Kinetic analysis of short-term effects of alpha-agonists on gluconeogenesis in isolated rat hepatocytes

Isolated hepatocytes from fasted rats were perifused with glycerol as gluconeogenic substrate. Stimulation of gluconeogenesis with phenylephrine (10(-5) M) as alpha-adrenergic agonist consisted of two distinct phases. The first phase was a transient stimulation of gluconeogenesis and was accompanied by transient changes in cytosolic and mitochondrial redox state; this phase was abolished by the transaminase inhibitor aminooxyacetate. The second phase was a stable stimulation of less magnitude, without change in redox state and insensitive to addition of aminooxyacetate. It is concluded that the first phase is due to a transient enhancement of flux through the malate/aspartate shuttle and that the stable phase is probably due to a stimulation of mitochondrial glycerol-3-phosphate dehydrogenase and glycerol kinase.     

Regulation of gluconeogenesis by glycerol and its phosphorylated derivatives

Glycerol, glycerol-3-phosphate (G3P), and dihydroxyacetone phosphate (DHAP) were evaluated as inhibitors of gluconeogenesis on rat liver enzymes in vitro, and for their effects on glucose formation in vivo in well-nourished and malnourished rats. DHAP was more potent as an inhibitor than G3P on fructose-1,6-diphosphatase (FDPase), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase). The I50 for DHAP was 2, 8, and 9 x 10(-3) M, respectively. No effect was observed on rat liver pyruvate carboxylase (PC). Glycerol was a weak inhibitor of FDPase and PEPCK, but did not inhibit PC and G6Pase. In vivo, when G3P was injected before a parenteral L-alanine (Ala) challenge, it produced a hypoglycemic effect in malnourished rats and a lesser, but noticeable, blood glucose level reduction in well-fed animals. Glycerol caused a smaller reduction in glucose formation from Ala. No comparable effects were observed after a fructose pretreatment. These results underscore the potential hypoglycemic effects of phosphorylated glycerol metabolites and identify the steps in gluconeogenesis where this action is exerted. The study also stresses the nutritional component in the glycerol intolerance syndrome, apparent from the far more severe effects observed in malnourished rats given G3P or glycerol prior to Ala.     

Glycerol metabolism in the neonatal rat

1. The possible role of glycerol as a precursor in neonatal gluconeogenesis in the rat was investigated by recording the activities of glycerol kinase and l-glycerol 3-phosphate dehydrogenase in the liver, kidney and other tissues around birth and during the neonatal period. 2. Blood glycerol concentrations in the neonatal rat are high. 3. There is a marked increase after birth in the ability of both liver and kidney slices to convert glycerol into glucose plus glycogen that correlates with the increase in glycerol kinase activity. 4. High hepatic and renal l-glycerol 3-phosphate dehydrogenase activities are also found in the neonatal period. 5. The marked capacity for neonatal gluconeogenesis from glycerol thus demonstrated and the role of glycerol kinase in its control are discussed.     

Aquaporin-9 protein is the primary route of hepatocyte glycerol uptake for glycerol gluconeogenesis in mice

It has been hypothesized that aquaporin-9 (AQP9) is part of the unknown route of hepatocyte glycerol uptake. In a previous study, leptin receptor-deficient wild-type mice became diabetic and suffered from fasting hyperglycemia whereas isogenic AQP9(-/-) knock-out mice remained normoglycemic. The reason for this improvement in AQP9(-/-) mice was not established before. Here, we show increased glucose output (by 123% ± 36% S.E.) in primary hepatocyte culture when 0.5 mM extracellular glycerol was added. This increase depended on AQP9 because it was absent in AQP9(-/-) cells. Likewise, the increase was abolished by 25 μM HTS13286 (IC(50) ~ 2 μM), a novel AQP9 inhibitor, which we identified in a small molecule library screen. Similarly, AQP9 deletion or chemical inhibition eliminated glycerol-enhanced glucose output in perfused liver preparations. The following control experiments suggested inhibitor specificity to AQP9: (i) HTS13286 affected solute permeability in cell lines expressing AQP9, but not in cell lines expressing AQPs 3, 7, or 8. (ii) HTS13286 did not influence lactate- and pyruvate-dependent hepatocyte glucose output. (iii) HTS13286 did not affect glycerol kinase activity. Our experiments establish AQP9 as the primary route of hepatocyte glycerol uptake for gluconeogenesis and thereby explain the previously observed, alleviated diabetes in leptin receptor-deficient AQP9(-/-) mice.     

