article; doi: 10.1172/JCI38650DS1) were unchanged under all conditions, consistent with previous results (11).

Glucagon action mediates changes in the hepatic energy state associated with metabolic stress

Given that the aforementioned stressors are associated with elevated glucagon levels, we performed fasting and exercise protocols in mice lacking the glucagon receptor (Gcgr^–/–) and wild-type littermate mice (Gcgr^+/+) to test the requirement for glucagon receptor activation in mediating the effects of metabolic stress on hepatic adenine nucleotides. Body weight was similar between genotypes (24.0 ± 1.0 vs. 23.5 ± 0.8 g for Gcgr^+/+ and Gcgr^–/– mice, respectively). In Gcgr^+/+ mice, fasting and exercise (Figure 2, A and B) altered ATP, ADP, and AMP levels similar to the results obtained in C57BL/6 mice (see Figure 1, A and B). Fasting- and exercise-induced changes in Gcgr^+/+ liver adenine nucleotides corresponded to a fall in hepatic energy charge from 0.69 ± 0.03 and 0.75 ± 0.08 in fed and sedentary control mice, respectively, to 0.47 ± 0.03 and 0.45 ± 0.05, respectively (P < 0.05 in both conditions). In Gcgr^–/– mice, however, fasting-induced changes in hepatic adenine nucleotides were attenuated (Figure 2A) and exercise-induced changes were absent (Figure 2B). In both conditions, changes in hepatic AMP/ATP ratios (Figure 2C) or energy charge were absent in Gcgr^–/– mice compared with Gcgr^+/+ mice. The absence of exercise-induced changes in the hepatic energy state of Gcgr^–/– mice did not alter exercise capacity (22.6 ± 2.1 vs. 24.9 ± 1.9 min). TAN in the liver did not change in any group (Figure 2, A and B). The gastrocnemius energy state did not change in either genotype, despite fasting or exhaustive work (data not shown).

Figure 2

Glucagon receptor is required for metabolic stress–induced reductions in hepatic energy state. Hepatic adenine nucleotides measured by HPLC in Gcgr^+/+ and Gcgr^–/– littermate mice (n = 8/group) following (A) an 18-hour overnight fast and (B) maximal treadmill exercise (starting at 10 m/min and adding 4 m/min every 3 minutes, after a 5-hour fast). Mice were compared with fed controls or with mice that remained sedentary on the treadmill for 30 minutes. (C) Hepatic AMP/ATP ratios for each condition shown in A and B. There were no differences between fed and 5-hour-fasted control mice. Data are presented as mean ± SEM. *P < 0.05 compared with all other groups. **P < 0.05 compared with Gcgr^+/+ control mice. ^#P < 0.05 compared with Gcgr^–/–.

PEPCK-C expression is essential for fasting-induced changes in the hepatic energy state

The requirement for gluconeogenic flux through PEPCK-C in provoking changes in the hepatic energy state was initially tested using an 18-hour fast as a stressor in Pck^lox/lox and Pck^lox/loxAlb-cre mice. As shown in Figure 3, hepatic levels of ATP, ADP, and AMP were decreased, unchanged, and increased, respectively, by an 18-hour fast in Pck^lox/lox mice, corresponding to a 3-fold increase in hepatic AMP/ATP ratios compared with fed controls (Figure 3). Fasting lowered the energy charge from 0.70 ± 0.04 in fed mice to 0.44 ± 0.07 in Pck^lox/lox mice (P < 0.05). In Pck^lox/loxAlb-cre littermates, fasting-induced changes in hepatic adenine nucleotides were absent (Figure 3).

Figure 3

Hepatic PEPCK-C is required for fasting-induced reduction in energy state. Hepatic adenine nucleotides measured by HPLC in Pck^lox/lox and littermate Pck^lox/loxAlb-cre mice (n = 8/group) following an 18-hour overnight fast. Mice were compared with fed controls. Hepatic AMP/ATP ratios for each group is shown on right. Data are presented as mean ± SEM. *P < 0.05 compared with fed Pck^lox/lox mice.

Studies using the hyperglucagonemic-euglycemic clamp

A physiological increment in glucagon levels under euglycemic conditions lowers the hepatic energy state. An in vivo clamp was developed to isolate hepatic glucagon action. Phloridzin and exogenous glucose were infused to create a euglycemic background. Glucagon was then infused, and the glucose infusion rate (GIR) was adjusted to maintain euglycemia. As shown in Figure 4A, plasma glucagon was significantly increased in Gcgr^+/+ mice. Glucagon levels in glucagon- and vehicle-infused Gcgr^–/– mice were greater than 8,000 ng/l, consistent with previous data (31). Clamp insulin levels were unchanged (Figure 4B), and the target euglycemia levels of approximately 8.5 mmol/l were achieved in all groups (Figure 4C). Insulin was also assessed during the non–steady-state period at 30 minutes in separate experiments and was not different from basal (insulin was 186 ± 18, 197 ± 21, 155 ± 11, and 151 ± 15 pmol/l at 0 minutes and 198 ± 31, 161 ± 23, 137 ± 17, and 160 ± 18 pmol/l at 30 minutes in Gcgr^+/+ plus saline, Gcgr^+/+ plus glucagon, Gcgr^–/– plus vehicle, and Gcgr^–/– plus glucagon mice) The GIR needed to maintain blood glucose at about 8.5 mmol/l is shown in Figure 4D.

Figure 4

The hyperglucagonemic-euglycemic clamp protocol permits an increment in glucagon levels without hyperglycemia or hyperinsulinemia. (A) Plasma glucagon, (B) plasma insulin, (C) blood glucose, and (D) GIR in 5-hour-fasted, 12-week-old Gcgr^+/+ and Gcgr^–/– littermate mice on a C57BL/6 background during a hyperglucagonemic-euglycemic clamp (n = 7–9/group). At –60 minutes (equilibration), mice were infused with phloridzin (80 μg/kg/min) and a variable GIR to achieve and maintain euglycemia (~8.5 mmol/l). At 0 minutes, glucagon (10 ng/kg/min) was infused during a 120-minute experimental period. Blood glucose was measured every 5 minutes during equilibration and every 10 minutes during the experimental period. Basal glucose and hormone levels are the mean ± SEM of samples taken at –15 and –5 min. Clamp hormones are the mean ± SEM of samples taken at 100 and 120 minutes. *P < 0.05 compared with Gcgr^+/+ vehicle-injected control mice.

