Real-time Detection of Hepatic Gluconeogenic and Glycogenolytic States Using Hyperpolarized [2-13C]Dihydroxyacetone

Glycogenolysis and gluconeogenesis are sensitive to nutritional state, and the net direction of flux is controlled by multiple enzymatic steps. This delicate balance in the liver is disrupted by a variety of pathological states including cancer and diabetes mellitus. Hyperpolarized carbon-13 magnetic resonance is a new metabolic imaging technique that can probe intermediary metabolism nondestructively. There are currently no methods to rapidly distinguish livers in a gluconeogenic from glycogenolytic state. Here we use the gluconeogenic precursor dihydroxyacetone (DHA) to deliver hyperpolarized carbon-13 to the perfused mouse liver. DHA enters gluconeogenesis at the level of the trioses. Perfusion conditions were designed to establish either a gluconeogenic or a glycogenolytic state. Unexpectedly, we found that [2-¹³C]DHA was metabolized within a few seconds to the common intermediates and end products of both glycolysis and gluconeogenesis under both conditions, including [2,5-¹³C]glucose, [2-¹³C]glycerol 3-phosphate, [2-¹³C]phosphoenolpyruvate (PEP), [2-¹³C]pyruvate, [2-¹³C]alanine, and [2-¹³C]lactate. [2-¹³C]Phosphoenolpyruvate, a key branch point in gluconeogenesis and glycolysis, was monitored in functioning tissue for the first time. Observation of [2-¹³C]PEP was not anticipated as the free energy difference between PEP and pyruvate is large. Pyruvate kinase is the only regulatory step of the common glycolytic-gluconeogenic pathway that appears to exert significant control over the kinetics of any metabolites of DHA. A ratio of glycolytic to gluconeogenic products distinguished the gluconeogenic from glycogenolytic state in these functioning livers.     

A transcriptional fusion of genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enolase in dinoflagellates

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enolase are enzymes essential for glycolysis and gluconeogenesis. Dinoflagellates possess several types of both GAPDH and enolase genes. Here, we identify a novel cytosolic GAPDH-enolase fusion protein in several dinoflagellate species. Phylogenetic analyses revealed that the GAPDH moiety of this fusion is weakly related to a cytosolic GAPDH previously reported in dinoflagellates, ciliates, and an apicomplexan. The enolase moiety has phylogenetic affinity with sequences from ciliates and apicomplexans, as expected for dinoflagellate genes. Furthermore, the enolase moiety has two insertions in a highly conserved region of the gene that are shared with ciliate and apicomplexan homologues, as well as with land plants, stramenopiles, haptophytes, and a chlorarachniophyte. Another glycolytic gene fusion in eukaryotes is the mitochondrion-targeted triose-phosphate isomerase (TPI) and GAPDH fusion in stramenopiles (i.e. diatoms and oomycetes). However, unlike the mitochondrial TPI-GAPDH fusion, the GAPDH-enolase fusion protein appears to exist in the same compartment as stand-alone homologues of each protein, and the metabolic reactions they catalyze in glycolysis and gluconeogenesis are not directly sequential. It is possible that the fusion is post-translationally processed to give separate GAPDH and enolase products, or that the fusion protein may function as a single bifunctional polypeptide in glycolysis, gluconeogenesis, or perhaps more likely in some previously unrecognized metabolic capacity.     

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.     

Influence of tamoxifen on gluconeogenesis and glycolysis in the perfused rat liver

The actions of tamoxifen, a selective estrogen receptor modulator used in chemotherapy and chemo-prevention of breast cancer, on glycolysis and gluconeogenesis were investigated in the isolated perfused rat liver. Tamoxifen inhibited gluconeogenesis from both lactate and fructose at very low concentrations (e.g., 5 μM). The opposite, i.e., stimulation, was found for glycolysis from both endogenous glycogen and fructose. Oxygen uptake was unaffected, inhibited or stimulated, depending on the conditions. Stimulation occurred in both microsomes and mitochondria. Tamoxifen did not affect the most important key-enzymes of gluconeogenesis, namely, phosphoenolpyruvate carboxykinase, pyruvate carboxylase, fructose 1,6-bisphosphatase and glucose 6-phosphatase. Confirming previous observations, however, tamoxifen inhibited very strongly NADH- and succinate-oxidase of freeze–thawing disrupted mitochondria. Tamoxifen promoted the release of both lactate dehydrogenase (mainly cytosolic) and fumarase (mainly mitochondrial) into the perfusate. Tamoxifen (200 μM) clearly diminished the ATP content and increased the ADP content of livers in the presence of lactate with a diminution of the ATP/ADP ratio from 1.67 to 0.79. The main causes for gluconeogenesis inhibition are probably: (a) inhibition of energy metabolism; (b) deviation of intermediates (malate and glucose 6-phosphate) for the production of NADPH required in hydroxylation and demethylation reactions; (c) deviation of glucosyl units toward glucuronidation reactions; (d) secondary inhibitory action of nitric oxide, whose production is stimulated by tamoxifen; (e) impairment of the cellular structure, especially the membrane structure. Stimulation of glycolysis is probably a compensatory phenomenon for the diminished mitochondrial ATP production. The multiple actions of tamoxifen at relatively low concentrations can represent a continuous burden to the overall hepatic functions during long treatment periods.     