Proportional activities of glycerol kinase and glycerol 3-phosphate dehydrogenase in rat hepatomas.

The activities of glycerol 3-phosphate dehydrogenase (EC 1.1.1.8), glycerol kinase (EC 2.7.1.30), lactate dehydrogenase (EC 1.1.1.27), "malic' enzyme (L-malate-NADP+ oxidoreductase; EC 1.1.1.40) and the beta-oxoacyl-(acyl-carrier protein) reductase component of the fatty acid synthetase complex were measured in nine hepatoma lines (8 in rats, 1 in mouse) and in the livers of host animals. With the single exception of Morris hepatoma 16, which had unusually high glycerol 3-phosphate dehydrogenase activity, the activities of glycerol 3-phosphate dehydrogenase and glycerol kinase were highly correlated in normal livers and hepatomas (r = 0.97; P less than 0.01). The activities of these two enzymes were not strongly correlated with the activities of any of the other three enzymes. The primary function of hepatic glycerol 3-phosphate dehydrogenase appears to be in gluconeogenesis from glycerol.     

Thyroid hormone and dehydroepiandrosterone permit gluconeogenic hormone responses in hepatocytes

The importance of the sn-glycerol- 3-phosphate (G-3-P) electron transfer shuttle in hormonal regulation of gluconeogenesis was examined in hepatocytes from rats with decreased mitochondrial G-3-P dehydrogenase activity (thyroidectomized) or increased G-3-P dehydrogenase activity [triiodothyronine (T(3)) or dehydroepiandrosterone (DHEA) treated]. Rates of glucose formation from 10 mM lactate, 10 mM pyruvate, or 2.5 mM dihydroxyacetone were somewhat less in hypothyroid cells than in cells from normal rats but gluconeogenic responses to calcium addition and to norepinephrine (NE), glucagon (G), or vasopressin (VP) were similar to the responses observed in cells from normal rats. However, with 2. 5 mM glycerol or 2.5 mM sorbitol, substrates that must be oxidized in the cytosol before conversion to glucose, basal gluconeogenesis was not appreciably altered by hypothyroidism but responses to calcium and to the calcium-mobilizing hormones were abolished. Injecting thyroidectomized rats with T(3) 2 days before preparing the hepatocytes greatly enhanced gluconeogenesis from glyc erol and restored the response to Ca(2+) and gluconeogenic hormones. Feeding dehydroepiandrosterone for 6 days depressed gluconeogenesis from lactate or pyruvate but substantially increased glucose production from glycerol in euthyroid cells and restored responses to Ca(2+) in hypothyroid cells metabolizing glycerol. Euthyroid cells metabolizing glycerol or sorbitol use the G-3-P and malate/aspartate shuttles to oxidize excess NADH generated in the cytosol. The transaminase inhibitor aminooxyacetate (AOA) decreased gluconeogenesis from glycerol 40%, but had little effect on responses to Ca(2+) and NE. However, in hypothyroid cells, with minimal G-3-P dehydrogenase, AOA decreased gluconeogenesis from glycerol more than 90%. Thus, the basal rate of gluconeogenesis from glycerol in the euthyroid cells is only partly dependent on electron transport from cytosol to mitochondria via the malate/aspartate shuttle and almost completely dependent in the hypothyroid state, and the hormone enhancement of the rate in euthyroid cells involves primarily the G-3-P cycle. These data are consistent with Ca(2+) being mobilized by gluconeogenic hormones and G-3-P dehydrogenase being activated by Ca(2+) so as to permit it to transfer reducing equivalents from the cytosol to the mitochondria.     

Gluconeogenesis by isolated guinea-pig liver parenchymal cells

1. Guinea-pig hepatocytes were prepared by collagenase digestion of the perfused liver. 2. The highest rates of gluconeogenesis were obtained from fructose, followed by pyruvate, xylitol and lactate, glycerol and propionate in that order. Maximum rates of gluconeogenesis were attained at 6-10mm substrate. 3. An initial 15-min lag period occurred during gluconeogenesis from lactate. This lag was abolished by preincubating the cells or by preincubation plus the addition of NH(4)Cl or lysine. 4. The lactate/pyruvate and 3-hydroxybutyrate/acetoacetate ratios were increased during the lag and adjusted to values favouring rapid gluconeogenesis from lactate after 15min. 5. The data suggest that the low glucose synthesis during the lag resulted from a limitation of the glutamate-aspartate shuttle and from the unusual redox state of the NAD(+) couple prevailing during this period. 6. At 0.1mm, amino-oxyacetate, a transaminase inhibitor, decreased gluconeogenesis from lactate by 80%, but had a negligible effect on glucose production from pyruvate. Gluconeogenesis from lactate was also inhibited (20%) by 10mm-dl-3-hydroxybutyrate.     

