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. IntroductionThe 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...

A portal venous 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside infusion that results in hepatic 5-aminoimidazole-4-carboxamide-1-␤-D-ribosyl-5-monophosphate (ZMP) concentrations of ϳ4 mol/g liver increases hepatic glycogenolysis and glucose output. ZMP is an AMP analog that mimics the regulatory actions of this nucleotide. The aim of this study was to measure hepatic AMP concentrations in response to increasing energy requirements to test the hypothesis that AMP achieves concentrations during exercise, consistent with a role in stimulation of hepatic glucose metabolism. Male C57BL/6J mice (27.4 Ϯ 0.4 g) were subjected to 35 min of rest [sedentary (SED), n ϭ 8], underwent short-term (ST, 35 min) moderate (20 m/min, 5% grade) exercise (n ϭ 8), or underwent treadmill exercise under similar conditions but until exhaustion (EXH, n ϭ 8). Hepatic AMP concentrations were 0.82 Ϯ 0.05, 1.17 Ϯ 0.11, and 2.52 Ϯ 0.16 mol/g liver in SED, ST, and EXH mice, respectively (P Ͻ 0.05). Hepatic energy charge was 0.66 Ϯ 0.01, 0.58 Ϯ 0.02, and 0.33 Ϯ 0.22 in SED, ST, and EXH mice, respectively (P Ͻ 0.05). Hepatic glycogen was 11.6 Ϯ 1.0, 8.8 Ϯ 2.2, and 0.0 Ϯ 0.1 mg/g liver in SED, ST, and EXH mice, respectively (P Ͻ 0.05). Hepatic AMPK (Thr 172 ) phosphorylation was 1.00 Ϯ 0.14, 1.96 Ϯ 0.16, and 7.44 Ϯ 0.63 arbitrary units in SED, ST, and EXH mice, respectively (P Ͻ 0.05). Thus exercise increases hepatic AMP concentrations. These data suggest that the liver is highly sensitive to metabolic demands, as evidenced by dramatic changes in cellular energy indicators (AMP) and sensors thereof (AMP-activated protein kinase). In conclusion, AMP is sensitively regulated, consistent with it having an important role in hepatic metabolism.adenosine 5Ј-monophosphate; adenosine 5Ј-monophosphate-activated protein kinase; glycogen ALMOST ALL CELLULAR PROCESSES are coupled to ATP breakdown, giving critical importance to the pathways that maintain appropriate ratios of AMP and ATP. It is not surprising that the cell has evolved sensitive mechanisms to detect, regulate, and compensate for changes in their levels. AMP-activated protein kinase (AMPK) is one such mechanism that acts to prevent deficits in energy metabolism during metabolic stresses, including exercise (9,10,23,31,33). AMP is well known to regulate, in addition to AMPK, other enzyme reactions that act to preserve cellular energy charge, including both liver (13,14) and muscle (26) glycogen phosphorylase and fructose-1,6-bisphosphatase (22). It was recently shown that the intraportal infusion (1 mg ⅐ kg Ϫ1 ⅐ min Ϫ1 ) of the purine nucleotide precursor 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside (AICAR) potently stimulates the breakdown of liver glycogen (5, 6, 24). Two aspects of this work were particularly noteworthy. First, AICAR-induced hepatic glycogen breakdown is AMPK independent because it occurs in the presence of marked hyperinsulinemia that suppresses the activation of AMPK (5). Second, the AICAR infusion was considerably less than previously used in vivo, creating hepa...

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AMP-activated protein kinase (AMPK) plays a key role in regulating metabolism, serving as a metabolic master switch. The aim of this study was to assess whether increased concentrations of the AMP analog, 5-aminoimidazole-4-carboxamide-1-␤-D-ribosyl-5-monophosphate, in the liver would create a metabolic response consistent with an increase in whole-body metabolic need. Dogs had sampling (artery, portal vein, hepatic vein) and infusion (vena cava, portal vein) catheters and flow probes (hepatic artery, portal vein) implanted >16 days before a study. Protocols consisted of equilibration (؊130 to ؊30 min), basal (؊30 to 0 min), and hyperinsulinemic-euglycemic or -hypoglycemic clamp periods (0 -150 min). At t ‫؍‬ 0 min, somatostatin was infused and glucagon was replaced in the portal vein at basal rates. An intraportal hyperinsulinemic (2 mU ⅐ kg ؊1 ⅐ min ؊1) infusion was also initiated at this time. Glucose was clamped at hypoglycemic or euglycemic levels in the presence (H-AIC, n ‫؍‬ 6; E-AIC, n ‫؍‬ 6) or absence (H-SAL, n ‫؍‬ 6; E-SAL, n ‫؍‬ 6) of a portal venous 5-aminoimidazole-4-carboxamide-ribofuranoside (AICAR) infusion (1 mg ⅐ kg ؊1 ⅐ min ؊1) initiated at t ‫؍‬ 60 min. In the presence of intraportal saline, glucose was infused into the vena cava to match glucose levels seen with intraportal AICAR. Glucagon remained fixed at basal levels, whereas insulin rose similarly in all groups. Glucose fell to 50 ؎ 2 mg/dl by t ‫؍‬ 60 min in hypoglycemic groups and remained at 105 ؎ 3 mg/dl in euglycemic groups. Endogenous glucose production (R a ) was similarly suppressed among groups in the presence of euglycemia or hypoglycemia before t ‫؍‬ 60 min and remained suppressed in the H-SAL and E-SAL groups. However, intraportal AICAR infusion stimulated R a to increase by 2.5 ؎