The crystal structure of glucose-6-phosphate isomerase from Leishmania mexicana reveals novel active site features.

Glucose-6-phosphate isomerase catalyzes the reversible aldose-ketose isomerization of D-glucose-6-phosphate to D-fructose-6-phosphate in glycolysis and gluconeogenesis, and in the recycling of hexose-6-phosphate in the pentose phosphate pathway. The unicellular protozoans, Trypanosoma brucei, T. cruzi and Leishmania spp., of the order Kinetoplastida are important human parasites responsible for African sleeping sickness, Chagas' disease and leishmaniases, respectively. In these parasites, glycolysis is an important (and in some cases the only) metabolic pathway for ATP supply. The first seven of the 10 enzymes that participate in glycolysis, as well as an important fraction of the enzymes of the pentose phosphate pathway, are compartmentalized in peroxisome-like organelles called glycosomes. The dependence of the parasites on glycolysis, the importance of the pentose phosphate pathway in defense against oxidative stress, and the unique compartmentalization of these pathways, point to the enzymes contained in the glycosome as potential targets for drug design. The present report describes the first crystallographic structure of a parasite (Leishmania mexicana) glucose-6-phosphate isomerase. A comparison of the atomic structure of L. mexicana, human and other mammalian PGIs, which highlights unique features of the parasite's enzyme, is presented.     

Localization and role of pyruvate kinase isoenzymes in the regulation of carbohydrate metabolism and pyruvate recycling in rat kidney cortex

This work was performed to gain more information on the role of pyruvate kinase isoenzymes in the regulation of renal carbohydrate metabolism. Immunohistochemically, pyruvate kinase type L is shown to be localized in the proximal tubule of the nephron and pyruvate kinase type M2 in the distal tubule and the collecting duct. a tight relationship between gluconeogenesis and pyruvate recycling was found. The rate of gluconeogenesis (8 mumol/g wet wt. per 30 min) was of the same order of magnitude as the rate of pyruvate recycling (10.92 mumol/g wet wt. per 30 min). Stimulation of gluconeogenesis from 20 mM lactate in kidney cortex slices of 24-h-starved rats by dibutyryl-cAMP, alanine and parathyroid hormone was connected with a decrease in pyruvate recycling; inhibition of gluconeogenesis due to a lack of Ca2+ in the incubation medium was linked with an increase in pyruvate recycling. The degradation of [6-14C]glucose to lactate, pyruvate, ketone bodies and CO2 and of [2-14C]lactate was unaffected by dibutyryl-cAMP, alanine, epinephrine, vasopressin or the omission of Ca2+ from the incubation medium. 1 mM dibutyryl-cAMP or 5 mM alanine did not alter the activities of oxaloacetate decarboxylase, 'malic' enzyme and malate dehydrogenase from rat kidney cortex. Since aerobic glycolysis in the distal tubules and the collecting ducts is not influenced by hormones, dibutyryl-cAMP and Ca2+, pyruvate kinase type M2 residing in this tissue is unlikely to be a control point of glycolysis. Since this tissue degrades only one-seventh of the glucose formed via gluconeogenesis, it does not contribute significantly to pyruvate recycling. Therefore, the decrease of pyruvate recycling in the presence of dibutyryl-cAMP and alanine in rat kidney cortex slices, leading to increased renal gluconeogenesis, has to be ascribed to the regulation of pyruvate kinase type L.     