Futile hydrogen cycling in liver cells from triiodothyronine treated rats

Aminooxyacetate does not inhibit gluconeogenesis from sorbitol or glycerol, and ethanol does not inhibit gluconeogenesis from L-lactate, in liver cells prepared from triiodothyronine (T3) treated rats. These results are in accord with the previously documented marked increase in α-glycerol phosphate shuttle activity induced by thyroid hormones. Aminooxyacetate inhibits gluconeogenesis from L-lactate in hepatocytes from T3 treated rats by only about 30% (vs 90% in hepatocytes from normal rats). Also pyruvate kinase flux during gluconeogenesis from L-lactate is markedly increased in liver cells from fasted, T3 treated, rats.     

Stimulation by glucose of gluconeogenesis in hepatocytes isolated from starved rats.

Control properties of the gluconeogenic pathway in hepatocytes isolated from starved rats were studied in the presence of glucose. The following observations were made. (1) Glucose stimulated the rate of glucose production from 20 mM-glycerol, from a mixture of 20 mM-lactate and 2 mM-pyruvate, or from pyruvate alone; no stimulation was observed with 20 mM-alanine or 20 mM-dihydroxyacetone. Maximal stimulation was obtained between 2 and 5 mM-glucose, depending on the conditions. At concentrations above 6 mM, gluconeogenesis declined again, so that at 10 mM-glucose the glucose production rate became equal to that in its absence. (2) With glycerol, stimulation of gluconeogenesis by glucose was accompanied by oxidation of cytosolic NADH and reduction of mitochondrial NAD+ and was insensitive to the transaminase inhibitor amino-oxyacetate; this indicated that glucose accelerated the rate of transport of cytosolic reducing equivalents to the mitochondria via the glycerol 1-phosphate shuttle. (3) With lactate plus pyruvate (10:1) as substrates, stimulation of gluconeogenesis by glucose was almost additive to that obtained with glucagon. From an analysis of the effect of glucose on the curves relating gluconeogenic flux and the steady-state intracellular concentrations of gluconeogenic intermediates under various conditions, in the absence and presence of glucagon, it was concluded that addition of glucose stimulated both phosphoenolpyruvate carboxykinase and pyruvate carboxylase activity.     

Stimulation of gluconeogenesis by intravenous lipids in preterm infants: response depends on fatty acid profile

In preterm infants, both hypo- and hyperglycemia are a frequent problem. Intravenous lipids can affect glucose metabolism by stimulation of gluconeogenesis by providing glycerol, which is a gluconeogenic precursor, and/or free fatty acids (FFA), which are stimulants of the rate of gluconeogenesis. In 25 preterm infants, glucose production and gluconeogenesis were measured using stable isotope techniques during a 6-h infusion of glucose only, glucose plus glycerol, or glucose plus an intravenous lipid emulsion. Two lipid emulsions differing in FFA composition were used: Intralipid ( approximately 60% polyunsaturated FFA) and Clinoleic (approximately 60% monounsaturated FFA). The rate of glucose infusion was 22 micromol x kg(-1) x min(-1) in all groups. During the study infusion, the FFA concentrations were higher in both lipid groups vs. the glycerol group (P < 0.001). Compared with baseline, the glucose production rate increased in the Intralipid group, whereas it decreased in the other groups (P = 0.002) due to a significant increase in gluconeogenesis in the Intralipid group (P = 0.016). The plasma glucose concentration was significantly higher during Intralipid infusion vs. the other groups (P = 0.046). Our conclusion was that Intralipid enhanced glucose production by increasing gluconeogenesis in preterm infants. This can be ascribed to the stimulatory effect of FFA in addition to any effect of glycerol alone. The lack of stimulation of gluconeogenesis in the Clinoleic vs. the Intralipid group suggests that different classes of fatty acids exert different effects on glucose kinetics in preterm infants.     