The increment in glucagon levels during the clamp resulted in adenine nucleotide levels comparable to metabolic stressors in Gcgr^+/+ and Gcgr^–/– mice. In Gcgr^+/+ mice, elevated glucagon was associated with decreased ATP, unchanged ADP, and increased AMP levels compared to all other groups (Figure 5A), such that hepatic AMP/ATP ratios were markedly increased (Figure 5A). Energy charge was significantly lowered, from 0.67 ± 0.01 in vehicle-infused Gcgr^+/+ to 0.46 ± 0.09 in glucagon-infused Gcgr^+/+ mice (P < 0.05). In Gcgr^–/– mice, adenine nucleotides were unchanged (Figure 5A) and the hepatic energy charge was 0.73 ± 0.04 and 0.71 ± 0.02 in vehicle- and glucagon-infused mice, respectively. Elevated glucagon in Gcgr^+/+ mice reduced the exogenous glucose requirement to maintain euglycemia (Figure 5B), due to increased hepatic glucose output and greater glycogen breakdown (Figure 5C) compared with vehicle-infused Gcgr^+/+ control mice. The glucose requirement was higher in Gcgr^–/– mice, but the glucagon infusion had no effect (Figure 5B). Urine glucose exceeded 27 mmol/l in each group, indicating a potent inhibition of renal glucose reuptake by phloridzin. There were no significant changes in hematocrit or catecholamines (Supplemental Figure 1), indicating that mice were unstressed during the clamp.

Figure 5

Glucagon lowers the hepatic energy state sufficient to activate AMPK. (A) Hepatic adenine nucleotides measured by HPLC, (B) total glucose infused, and (C) hepatic glycogen in 5-hour-fasted 12-week-old Gcgr^+/+ and Gcgr^–/– littermate mice on a C57BL/6 background following a hyperglucagonemic-euglycemic clamp (n = 7–9/group). Hepatic AMP/ATP ratios are shown for each group in the inset of A. (D–I) Representative immunoblots for p-AMPKα^Thr172/total AMPKα, p-AMPKα1^Thr172/AMPKα1, p-AMPKα2^Thr172/AMPKα2, p-ACC^Ser79/ACC, PEPCK-C, and p-LKB1^Ser428/LKB1 content. All blots are normalized to GAPDH. Data are mean ± SEM. The numbers beneath each lane are arbitrary units normalized to vehicle-infused Gcgr^+/+ mice. *P < 0.05 compared with all other groups. ^†P < 0.05 compared with Gcgr^+/+ mice.

Increased AMP and decreased ATP levels in Gcgr^+/+ mouse livers infused with glucagon corresponded to increased phosphorylation of total AMPKα (Figure 5D) due to increased phosphorylation of both α1 and α2 isoforms (Figure 5, E and F). A downstream target of AMPK, acetyl-CoA carboxylase (ACC), also showed increased phosphorylation in the liver (Figure 5G). Increased AMP/ATP ratios in glucagon-infused Gcgr^+/+ mouse liver also corresponded to increased PEPCK-C protein content (Figure 5F). In contrast, phosphorylation of AMPKα and ACC as well as total PEPCK-C protein content were unchanged in Gcgr^–/– mice infused with glucagon or vehicle (Figure 5, D–F). Total AMPKα, ACC, and PEPCK-C protein content did not differ between Gcgr^–/– and Gcgr^+/+ mice (Figure 5, D–F). There was also no change in the phosphorylation status or total protein content of LKB1, an upstream kinase of AMPK (32), in any group (Figure 5G), supporting a role for allosteric activation of the enzyme by adenine nucleotides. The gastrocnemius energy state was unaltered during the clamp (Supplemental Table 2).

Glucagon-stimulated transition to a hepatic energy–depleted state requires PEPCK-C expression. Hyperglucagonemic-euglycemic clamps were performed in Pck^lox/lox and Pck^lox/loxAlb-cre mice to test the hypothesis that flux through PEPCK-C is required for glucagon action to induce the transition to a hepatic energy–depleted state. As shown in Figure 6A, plasma glucagon levels were elevated in glucagon-infused Pck^lox/lox and Pck^lox/loxAlb-cre mice. Glucagon levels remained similar to basal levels in vehicle-infused mice (Figure 6A). Insulin was unchanged in all groups (Figure 6B). Target euglycemia was achieved in each group (Figure 6C), and the required GIR is shown in Figure 6D.

Figure 6

Liver-specific deletion of PEPCK-C blunts the effect of glucagon during a hyperglucagonemic-euglycemic clamp. (A) Plasma glucagon, (B) plasma insulin, (C) blood glucose, and (D) GIR in 5-hour-fasted, 12-week-old Pck^lox/lox and littermate Pck^lox/loxAlb-cre mice during a hyperglucagonemic-euglycemic clamp (n = 7–9/group). At –60 min (equilibration), mice were infused with phloridzin (80 μg/kg/min) and a variable GIR to achieve and maintain euglycemia (~8.0 mmol/l). At 0 minutes, glucagon (10 ng/kg/min) was infused during a 90-minute experimental period. Blood glucose was measured every 5 minutes during equilibration and every 10 minutes during the experimental period. Basal glucose and hormone levels are the mean ± SEM of samples taken at –15 and –5 minutes. Clamp hormones are the mean ± SEM of samples taken at 70 and 90 minutes. *P < 0.05 compared with Pck^lox/lox vehicle control mice.

In Pck^lox/lox mice, elevated glucagon was associated with decreased ATP, unchanged ADP, and increased AMP levels compared with all other groups (Figure 7A). Glucagon-induced changes in Pck^lox/lox mice corresponded to increased hepatic AMP/ATP ratios (Figure 7A) and an energy charge of 0.32 ± 0.04 (P < 0.05 vs. vehicle-infused Pck^lox/lox mice). Glucagon had no effect in Pck^lox/loxAlb-cre to induce changes in hepatic adenine nucleotides (Figure 7A).