Futile cycles revisited: a markov chain model of simultaneous glycolysis and gluconeogenesis

We have used a random walk model of glycolysis and gluconeogenesis to investigate the bioenergetic implications of considering the cell cytoplasm to be a uniform well-mixed compartment. Radiotracer studies conducted on hepatocytes harvested from fasted rats and incubated with 40 mM glucose and 10 mM lactate demonstrated simultaneous glycolysis and gluconeogenesis, with net glycolysis. Tracer introduced as glycerol was incorporated both into glucose (via gluconeogenesis) and into pyruvate (via glycolysis). The data allow us to place a lower bound on the energetic cost of futile cycles involving adenosine triphosphate (ATP) hydrolysis in the early phosphorylation steps of glycolysis. Applying the Markov Chain model for glucose undergoing metabolism to pyruvate, the expected number of ATP molecules hydrolysed is not less than 15 ATP molecules per glucose molecule. The data suggest that, in hepatocytes under the circumstances of this experiment, either glycolysis is a net consumer of ATP, or glycolysis and gluconeogenesis are compartmentalized to a greater extent than is generally supposed.     

Induction in primary culture of 'gluconeogenic' and 'glycolytic' hepatocytes resembling periportal and perivenous cells

Adult rat hepatocytes were kept in primary culture for 48 h under different hormonal conditions to induce an enzyme pattern which with respect to carbohydrate metabolism approximated that of periportal and perivenous hepatocytes in vivo. 1. Glucagon-treated cells compared with control cells possessed a lower activity of glucokinase, a 4.5-fold higher activity of phosphoenolpyruvate carboxykinase and unchanged levels of glucose-6-phosphatase, phosphofructokinase, fructose-bisphosphatase and pyruvate kinase; they resembled in a first approximation the periportal cell type and are called for simplicity 'periportal'. Inversely, insulin-treated cells compared with control cells contained a 2.2-fold higher activity of glucokinase, a slightly decreased activity of phosphoenolpyruvate carboxykinase, increased activities of phosphofructokinase and pyruvate kinase and unaltered levels of glucose-6-phosphatase and fructose-bisphosphatase; they resembled perivenous cells and are called simply 'perivenous'. Gluconeogenesis and glycolysis were studied under various substrate and hormone concentrations. 2. Physiological concentrations of glucose (5 mM) and lactate (2 mM) gave about 80% saturation of gluconeogenesis from lactate and less than 15% saturation of glycolysis at a simultaneous 40% inhibition of the glycolytic rate by lactate. 3. Comparison of the two cell types showed that under identical assay conditions (5 mM glucose, 2 mM lactate, 0.5 nM insulin, 0.1 muM dexamethasone) gluconeogenesis was 1.5-fold faster in the 'periportal' cells and glycolysis was 2.4-fold faster in the 'perivenous' cells. 4. Metabolic rates were under short-term hormonal control. Insulin increased glycolysis three fold in both cell types with a half-maximal effect at about 0.4 nM, but did not influence the gluconeogenic rate. Glucagon inhibited glycolysis by 70% with a half-maximal effect at about 0.1 nM. Gluconeogenesis was stimulated by glucagon (half-maximal dose: 0.5 nM) 1.8-fold only in 'periportal' cells containing high phosphoenolpyruvate carboxykinase activity, not in the 'perivenous' cells with a low level of this enzyme. 5. A comparison of the two cell types showed that with maximally stimulating hormone concentrations gluconeogenesis was threefold faster in 'periportal' cells and glycolysis was eightfold faster in 'perivenous' cells. The results support the view that periportal and perivenous hepatocytes in vivo catalyse gluconeogenesis and glycolysis at inverse rates.     

The selective control of glycolysis,gluconeogenesis and glycogenesis by temporal insulin patterns

Insulin governs systemic glucose metabolism, including glycolysis, gluconeogenesis and glycogenesis, through temporal change and absolute concentration. However, how insulin‐signalling pathway selectively regulates glycolysis, gluconeogenesis and glycogenesis remains to be elucidated. To address this issue, we experimentally measured metabolites in glucose metabolism in response to insulin. Step stimulation of insulin induced transient response of glycolysis and glycogenesis, and sustained response of gluconeogenesis and extracellular glucose concentration (GLC _ex ). Based on the experimental results, we constructed a simple computational model that characterises response of insulin‐signalling‐dependent glucose metabolism. The model revealed that the network motifs of glycolysis and glycogenesis pathways constitute a feedforward (FF) with substrate depletion and incoherent feedforward loop (iFFL), respectively, enabling glycolysis and glycogenesis responsive to temporal changes of insulin rather than its absolute concentration. In contrast, the network motifs of gluconeogenesis pathway constituted a FF inhibition, enabling gluconeogenesis responsive to absolute concentration of insulin regardless of its temporal patterns. GLC _ex was regulated by gluconeogenesis and glycolysis. These results demonstrate the selective control mechanism of glucose metabolism by temporal patterns of insulin.     