Lipolysis and glycerol gluconeogenesis in simultaneously fasting and lactating northern elephant seals

Adult female elephant seals (Mirounga angustirostris) combine long-term fasting with lactation and molting. Glycerol gluconeogenesis has been hypothesized as potentially meeting all of the glucose requirements of the seals during these fasts. To test this hypothesis, a primed constant infusion of [2-(14)C]glycerol was administered to 10 ten adult female elephant seals at 5 and 21-22 days postpartum and to 10 additional adult females immediately after the molt. Glycerol kinetics, rates of lipolysis, and the contribution of glycerol to glucose production were determined for each period. Plasma metabolite levels as well as insulin, glucagon, and cortisol were also measured. Glycerol rate of appearance was not significantly correlated with mass (P = 0.14, r2 = 0.33) but was significantly related to the percentage of glucose derived from glycerol (P < 0.01, r2 = 0.81) during late lactation. The contribution of glycerol to glucose production was <3% during each fasting period, suggesting a lower contribution to gluconeogenesis than is observed in other long-term fasting mammals. Because of a high rate of endogenous glucose production in fasting elephant seals, it is likely that glycerol gluconeogenesis still makes a substantial contribution to the substrate needs of glucose-dependent tissues. The lack of a relationship between glucoregulatory hormones and glycerol kinetics, glycerol gluconeogenesis, and metabolites supports the proposition that fasting elephant seals do not conform to the traditional insulin-glucagon model of substrate metabolism.     

13C-NMR study of glycerol metabolism in rabbit renal cells of proximal convoluted tubules

Perchloric acid extracts of rabbit renal proximal convoluted tubular cells (PCT) incubated with [2-13C]glycerol and [1,3-13C]glycerol were investigated by 13C-NMR spectroscopy. These 13C-NMR spectra enabled us to determine cell metabolic pathways of glycerol in PCT cells. The main percentage of 13C-label, arising from 13C-enriched glycerol, was found in glucose, lactate, glutamine and glutamate. So far it can be concluded that glycerol is a suitable substrate for PCT cells and is involved in gluconeogenesis and glycolysis as well in the Krebs cycle intermediates. Label exchange and label enrichment in 13C-labelled glucose, arising from [2-13C]glycerol and [1,3-13C]glycerol, is explained by label scrambling through the pentose shunt and a label exchange in the triose phosphate pool. From relative enrichments it is estimated that the ratio of the pyruvate kinase flux to the gluconeogenetic flux is 0.97:1 and that the ratio of pyruvate carboxylase activity relative to pyruvate dehydrogenase activity is 2.0:1. Our results show that 13C-NMR spectroscopy, using 13C-labelled substrates, is a powerful tool for the examination of renal metabolism.     

The r?le of mitochondrial pyruvate transport in the stimulation by glucagon and phenylephrine of gluconeogenesis from L-lactate in isolated rat hepatocytes.

The sensitivity of glucose production from L-lactate by isolated liver cells from starved rats to inhibition by alpha-cyano-4-hydroxycinnamate was studied. A small percentage of the maximal rate of gluconeogenesis was insensitive to inhibition by alpha-cyano-4-hydroxycinnamate, and evidence is presented to show that this is due to pyruvate entry into the mitochondria as alanine. After subtraction of this rate, Dixon plots of the reciprocal of the rate of gluconeogenesis against inhibitor concentration were linear both in the absence and presence of glucagon, phenylephrine or valinomycin, each of which stimulated gluconeogenesis by 30-50%. Pyruvate kinase activity was decreased by glucagon, but not by phenylephrine or valinomycin. Inhibition of gluconeogenesis by quinolinate (inhibitor of phosphoenolpyruvate carboxykinase) or monochloroacetate (probably inhibiting pyruvate carboxylation) caused a significant deviation from linearity of the Dixon plot obtained with alpha-cyano-4-hydroxycinnamate. Amytal, however, inhibited gluconeogenesis without affecting the linearity of this plot. These data, coupled with a computer simulation study, suggest that pyruvate transport may control gluconeogenesis from L-lactate and that hormones may stimulate this process through an effect on the respiratory chain. An additional role for pyruvate kinase and pyruvate carboxylase is quite compatible with the data presented.     

Implications of the simultaneous occurrence of hepatic glycolysis from glucose and gluconeogenesis from glycerol.