Figure 7

Hepatic PEPCK-C is required to mediate metabolic stress– and glucagon-induced reductions in hepatic energy state. (A) Hepatic adenine nucleotides measured by HPLC in 5-hour-fasted 12-week-old Pck^lox/lox and littermate Pck^lox/loxAlb-cre mice following a hyperglucagonemic-euglycemic clamp (n = 7–9/group). Hepatic AMP/ATP ratios are shown for each group on the right. (B–E) Representative immunoblots for p-AMPKα^Thr172/total AMPKα, p-ACC^Ser79/ACC, PEPCK-C, and p-LKB1^Ser428/LKB1 content. The numbers beneath each lane are arbitrary units normalized to vehicle-infused Pck^lox/loxAlb-cre mice. (F) Hepatic PEPCK-C protein content relative to hepatic AMP/ATP ratios in mice from all metabolic stress and/or clamp conditions in C57BL/6, 129X1/Sv, Gcgr^+/+, Gcgr^–/–, Pck^lox/lox, and Pck^lox/loxAlb-cre mice. PEPCK-C protein content was determined by immunoblot and normalized to C57BL/6 mice in the fed state. Data are mean ± SEM. *P < 0.05 compared with all other groups.

Increased AMP and decreased ATP levels in Pck^lox/lox mouse livers infused with glucagon corresponded to increased phosphorylation of total AMPKα, phosphorylation of its downstream target, ACC, and increased PEPCK-C protein content (Figure 7, B–D). In contrast, phosphorylation of AMPKα and ACC was unchanged in Pck^lox/loxAlb-cre mice infused with glucagon or vehicle (Figure 7, B and C). Neither AMPKα nor ACC protein content differed between genotypes. There was also no change in the phosphorylation status or total protein content of LKB1 in any group (Figure 7E).

The link between hepatic PEPCK-C protein and energy state was further defined by determining the correlation between changes in protein content and hepatic AMP/ATP ratios in mice subjected to metabolic stressors and hyperglucagonemic-euglycemic clamps. As shown in Figure 7F, this correlation is r = 0.63.

The goal of these studies was to define the physiological regulation of the hepatic energy state. This is a fundamental issue in metabolic regulation because adenine nucleotides, which define the energy state, are critically coupled to energetic processes and modulate key regulatory enzymes (e.g., AMPK). Moreover, metabolic pathways in the liver are a hub for whole-body metabolism, and regulatory changes linked to alterations in the hepatic energy state have potentially robust consequences in health and disease. In order to examine the site(s) involved in the regulation of the hepatic energy state, it was necessary to develop a well-controlled model system in which liver metabolism could be studied in vivo. Isolated hepatocytes and perfused liver preparations are valuable model systems. However, they are disruptive to the energy state and require an artificially high oxygen content (usually 95% O₂) to use. This obviated their utility in the current study. Our current findings show that (a) a number of physiological metabolic stressors induce the transition to a hepatic energy–depleted state; (b) hepatic glucagon action and associated flux through PEPCK-C are critical in mediating the stress-induced transition to a hepatic energy–depleted state; and (c) glucagon action amplifies AMPK signaling in the liver through a mechanism that is independent of increased LKB1 activation.

Experiments testing the effect(s) of fasting, exercise, hypoglycemia, diabetes, and a high-fat diet in C57BL/6 mice demonstrate that the transition to a hepatic energy–depleted state occurs across a range of metabolic stressors. These comprehensive results illustrate that adenine nucleotides in the mouse liver are highly sensitive to acute physiological stressors as well as metabolic disease and that changes are not solely isolated to exercise. These findings are important because the magnitude of the changes in hepatic energy state suggests that regulatory consequences are likely, considering the coupling of adenine nucleotides to metabolic pathways in the liver (1). This notion is reinforced by findings that hepatic AMPK phosphorylation is increased following exercise (11, 33, 34) and fasting (34, 35). The existing literature on changes in liver adenine nucleotides in response to metabolic stressors is difficult to interpret (22, 36–43). Previous studies in rats have generally reported stress-induced reductions in hepatic ATP (36–43), although one study found that this did not occur in response to varying fast durations (22). A number of these earlier studies have also shown that hepatic AMP levels increase in response to stress (22, 36, 37, 41), while others have reported no change (42, 43). Many of these earlier studies do not report ADP and/or AMP levels (38–40), estimate adenine nucleotide concentrations using enzymatic methods (22, 36–40, 42, 43), or report reductions in TAN (41), thus complicating a full assessment of the energy state. The current findings, which used HPLC to measure adenine nucleotides, are therefore valuable to show that physiological stress conditions do indeed provoke changes in adenine nucleotides corresponding to a reduced energy state. Changes are characterized by decreases in ATP, no change in ADP, and increases in AMP, such that TAN does not vary. It is important to note that the adenine nucleotide levels reported in the present study are consistent with other data in the mouse (44, 45). It is also unlikely that any of the observed differences are due to tissue handling or analytical differences because the control and experimental groups were studied contemporaneously.

The present studies focusing on adenine nucleotides in the liver also show that changes in response to metabolic stress are in marked contrast to that which occurs in skeletal muscle. This is particularly surprising during exercise because skeletal muscle is performing exhaustive work and AMPK is activated under these circumstances (46, 47). This finding is, however, consistent with the results of a previous study (11). The multifaceted metabolic roles of the liver and the absence of creatine kinase may underlie these contrasting effects. Measurement of skeletal muscle adenine nucleotides is important because they provide further support that results in the liver are not artifacts of anoxia due to technical issues. Anoxia has been previously shown to alter hepatic adenine nucleotides in a manner consistent with a lowered energy state (48). Skeletal muscle is also prone to anoxia, and this condition would reduce the energy state. Skeletal muscle was removed after the liver in each experiment, and adenine nucleotides deviated little, if any, in response to a variety of conditions and a variety of models.