Glycolysis during gluconeogenesis in cotyledons of Cucurbita pepo

The aim of this work was to investigate the extent of glycolysis during gluconeogenesis in the germination of marrow (Cucurbita pepo L. var. medullosa Alef.). The activities of phosphofructokinase (E.C. 2.7.1.11) in extracts of cotyledons, of seeds, and seedlings grown in the dark for 2, 5, and 8 days were 3·5, 4·8, 9·4, and 11·8 nmol substrate consumed per cotyledon per min, respectively. The comparable figures for pyruvate kinase (E.C. 2.7.1.41) were 16·3, 72·3, 974, and 1485. The patterns of ¹⁴CO₂ production from [1-¹⁴C], [2-¹⁴C], [3,4-¹⁴C], and [6-¹⁴C]glucose indicated that at all the above stages of germination glycolysis was appreciable and predominated over the pentose phosphate pathway. These patterns, and the distribution of label from [1-¹⁴C], and [3-¹⁴C]pyruvate supplied to 5-day-old cotyledons, indicated that the pyruvate formed in glycolysis was converted to acetyl units that were used primarily in biosyntheses. It is concluded that glycolysis occurred at all the stages of germination examined and was particularly active during gluconeogenesis. It is suggested that the significance of this glycolysis is the provision of intermediates for biosyntheses, a need that may not be met by corresponding gluconeogenic intermediates as these may be retained within organelles.     

Development of gluconeogenesis from dihydroxyacetone in rat hepatocytes during a feeding cycle and starvation.

Pyruvate kinase activity and the rates of gluconeogenesis and glycolysis in rat hepatocytes were evaluated by production of glucose and lactate + pyruvate from dihydroxyacetone during a feeding cycle or progressive starvation. In fed rats, during daylight (low food intake) and until darkness, gluconeogenesis progressively increased and glycolysis decreased slightly, but gluconeogenesis never exceeded glycolysis. During nocturnal feeding, gluconeogenesis and glycolysis returned to their morning rates. After 8 h starvation, an equal proportion of dihydroxyacetone was converted into glucose and into lactate + pyruvate. When glycogen was depleted (11 h of starvation), gluconeogenesis was maximal and glycolysis minimal. In fed and starved rats, the concentration of fructose 1,6-bisphosphate was the same. The activity ratio of pyruvate kinase (ratio of velocity at 0.5 mM-phosphoenolpyruvate to the maximum catalytic activity obtained with 4mM-phosphoenolpyruvate) was high in crude extracts of cells incubated with dihydroxyacetone and low in (NH4)2SO4-treated extracts, but remained unchanged during the whole experiment. There was no correlation between the rates of gluconeogenesis and glycolysis from dihydroxyacetone and the activity ratio of pyruvate kinase.     

Regulation of hepatic glycolysis and gluconeogenesis by atrial natriuretic peptide.

Recently we reported the presence of both the guanylyl cyclase-linked (116 kDa) and the ANF-C (66 kDa) atrial natriuretic peptide receptors in the rat liver. Since ANF 103-125 (atriopeptin II) stimulates cGMP production in livers and because cGMP has previously been shown to mimic the actions of cAMP in regulating hepatic carbohydrate metabolism, studies were performed to investigate the effects of atriopeptin II on hepatic glycolysis and gluconeogenesis. Additionally, employing analogs of atrial natriuretic hormone [des-(Q116, S117, G118, L119, G120) ANF 102-121 (C-ANF) and des-(C105,121) ANF 104-126 (analog I)] which bind only the ANF-C receptors, the role of the ANF-C receptors in the hepatic actions of atriopeptin II was evaluated. In perfused livers of fed rats atriopeptin II, but not C-ANF and analog I, inhibited hepatic glycolysis and stimulated glucose production. Moreover, analog I did not alter the ability of atriopeptin II to inhibit hepatic glycolysis. Atriopeptin II, but not C-ANF and analog I, also stimulated cGMP production in perfused rat livers. Furthermore, while atriopeptin II inhibited the activity ratio of pyruvate kinase by 30%, C-ANF did not alter hepatic pyruvate kinase activity. Finally, in rat hepatocytes, atriopeptin II stimulated the synthesis of [14C]glucose from [2-14C]pyruvate by 50% and this effect of atriopeptin II was mimicked by the exogenously supplied cGMP analog, 8-bromo cGMP. Thus atriopeptin II increases hepatic gluconeogenesis and inhibits glycolysis, in part by inhibiting pyruvate kinase activity, and the effects of atriopeptin II are mediated via activation of guanylyl cyclase-linked ANF receptors which elevate cGMP production.     