Glycolysis from [6-(3)H]glucose and gluconeogenesis from [U-(14)C]glycerol were examined in isolated hepatocytes from fasted rats. A 5 mm bolus of glycerol inhibited phosphorylation of 40 mm glucose by 50% and glycolysis by more than 60%, and caused cellular ATP depletion and glycerol 3-phosphate accumulation. Gluconeogenesis from 5 mm glycerol was unaffected by the presence of 40 mm glucose. When nonsaturating concentrations of glycerol (< 200 microm) were maintained in the medium by infusion of glycerol, cellular ATP concentrations remained normal. The rate of uptake of infused glycerol was unaffected by 40 mm glucose, but carbohydrate synthesis from glycerol was inhibited 25%, a corresponding amount of glycerol being diverted to glycolytic products, whereas 10 mm glucose had no inhibitory effect on conversion of infused glycerol into carbohydrate. Glycerol infusion depressed glycolysis from 10 mm and 40 mm glucose by 15 and 25%, respectively; however, the overall rates of glycolysis were unchanged because of a concomitant increase in glycolysis from the infused glycerol. These studies show that exposure of hepatocytes to glucose and low quasi-steady-state concentrations of glycerol result in the simultaneous occurrence, at substantial rates, of glycolysis from glucose and gluconeogenesis from the added glycerol. We interpret our results as demonstrating that, in hepatocytes from normal rats, segments of the pathways of glycolysis from glucose and gluconeogenesis from glycerol are compartmentalized and that this segregation prevents substantial cross-over of phosphorylated intermediates from one pathway to the other. The competition between glucose and glycerol implies that glycolysis and phosphorylation of glycerol take place in the same cells, and that the occurrence of simultaneous glycolysis and gluconeogenesis may indicate channelling within the cytoplasm of individual hepatocytes.     

Blockade of fatty acid oxidation mimics phase II-phase III transition in a fasting bird,the king penguin

This study tests the hypothesis that the metabolic and endocrine shift characterizing the phase II-phase III transition during prolonged fasting is related to a decrease in fatty acid (FA) oxidation. Changes in plasma concentrations of various metabolites and hormones and in lipolytic fluxes, as determined by continuous infusion of [2-(3)H]glycerol and [1-(14)C]palmitate, were examined in vivo in spontaneously fasting king penguins in the phase II status (large fat stores, protein sparing) before, during, and after treatment with mercaptoacetate (MA), an inhibitor of FA oxidation. MA induced a 7-fold decrease in plasma beta-hydroxybutyrate and a 2- to 2.5-fold increase in plasma nonesterified fatty acids (NEFA), glycerol, and triacylglycerols. MA also stimulated lipolytic fluxes, increasing the rate of appearance of NEFA and glycerol by 60-90%. This stimulation might be partly mediated by a doubling of circulating glucagon, with plasma insulin remaining unchanged. Plasma glucose level was unaffected by MA treatment. Plasma uric acid increased 4-fold, indicating a marked acceleration of body protein breakdown, possibly mediated by a 2.5-fold increase in circulating corticosterone. Strong similarities between these changes and those observed at the phase II-phase III transition in fasting penguins support the view that entrance into phase III, and especially the end of protein sparing, is related to decreased FA oxidation, rather than reduced NEFA availability. MA could be therefore a useful tool for understanding mechanisms underlying the phase II-phase III transition in spontaneously fasting birds and the associated stimulation of feeding behavior.     

Microbial synthesis of polyhydroxybutyrate from glycerol: Gluconeogenesis, molecular weight and material properties of biopolyester

Glycerol is considered as an ideal feedstock for producing bioplastics via bacterial fermentation due to its ubiquity, low price, and high degree of reduction substrate. In this work, we study the yield and cause of limitation in poly(3‐hydroxybutyrate) (PHB) production from glycerol. Compared to glucose‐based PHB production, PHB produced by Cupriavidus necator grown on glycerol has a low productivity (0.92 g PHB/L/h) with a comparably low maximum specific growth rate of 0.11 h^?1. We found that C. necator can synthesize glucose from glycerol and that the lithotrophical utilization of glycerol (non‐fermentative substrate) or gluconeogenesis is an essential metabolic pathway for biosynthesis of cellular components. Here, we show that gluconeogenesis affects the reduction of cell mass, the productivity of biopolymer product, and the molecular chain size of intracellular PHB synthesized from glycerol by C. necator. We use NMR spectroscopy to show that the isolated PHB is capped by glycerol. We then characterized the physical properties of the isolated glycerol‐based PHB with differential scanning calorimetry and tensile tests. We found that although the final molecular weight of the glycerol‐based PHB is lower than those of glucose‐based and commercial PHB, the thermal and mechanical properties of the biopolymers are similar. Biotechnol. Bioeng. 2012; 109: 2808–2818. © 2012 Wiley Periodicals, Inc.     