Glucagon action was considered a mechanism to explain changes in the hepatic energy state based on independent evidence that glucagon levels rise during metabolic stress (17–24), activate AMPK (25, 26), and potently stimulate energy-requiring processes in the liver (27–29, 49). Glucagon binding to its receptor increases adenosine cAMP levels in the liver, leading to stimulation of PKA activity. PKA triggers numerous metabolic changes in the liver, including acute effects to increase glycogen breakdown and stimulate gluconeogenic flux through PEPCK-C. These combined pathways represent the canonical actions of glucagon to increase hepatic glucose production (49). Glucagon action is, however, also shown to stimulate metabolic pathways in the liver, such as fat oxidation and ureagenesis, which are fundamental to fueling gluconeogenesis in the liver (27–29). Energetic requirements of gluconeogenesis and activation of substrates in the liver are proposed to underlie reductions in the energy state because several steps in these interrelated processes require hydrolysis of ATP to ADP. AMP and ATP levels in the liver would rise and fall, respectively, due to adenylate kinase activity (2 ADP converted to 1 ATP and 1 AMP), thus lowering the energy state (50). Activation of fatty acids and amino acids in the liver also entails hydrolysis of ATP to AMP, thus directly reducing the energy state. Studies in adipocytes suggest that epinephrine and exercise provoke dramatic changes in adenine nucleotides that activate AMPK (51, 52). Such effects were blocked in isolated adipocytes when acetyl-CoA synthetase, the enzyme required to activate fatty acids, was inhibited, suggesting that this step lowers the energy state (51). The fact that ADP levels do not vary in the current physiological perturbations also suggests that oxidative uncoupling in the mitochondria does not contribute to changes in the energy state.

The requirement for glucagon receptor activation to provoke hepatic energy depletion was first examined using acute metabolic stressors in mice lacking the glucagon receptor. Remarkably, in Gcgr^–/– mice, the exercise-induced hepatic energy–depleted state was abolished and the response to fasting was largely attenuated compared with Gcgr^+/+ littermates. Gcgr^–/– mice have a complicated phenotype, including reduced blood glucose, lower insulin, supra-physiological levels of glucagon, and diminished hepatic fat oxidation (26, 31). Gcgr^–/– mice do, however, retain the capacity to increase cAMP levels in response to epinephrine (31), suggesting that the limited or absent changes in the energy state following metabolic stress is due to loss of the receptor rather than defective pathways in the liver. The action of glucagon to lower the hepatic energy state was further supported by hypoglycemic clamp results in 129X1/Sv mice. In this strain, the blunted glucagon response to hypoglycemia was associated with a higher hepatic energy state compared with C57BL/6 mice, which showed a marked glucagon response and lowered hepatic energy state (30). Taken together, these findings suggest that factors other than glucagon have a limited role in mediating the transition to a hepatic energy–depleted state. Moreover, these results provide in vivo evidence using 2 different model systems, demonstrating that changes in the hepatic energy state are mediated by glucagon receptor activation in the liver.

The action of glucagon to induce changes in the hepatic energy state was isolated in these studies using what is to our knowledge a novel hyperglucagonemic-euglycemic clamp. This powerful in vivo technique permits a selective increase in glucagon to physiological levels without increases in blood glucose and insulin. This is critical to fully define the sensitivity of hepatic glucagon receptor activation. This technique was first performed in Gcgr^+/+ mice to isolate the effect of glucagon on adenine nucleotides. Gcgr^–/– mice were also studied to validate that the clamp technique had no effects other than glucagon receptor activation. In Gcgr^+/+ mice, clamp glucagon was successfully increased to levels observed in mice performing maximal treadmill exercise (18). The elevation in glucagon during the clamp in Gcgr^+/+ mice corresponded to greater stimulation of liver metabolism based on increased glycogen breakdown and a lower GIR requirement to maintain target glycemic levels compared with vehicle-infused Gcgr^+/+ mice. Stimulation of liver metabolism by glucagon in Gcgr^+/+ mice was indeed linked to a lower hepatic energy state. Increased hepatic AMP/ATP ratios corresponded to increased total PEPCK-C protein content as well as greater phosphorylation of AMPK, indicative of increased enzyme activity. Phosphorylation of ACC, a downstream target of AMPK critically involved in regulating fat oxidation, was also increased in Gcgr^+/+ mice, further supporting increased AMPK activity due to elevated glucagon levels. Increased glucagon did not, however, correspond to changes in LKB1 activation, suggesting that changes in AMPK were not strictly due to activity of this upstream kinase, as previously suggested from a study conducted in vitro (25). As expected, glucagon had no effect on the hepatic energy state in Gcgr^–/– mice. There were also no differences in hepatic p-AMPK^Thr172/AMPK, p-ACC^Ser79/ACC, and PEPCK-C between Gcgr^–/– mice and vehicle-infused Gcgr^+/+ mice, indicating that loss of that specific receptor was responsible for effects observed in response to fasting and exercise in Gcgr^–/– mice. Insulin did tend to increase during the clamp in Gcgr^–/– mice, but these levels were appropriate for the increase in blood glucose.

The hypothesis that glucagon action mediates the transition to a hepatic energy–depleted state was further refined using mice with liver-specific deletion of PEPCK-C. As previously noted, glucagon action is well characterized to stimulate gluconeogenesis via increased substrate flux through PEPCK-C. This capacity of the liver to produce glucose de novo results in a net loss of ATP and is critically linked to additional processes that require energy. It is important to note that Pck^lox/loxAlb-cre mice are a complex genetic mouse model that maintains near normal blood glucose despite the absence of this key gluconeogenic enzyme (53–55). Liver-specific deletion of PEPCK-C results in hepatic lipid accumulation and diminished TCA cycle flux indicative of an altered energetic phenotype. This is a highly valuable mouse model in the current studies because the genetic manipulation is liver specific, and sophisticated metabolic flux analyses by Burgess and colleagues have shown in great detail that PEPCK-C–mediated gluconeogenesis is coupled to oxidative and anapleurotic processes (53, 54), which are key in maintaining a hepatic energy state. In these mice, the hepatic energy state was preserved in response to an overnight fast, supporting the notion that PEPCK-C–mediated gluconeogenesis is needed to induce changes in the energy state. Hyperglucagonemic-euglycemic clamp results further clarify a critical role for PEPCK-C to mediate the glucagon-induced transition to a hepatic energy–depleted state and offer further mechanistic insight. This is based on findings that elevated glucagon levels fail to alter hepatic adenine nucleotides in mice with liver-specific deletion of PEPCK-C. The phosphorylation status of LKB1, AMPK, and ACC were also unaltered by higher glucagon levels in Pck^lox/loxAlb-cre mice. This finding again suggests that energetic changes in the liver, resulting in increased AMP-mediated allosteric effects, lead to activation of AMPK rather than a direct pathway involving PKA-mediated covalent modification of LKB1 (25). Our results, which indicate that allosteric modulation of AMPK by adenine nucleotides is the principal regulatory event, are strengthened by evidence that LKB1 is a constitutively active upstream kinase (56, 57). Additional data show that binding of AMP to AMPK increases enzyme activity by inhibiting dephosphorylation (58–60).