Is there a general paradigm of cyclic AMP action in eukaryotes?

Summary The cyclic AMP control system in eukaryotes has been highly conserved evolutionarily in four of its central properties. Such conservation suggests conservation of the regulatory function of cyclic AMP. Conservation is seen in the properties of adenylate cyclase, cyclic AMP-dependent protein kinase and, among diverse lower eukaryotes, the control of endogenous cyclic AMP levels. A conserved regulatory response to cyclic AMP is the stimulation of glycolysis and inhibition of gluconeogenesis. The control of glycolysis and gluconeogenesis is proposed to be evidence of general pattern of cyclic AMP action in many lower and higher eukaryotic cells.     

The Effect of Quercetin on the Accumulation of Carbohydrates and Amino Acids in Tomato Fruits

We studied the effects of low concentrations of quercetin on the contents of sugars and amino acids in ripe tomato (Lycopersicon esculentumMill.) fruits. In treated plants, the content of glucose increased by 1.5–4.5 times, whereas the total content of amino acids decreased by 1.5 times. The glucogenic amino acids, glutamic and aspartic acid, decreased most substantially, viz. by 1.7 and 1.6 times, respectively. The mechanism of the quercetin-induced enhancement of gluconeogenesis and suppression of glycolysis, both resulting in the accumulation of glucose, are discussed.     

Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Carbohydrate metabolism

1. The effects of the hypoglycaemic compound, pent-4-enoic acid, and of four structurally related non-hypoglycaemic compounds (pent-2-enoic acid, pentanoic acid, cyclopropanecarboxylic acid and cyclobutanecarboxylic acid), on glycolysis, glucose oxidation and gluconeogenesis in some rat tissues were determined. 2. None of the compounds at low concentrations inhibited glycolysis by particle-free supernatant fractions from rat liver, skeletal muscle and intestinal mucosa, though there was inhibition by cyclopropanecarboxylic acid and cyclobutanecarboxylic acid at 3mm concentration. 3. Pent-4-enoic inhibited the oxidation of [1-(14)C]palmitate by rat liver slices, but did not increase the oxidation of [U-(14)C]glucose. 4. Pent-4-enoic acid (0.01mm) strongly inhibited gluconeogenesis by rat kidney slices from pyruvate or succinate, but none of the other compounds inhibited significantly at low concentrations. 5. There was also some inhibition of gluconeogenesis in kidney slices from rats injected with pent-4-enoic acid. 6. The mechanism of the hypoglycaemic effect of pent-4-enoic acid is discussed; it is suggested that there is an inhibition of fatty acid and ketone-body oxidation and of gluconeogenesis so that glucose reserves become exhausted, leading to hypoglycaemia. 7. The mechanism of the hypoglycaemic action of pent-4-enoic acid appears to be similar to that of hypoglycin.     

Caveolae and the organization of carbohydrate metabolism in vascular smooth muscle

We have previously found that glycolysis and gluconeogenesis occur in separate "compartments" of the VSM cell. These compartments may result from spatial separation of glycolytic and gluconeogenic enzymes (Lloyd and Hardin [1999] Am J Physiol Cell Physiol. 277:C1250-C1262). We have also found that an intact plasma membrane is essential for compartmentation to exist (Lloyd and Hardin [2000] Am J Physiol Cell Physiol. 278:C803-C811), suggesting that glycolysis and gluconeogenesis may be associated with distinct plasma membrane microdomains. Caveolae are one such microdomain, in which proteins of related function colocalize. Thus, we hypothesized that membrane-associated glycolysis occurs in association with caveolae, while gluconeogenesis is localized to non-caveolae domains. To test this hypothesis, we disrupted caveolae in vascular smooth muscle (VSM) of pig cerebral microvessels (PCMV) with beta methyl-cyclodextrin (CD) and examined the metabolism of [2-(13)C]glucose (a glycolytic substrate) and [1-(13)C]fructose 1,6-bisphosphate (FBP, a gluconeogenic substrate in PCMV) using (13)C nuclear magnetic resonance spectroscopy. Caveolar disruption reduced flux of [2-(13)C]glucose to [2-(13)C]lactate, suggesting that caveolar disruption partially disrupted the glycolytic pathway. Caveolae disruption may also have resulted in a breakdown of compartmentation, since conversion of [1-(13)C]FBP to [3-(13)C]lactate was increased by CD treatment. Alternatively, the increased [3-(13)C]lactate production may reflect changes in FBP uptake, since conversion of [1-(13)C]FBP to [3-(13)C]glucose was also elevated in CD-treated cells. Thus, a link between caveolar organization and metabolic organization may exist.