Glycerol loss to water exceeds glycerol catabolism via glycerol kinase in freeze-resistant rainbow smelt (Osmerus mordax)

Rainbow smelt accumulate high amounts of glycerol in winter. In smelt, there is a predictable profile of plasma glycerol levels that starts to increase in November (<5 μmol/ml), peaks in mid-February (>200 μmol/ml), and thereafter decreases to reach the initial levels in the beginning of May. The aim of this study was to investigate the respective role of the two main mechanisms that might be involved in glycerol clearance from mid-February: 1) breakdown of glycerol to glycerol-3-phosphate through the action of the glycerol kinase (GK) and 2) direct loss toward the environment. Over the entire glycerol cycle, loss to water represents a daily loss of ～10% of the total glycerol content of fish. GK activities were very low in all tissues investigated and likely have a minor quantitative role in the glycerol cycle. These results suggest that glycerol levels are dictated by the rate of glycerol synthesis (accelerated and deactivated during the accumulation and decrease stages, respectively). Although not important in glycerol clearance, GK in liver might have an important metabolic function for other purposes, such as gluconeogenesis, as evidenced by the significant increase of activity at the end of the cycle.     

Lethal hypoglycemic ketosis and glyceroluria in mice lacking both the mitochondrial and the cytosolic glycerol phosphate dehydrogenases

The activities of either the mitochondrial or cytosolic glycerol phosphate dehydrogenase (mGPD, cGPD) plus that of glycerol kinase are required for the use of glycerol in aerobic metabolism and gluconeogenesis. A knockout mouse lacking mGPD has reduced body weight and fertility but shows remarkably normal liver and muscle metabolite levels. The BALB/cHeA mouse strain, which lacks cGPD, breeds well and is phenotypically normal, although it demonstrates metabolite abnormalities in certain tissues. Crosses were made between these two strains, and mice were generated that lacked both dehydrogenases. These mice, although active and nursing well for several days, failed to grow, and usually died within the first week. Liver glycerol phosphate levels were elevated 30-fold, whereas liver ATP, ADP, and AMP levels were reduced by 30-40%. Plasma glycerol was elevated 30- to 50-fold to 30-50 mm, and urine glycerol exceeded 0.45 m (4% w/v). GPD-deficient mice were hypoglycemic, had a 50% increase in plasma free fatty acids, and developed ketonuria within the first day of life. Uncoupling protein-1 mRNA in brown adipose tissue was reduced 60%. These mice share some features of both glycerol kinase deficiency and hereditary fructose intolerance, suggesting the phenotype may be due to the combined effects of the loss of a gluconeogenic substrate, the osmotic effects of glycerol, and the metabolic effects of the accumulation of a phosphorylated metabolite.     

Lipolytic and metabolic response to glucagon in fasting king penguins: phase II vs. phase III

This study aims to determine how glucagon intervenes in the regulation of fuel metabolism, especially lipolysis, at two stages of a spontaneous long-term fast characterized by marked differences in lipid and protein availability and/or utilization (phases II and III). Changes in the plasma concentration of various metabolites and hormones, and in lipolytic fluxes as determined by continuous infusion of [2-3H]glycerol and [1-14C]palmitate, were examined in vivo in a subantarctic bird (king penguin) before, during, and after a 2-h glucagon infusion. In the two fasting phases, glucagon infusion at a rate of 0.025 microg. kg(-1). min(-1) induced a three- to fourfold increase in the plasma concentration and in the rate of appearance (Ra) of glycerol and nonesterified fatty acids, the percentage of primary reesterification remaining unchanged. Infusion of glucagon also resulted in a progressive elevation of the plasma concentration of glucose and beta-hydroxybutyrate and in a twofold higher insulinemia. These changes were not significantly different between the two phases. The plasma concentrations of triacylglycerols and uric acid were unaffected by glucagon infusion, except for a 40% increase in plasma uric acid in phase II birds. Altogether, these results indicate that glucagon in a long-term fasting bird is highly lipolytic, hyperglycemic, ketogenic, and insulinogenic, these effects, however, being similar in phases II and III. The maintenance of the sensitivity of adipose tissue lipolysis to glucagon could suggest that the major role of the increase in basal glucagonemia observed in phase III is to stimulate gluconeogenesis rather than fatty acid delivery.