Taken together, the present study defines an important metabolic pathway whereby glucagon receptor activation and subsequent stimulation of gluconeogenic flux through PEPCK-C induce a lowered hepatic energy state sufficient to activate AMPK in the liver. A modest positive correlation was found between hepatic PEPCK-C protein content and changes in AMP/ATP ratios. These results support a link between the energetics of gluconeogenesis and associated pathways (i.e., substrate activation steps) and change in hepatic energy state. This link is also supported by a recent study, which found that adenoviral-mediated silencing of PEPCK-C in db/db mice lowered hepatic glucose production and improved hepatic energy status (45). It is important to note that all studies were done in vivo using genetically modified mice and what we believe is a novel clamp protocol to isolate the physiology of glucagon, the glucagon receptor, and PEPCK-C. While each approach may have inherent limitations, all findings converge on the conclusion that glucagon receptor activation leads to a lowered hepatic energy state. These studies also add to the concept of AMPK as a metabolic sensor sensitive to changes in AMP/ATP ratios. These data are noteworthy because few studies focusing on AMPK in the liver have actually measured adenine nucleotides. We propose that the fall in hepatic energy state during acute metabolic stressors is a normal physiological response mediated by glucagon receptor signaling, in which AMPK is stimulated in an orchestrated adaptive effort to increase energy production in the liver.

It is important to consider that glucagon-stimulated changes to lower the hepatic energy state and activate AMPK also likely have long-term transcriptional consequences. This notion is based on strong evidence that AMPK regulates a wide range of transcriptional processes (61). Low hepatic energy levels have also been recently linked to a pathway involving NAD⁺-dependent sirtuin 1–mediated (SIRT1-mediated) deacetylation of PPARγ coactivator 1α (PGC1α) and expression of forkhead box O1 (FOXO1) (62). This pathway has been extended in myocytes to show an interplay between AMPK and SIRT1 to modulate the FOXO family of transcription factors and PGC1α-dependent genes involved in mitochondrial and lipid metabolism (63). In support of our view that changes in adenine nucleotides have a profound regulatory impact, Cantó et al. (63) have proposed that changes in AMP/ATP ratios are the initial metabolic signal triggering transcriptional changes. Similar effects in the liver might contribute to attenuation of exercise-induced reductions in hepatic AMP following long-term training (40) and/or improved efficiency with which the liver converts gluconeogenic substrate into glucose following glucagon stimulation (64, 65). An additional study has, however, dissociated the role of SIRT1 from modulation of PGC1α-dependent genes in vivo in skeletal muscle and heart (66). Future studies are needed to elucidate key components of this pathway and clarify its role in the liver.

An additional important finding is that the hepatic energy state is lowered in high-fat diet–fed and diabetic mice that are not acutely stressed. This suggests that high AMP/ATP levels are part of the metabolic disease milieu, which is consistent with evidence in obese humans showing that hepatic ATP levels are reduced (67, 68). It is noteworthy in this context that ADP was reduced and AMP increased to levels inconsistent with the energetics of adenylate kinase and activation of substrate that have been proposed to mediate the changes seen in response to physiological stress (1, 8, 51). These observations suggest the possibility that hepatic mitochondrial dysfunction (69), hypoxia (70), or additional impairments underlie a lowered energy state in metabolic disease. In addition, if the increase in hepatic AMP/ATP ratios in metabolic disease corresponds to activation of AMPK, then the characteristic development of fatty liver is a paradox. An alternative is that the sensitivity of AMPK to AMP/ATP ratios is diminished in metabolic disease. This notion is supported by recent evidence in ethanol-fed rat liver (71) and the fact that AMPK is not activated by exercise in skeletal muscle of obese individuals with and without type 2 diabetes (72). This finding has therapeutic implications, as AMPK, the glucagon receptor, and PEPCK-C are current or proposed drug targets (45, 49, 73). It is currently unknown whether these interventions would affect AMPK sensitivity to the energy state or downstream AMPK targets. In summary, these studies extend our understanding of metabolic control in the liver to include significant changes in the energy state and provide a more precise understanding of how the liver regulates substrate metabolism.

View Supplemental data

We would like to thank Wanda Snead, Bakula Trivedi, and Eric Allen in the Vanderbilt University Mouse Metabolic Phenotyping Center Hormone and Analytical Core for measuring glucagon, insulin, and catecholamines. We would also like to thank Julio Ayala for technical advice and review of the manuscript. This work was supported by NIH grants DK50277 and DK59637 (to D.H. Wasserman) and by an NIH grant to the Vanderbilt University Molecular Endocrinology Training Program.

Address correspondence to: Eric Berglund, Vanderbilt University Medical Center, 2200 Pierce Ave., 823 Light Hall, Nashville, Tennessee 37232, USA. Phone: (615) 343-0580; Fax: (615) 343-1065; E-mail: berglunde@gmail.com.

Conflict of interest: The authors have declared that no conflict of interest exists.

Nonstandard abbreviations used: ACC, acetyl-CoA carboxylase; GIR, glucose infusion rate; PEPCK-C, cytosolic phosphoenolpyruvate carboxykinase; STZ, streptozotocin; TAN, total adenine nucleotide.

Reference information: J. Clin. Invest.119:2412–2422 (2009). doi:10.1172/JCI38650

1. Hardie, D.G., Hawley, S.A. 2001. AMP-activated protein kinase: the energy charge hypothesis revisited. Bioessays. 23:1112-1119.
   View this article via: CrossRef PubMed Google Scholar
2. Cheung, P.C., Salt, I.P., Davies, S.P., Hardie, D.G., Carling, D. 2000. Characterization of AMP-activated protein kinase gamma-subunit isoforms and their role in AMP binding. Biochem. J. 346:659-669.
   View this article via: CrossRef PubMed Google Scholar
3. Cohen, P.F., Colman, R.F. 1972. Diphosphopyridine nucleotide dependent isocitrate dehydrogenase from pig heart. Charactgerization of the active substrate and modes of regulation. Biochemistry. 11:1501-1508.
   View this article via: CrossRef PubMed Google Scholar
4. Ercan, N., Gannon, M.C., Nuttall, F.Q. 1996. Allosteric regulation of liver phosphorylase a: revisited under approximated physiological conditions. Arch. Biochem. Biophys. 328:255-264.
   View this article via: CrossRef PubMed Google Scholar
5. Ercan-Fang, N., et al. 2002. Integrated effects of multiple modulators on human liver glycogen phosphorylase a. Am. J. Physiol. Endocrinol. Metab. 283:E29-E37.
   View this article via: PubMed Google Scholar
6. Nelson, S.W., Honzatko, R.B., Fromm, H.J. 2002. Hybrid tetramers of porcine liver fructose-1,6-bisphosphatase reveal multiple pathways of allosteric inhibition. J. Biol. Chem. 277:15539-15545.
   View this article via: CrossRef PubMed Google Scholar
7. Hardie, D.G., Salt, I.P., Hawley, S.A., Davies, S.P. 1999. AMP-activated protein kinase: an ultrasensitive system for monitoring cellular energy charge. Biochem. J. 338:717-722.
   View this article via: CrossRef PubMed Google Scholar
8. Atkinson, D.E. 1977.Cellular energy metabolism and its regulation. Academic Press. New York, New York, USA. 304 pp.
9. Ramaiah, A., Hathaway, J.A., Atkinson, D.E. 1964. Adenylate as a metabolic regulator. Effect on yeast phosphofructokinase kinetics. J. Biol. Chem. 239:3619-3622.
   View this article via: PubMed Google Scholar
10. Hardie, D.G., Hawley, S.A., Scott, J.W. 2006. AMP-activated protein kinase — development of the energy sensor concept. J. Physiol. 574:7-15.
    View this article via: CrossRef PubMed Google Scholar
11. Camacho, R.C., et al. 2006. Energy state of the liver during short-term and exhaustive exercise in C57BL/6J mice. Am. J. Physiol. Endocrinol. Metab. 290:E405-E408.
    View this article via: CrossRef PubMed Google Scholar
12. Ponticos, M., et al. 1998. Dual regulation of the AMP-activated protein kinase provides a novel mechanism for the control of creatine kinase in skeletal muscle. EMBO J. 17:1688-1699.
    View this article via: CrossRef PubMed Google Scholar
13. Sauer, U., Schlattner, U. 2004. Inverse metabolic engineering with phosphagen kinase systems improves the cellular energy state. Metab. Eng. 6:220-228.
    View this article via: CrossRef PubMed Google Scholar
14. Camacho, R.C., Lacy, D.B., James, F.D., Donahue, E.P., Wasserman, D.H. 2005. 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside renders glucose output by the liver of the dog insensitive to a pharmacological increment in insulin. Am. J. Physiol. Endocrinol. Metab. 289:E1039-E1043.
    View this article via: CrossRef PubMed Google Scholar
15. Camacho, R.C., et al. 2005. Portal venous 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside infusion overcomes hyperinsulinemic suppression of endogenous glucose output. Diabetes. 54:373-382.
    View this article via: CrossRef PubMed Google Scholar
16. Pencek, R.R., et al. 2005. 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside causes acute hepatic insulin resistance in vivo. Diabetes. 54:355-360.
    View this article via: CrossRef PubMed Google Scholar
17. Alford, F.P., Zimmet, P., Chisholm, D.J., Whitehouse, S. 1983. Influence of obesity on basal glucagon levels in non-diabetic and diabetic Nauruans. Clin. Endocrinol. (Oxf.). 19:721-725.
    View this article via: CrossRef PubMed Google Scholar
18. Fueger, P.T., et al. 2007. Glucose kinetics and exercise tolerance in mice lacking the GLUT4 glucose transporter. J. Physiol. 582:801-812.
    View this article via: CrossRef PubMed Google Scholar
19. Galbo, H., Holst, J.J., Christensen, N.J. 1975. Glucagon and plasma catecholamine responses to graded and prolonged exercise in man. J. Appl. Physiol. 38:70-76.
    View this article via: PubMed Google Scholar
20. Gerich, J.E., et al. 1974. Characterization of the glucagon response to hypoglycemia in man. J. Clin. Endocrinol. Metab. 38:77-82.
    View this article via: PubMed Google Scholar
21. Marliss, E.B., Aoki, T.T., Unger, R.H., Soeldner, J.S., Cahill (Jr.), G.F. 1970. Glucagon levels and metabolic effects in fasting man. J. Clin. Invest. 49:2256-2270.
    View this article via: JCI CrossRef PubMed Google Scholar
22. Seitz, H.J., Muller, M.J., Krone, W., Tarnowski, W. 1977. Coordinate control of intermediary metabolism in rat liver by the insulin/glucagon ratio during starvation and after glucose refeeding. Regulatory significance of long-chain acyl-CoA and cyclic AMP. Arch. Biochem. Biophys. 183:647-663.
    View this article via: CrossRef PubMed Google Scholar
23. Starke, A.A., Erhardt, G., Berger, M., Zimmermann, H. 1984. Elevated pancreatic glucagon in obesity. Diabetes. 33:277-280.
    View this article via: CrossRef PubMed Google Scholar
24. Unger, R.H., Orci, L. 1976. Physiology and pathophysiology of glucagon. Physiol. Rev. 56:778-826.
    View this article via: PubMed Google Scholar
25. Kimball, S.R., Siegfried, B.A., Jefferson, L.S. 2004. Glucagon represses signaling through the mammalian target of rapamycin in rat liver by activating AMP-activated protein kinase. J. Biol. Chem. 279:54103-54109.
    View this article via: CrossRef PubMed Google Scholar
26. Longuet, C., et al. 2008. The glucagon receptor is required for the adaptive metabolic response to fasting. Cell Metab. 8:359-371.
    View this article via: CrossRef PubMed Google Scholar
27. Krishna, M.G., et al. 2000. Glucagon response to exercise is critical for accelerated hepatic glutamine metabolism and nitrogen disposal. Am. J. Physiol. Endocrinol. Metab. 279:E638-E645.
    View this article via: PubMed Google Scholar
28. Wasserman, D.H., Lacy, D.B., Goldstein, R.E., Williams, P.E., Cherrington, A.D. 1989. Exercise-induced fall in insulin and increase in fat metabolism during prolonged muscular work. Diabetes. 38:484-490.
    View this article via: CrossRef PubMed Google Scholar
29. Wasserman, D.H., et al. 1989. Glucagon is a primary controller of hepatic glycogenolysis and gluconeogenesis during muscular work. Am. J. Physiol. 257:E108-E117.
    View this article via: PubMed Google Scholar
30. Berglund, E.D., et al. 2008. Glucose metabolism in vivo in four commonly used inbred mouse strains. Diabetes. 57:1790-1799.
    View this article via: CrossRef PubMed Google Scholar
31. Gelling, R.W., et al. 2003. Lower blood glucose, hyperglucagonemia, and pancreatic alpha cell hyperplasia in glucagon receptor knockout mice. Proc. Natl. Acad. Sci. U. S. A. 100:1438-1443.
    View this article via: CrossRef PubMed Google Scholar
32. Hawley, S.A., et al. 2003. Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade. J. Biol. 2:28.
    View this article via: PubMed Google Scholar
33. Carlson, C.L., Winder, W.W. 1999. Liver AMP-activated protein kinase and acetyl-CoA carboxylase during and after exercise. J. Appl. Physiol. 86:669-674.
    View this article via: PubMed Google Scholar
34. Witters, L.A., Kemp, B.E. 1992. Insulin activation of acetyl-CoA carboxylase accompanied by inhibition of the 5ι-AMP-activated protein kinase. J. Biol. Chem. 267:2864-2867.
    View this article via: PubMed Google Scholar
35. Munday, M.R., Milic, M.R., Takhar, S., Holness, M.J., Sugden, M.C. 1991. The short-term regulation of hepatic acetyl-CoA carboxylase during starvation and re-feeding in the rat. Biochem. J. 280:733-737.
    View this article via: PubMed Google Scholar
36. Bravo, C., et al. 2001. Metabolic changes induced by cold stress in rat liver mitochondria. J. Bioenerg. Biomembr. 33:289-301.
    View this article via: CrossRef PubMed Google Scholar
37. Dohm, G.L., Newsholme, E.A. 1983. Metabolic control of hepatic gluconeogenesis during exercise. Biochem. J. 212:633-639.
    View this article via: PubMed Google Scholar
38. Ghanbari-Niaki, A., Bergeron, R., Latour, M.G., Lavoie, J.M. 1999. Effects of physical exercise on liver ATP levels in fasted and phosphate-injected rats. Arch. Physiol. Biochem. 107:393-402.
    View this article via: PubMed Google Scholar
39. Ghanbari-Niaki, A., Desy, F., Lavoie, J.M. 1999. Effects of phosphate injection on metabolic and hormonal responses to exercise in fructose-injected rats. Physiol. Behav. 67:747-752.
    View this article via: CrossRef PubMed Google Scholar
40. Houghton, C.R., Hawkins, R.A., Williamson, D.H., Krebs, H.A. 1971. The effects of physical training on the metabolic response to short-term severe exercise in the rat. Biochem. J. 124:57P.
    View this article via: PubMed Google Scholar
41. Mikami, T., Kitagawa, J. 2006. Intense exercise induces the degradation of adenine nucleotide and purine nucleotide synthesis via de novo pathway in the rat liver. Eur. J. Appl. Physiol. 96:543-550.
    View this article via: CrossRef PubMed Google Scholar
42. Start, C., Newsholme, E.A. 1968. The effects of starvation and alloxan-diabetes on the contents of citrate and other metabolic intermediates in rat liver. Biochem. J. 107:411-415.
    View this article via: PubMed Google Scholar
43. Williamson, D.H., Veloso, D., Ellington, E.V., Krebs, H.A. 1969. Changes in the concentrations of hepatic metabolites on administration of dihydroxyacetone or glycerol to starved rats and their relationship to the control of ketogenesis. Biochem. J. 114:575-584.
    View this article via: PubMed Google Scholar
44. Burgess, S.C., et al. 2006. Diminished hepatic gluconeogenesis via defects in tricarboxylic acid cycle flux in peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha)-deficient mice. J. Biol. Chem. 281:19000-19008.
    View this article via: CrossRef PubMed Google Scholar
45. Gomez-Valades, A.G., et al. 2008. Pck1 gene silencing in the liver improves glycemia control, insulin sensitivity, and dyslipidemia in db/db mice. Diabetes. 57:2199-2210.
    View this article via: CrossRef PubMed Google Scholar
46. Klein, D.K., et al. 2007. Lack of AMPKalpha2 enhances pyruvate dehydrogenase activity during exercise. Am. J. Physiol. Endocrinol. Metab. 293:E1242-E1249.
    View this article via: CrossRef PubMed Google Scholar
47. Winder, W.W., Hardie, D.G. 1996. Inactivation of acetyl-CoA carboxylase and activation of AMP-activated protein kinase in muscle during exercise. Am. J. Physiol. 270:E299-E304.
    View this article via: PubMed Google Scholar
48. Faupel, R.P., Seitz, H.J., Tarnowski, W., Thiemann, V., Weiss, C. 1972. The problem of tissue sampling from experimental animals with respect to freezing technique, anoxia, stress and narcosis. A new method for sampling rat liver tissue and the physiological values of glycolytic intermediates and related compounds. Arch. Biochem. Biophys. 148:509-522.
    View this article via: CrossRef PubMed Google Scholar
49. Jiang, G., Zhang, B.B. 2003. Glucagon and regulation of glucose metabolism. Am. J. Physiol. Endocrinol. Metab. 284:E671-E678.
    View this article via: PubMed Google Scholar
50. Richter, E.A., Ruderman, N.B. 2009. AMPK and the biochemistry of exercise: implications for human health and disease. Biochem. J. 418:261-275.
    View this article via: CrossRef PubMed Google Scholar
51. Gauthier, M.S., et al. 2008. AMP-activated protein kinase is activated as a consequence of lipolysis in the adipocyte: potential mechanism and physiological relevance. J. Biol. Chem. 283:16514-16524.
    View this article via: CrossRef PubMed Google Scholar
52. Koh, H.J., et al. 2007. Adrenaline is a critical mediator of acute exercise-induced AMP-activated protein kinase activation in adipocytes. Biochem. J. 403:473-481.
    View this article via: CrossRef PubMed Google Scholar
53. Burgess, S.C., et al. 2004. Impaired tricarboxylic acid cycle activity in mouse livers lacking cytosolic phosphoenolpyruvate carboxykinase. J. Biol. Chem. 279:48941-48949.
    View this article via: CrossRef PubMed Google Scholar
54. Burgess, S.C., et al. 2007. Cytosolic phosphoenolpyruvate carboxykinase does not solely control the rate of hepatic gluconeogenesis in the intact mouse liver. Cell Metab. 5:313-320.
    View this article via: CrossRef PubMed Google Scholar
55. She, P., et al. 2000. Phosphoenolpyruvate carboxykinase is necessary for the integration of hepatic energy metabolism. Mol. Cell. Biol. 20:6508-6517.
    View this article via: CrossRef PubMed Google Scholar
56. Sakamoto, K., Goransson, O., Hardie, D.G., Alessi, D.R. 2004. Activity of LKB1 and AMPK-related kinases in skeletal muscle: effects of contraction, phenformin, and AICAR. Am. J. Physiol. Endocrinol. Metab. 287:E310-E317.
    View this article via: CrossRef PubMed Google Scholar
57. Woods, A., et al. 2003. LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr. Biol. 13:2004-2008.
    View this article via: CrossRef PubMed Google Scholar
58. Davies, S.P., Helps, N.R., Cohen, P.T., Hardie, D.G. 1995. 5ι-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase. Studies using bacterially expressed human protein phosphatase-2C alpha and native bovine protein phosphatase-2AC. FEBS Lett. 377:421-425.
    View this article via: CrossRef PubMed Google Scholar
59. Sanders, M.J., Grondin, P.O., Hegarty, B.D., Snowden, M.A., Carling, D. 2007. Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade. Biochem. J. 403:139-148.
    View this article via: CrossRef PubMed Google Scholar
60. Suter, M., et al. 2006. Dissecting the role of 5ι-AMP for allosteric stimulation, activation, and deactivation of AMP-activated protein kinase. J. Biol. Chem. 281:32207-32216.
    View this article via: CrossRef PubMed Google Scholar
61. McGee, S.L., Hargreaves, M. 2008. AMPK and transcriptional regulation. Front. Biosci. 13:3022-3033.
    View this article via: CrossRef PubMed Google Scholar
62. Feige, J.N., et al. 2008. Specific SIRT1 activation mimics low energy levels and protects against diet-induced metabolic disorders by enhancing fat oxidation. Cell Metab. 8:347-358.
    View this article via: CrossRef PubMed Google Scholar
63. Canto, C., et al. 2009. AMPK regulates energy expenditure by modulating NAD(+) metabolism and SIRT1 activity. Nature. 458:1056-1060.
    View this article via: CrossRef PubMed Google Scholar
64. McGuinness, O.P., Burgin, K., Moran, C., Bracy, D., Cherrington, A.D. 1994. Role of glucagon in the metabolic response to stress hormone infusion in the conscious dog. Am. J. Physiol. 266:E438-E447.
    View this article via: PubMed Google Scholar
65. Stevenson, R.W., et al. 1987. Similar dose responsiveness of hepatic glycogenolysis and gluconeogenesis to glucagon in vivo. Diabetes. 36:382-389.
    View this article via: CrossRef PubMed Google Scholar
66. Gurd, B.J., Yoshida, Y., Lally, J., Holloway, G.P., Bonen, A. 2009. The deacetylase enzyme SIRT1 is not associated with oxidative capacity in rat heart and skeletal muscle and its overexpression reduces mitochondrial biogenesis. J. Physiol. 587:1817-1828.
    View this article via: CrossRef PubMed Google Scholar
67. Nair, S., Chacko, V.P., Arnold, C., Diehl, A.M. 2003. Hepatic ATP reserve and efficiency of replenishing: comparison between obese and nonobese normal individuals. Am. J. Gastroenterol. 98:466-470.
    View this article via: PubMed Google Scholar
68. Solga, S.F., et al. 2008. Hepatic fat and adenosine triphosphate measurement in overweight and obese adults using 1H and 31P magnetic resonance spectroscopy. Liver Int. 28:675-681.
    View this article via: PubMed Google Scholar
69. Lowell, B.B., Shulman, G.I. 2005. Mitochondrial dysfunction and type 2 diabetes. Science. 307:384-387.
    View this article via: CrossRef PubMed Google Scholar
70. Mantena, S.K., et al. 2009. High fat diet induces dysregulation of hepatic oxygen gradients and mitochondrial function in vivo. Biochem. J. 417:183-193.
    View this article via: CrossRef PubMed Google Scholar
71. Garcia-Villafranca, J., Guillen, A., Castro, J. 2008. Ethanol consumption impairs regulation of fatty acid metabolism by decreasing the activity of AMP-activated protein kinase in rat liver. Biochimie. 90:460-466.
    View this article via: CrossRef PubMed Google Scholar
72. Sriwijitkamol, A., et al. 2007. Effect of acute exercise on AMPK signaling in skeletal muscle of subjects with type 2 diabetes: a time-course and dose-response study. Diabetes. 56:836-848.
    View this article via: CrossRef PubMed Google Scholar
73. Zhou, G., et al. 2001. Role of AMP-activated protein kinase in mechanism of metformin action. J. Clin. Invest. 108:1167-1174.
    View this article via: JCI PubMed Google Scholar
74. Ayala, J.E., Bracy, D.P., McGuinness, O.P., Wasserman, D.H. 2006. Considerations in the design of hyperinsulinemic-euglycemic clamps in the conscious mouse. Diabetes. 55:390-397.
    View this article via: CrossRef PubMed Google Scholar
75. Chan, T.M., Exton, J.H. 1976. A rapid method for the determination of glycogen content and radioactivity in small quantities of tissue or isolated hepatocytes. Anal. Biochem. 71:96-105.
    View this article via: CrossRef PubMed Google Scholar
76. Morgan, C.R., Lazarow, A. 1962. Immunoassay of insulin using a two-antibody system. Proc. Soc. Exp. Biol. Med. 110:29-32.
    View this article via: PubMed Google Scholar
77. Macdonald, I.A., Lake, D.M. 1985. An improved technique for extracting catecholamines from body fluids. J. Neurosci. Methods. 13:239-248.
    View this article via: PubMed Google Scholar