Active glycolysis and glycogenolysis in early stages of primary cultured hepatocytes. Role of AMP and fructose 2.6-bisphosphate

Summary This study examines the factors involved in the rapid glycolysis and glycogenolysis that occur during the first stages of hepatocyte culture: a) Shortly after seeding glycolysis, estimated as lactate released to culture medium, increased 10 times in comparison to that reported in vivo. By 8 to 9 h of culture, hepatocytes were nearly glycogen-depleted even in the presence of insulin. b) 6-Phosphofructo-2-kinase remained 100% active during this period. The proportion of the initial active phosphorylase (87%) decreased to 57% by 7 h of culture. c) Fructose 2,6-bisphosphate content was initially similar to that found in liver of fed animals, decreased after seeding and increased thereafter up to four times the initial concentration. In spite of changes in the concentration of this activator, the glycolytic rate remained high and constant. d) ADP and AMP increased sharply after cell plating, reaching values 1.7 and 3.5 times higher. The rise in AMP levels may be involved in the activation of glycolysis and glycogenolysis, because this metabolite is known to act as an allosteric activator of phosphofrucktokinase and glycogen phosphorylase. This metabolic situation resembles that of cells under hypoxia. Part of this work was presented at the 38th Annual Meeting of the Tissue Culture Association, Washington, DC, May 1987.     

Purification and characterization of protein kinase A from liver of the freeze-tolerant wood frog: role in glycogenolysis during freezing

Freeze tolerance by various amphibians includes cryoprotectant production in the form of glucose. Activation of the catalytic subunit of liver cAMP-dependent protein kinase (PKAc) facilitates activation of glycogenolysis, a critical biochemical process necessary for production of glucose. Here, we purified PKAc from Rana sylvatica liver to determine the extent to which cold temperature, which stimulates cryoprotectant production, affected PKAc activity and function. PKAc was purified to greater than 95% homogeneity, with a final specific activity of 71 nmol phosphate transferred/min/mg protein. The molecular weight of frog liver PKAc was 47.6 +/- 1.1 kDa and K(m) values for the phosphate acceptor kemptide and Mg-ATP were 9.0 +/- 0.1 and 51.8 +/- 1.0 microM at 22 degrees C, respectively. K(m) values for both substrates dropped significantly at 5 degrees C. The enzyme was sensitive to specific inhibitors of mammalian PKAc (PKA(i), H89) but was only moderately inhibited by high salt concentrations. Furthermore, salt inhibition was reduced at low temperature. The effect of temperature on enzyme activity indicated a conformational change in PKAc at 10 +/- 2 degrees C, with calculated activation energies of 51 +/- 4 kJ/mol at temperatures above 10 degrees C and 110 +/- 9 kJ/mol below 10 degrees C. PKAc in wood frog liver plays a crucial role in mediating the freeze-induced glycogenolysis that is responsible for the production of 200-300 mM levels of glucose as a cryoprotectant. Differential effects of low temperature on enzyme function, increased substrate affinity and reduced ion inhibition, appear to be central to this role.     

Liver parenchyma heterogeneity in the response to extracellular NAD+

The perfused rat liver responds intensely to NAD+ infusion (20-100 microM). Increases in portal perfusion pressure and glycogenolysis and transient inhibition of oxygen consumption are some of the effects that were observed. The aim of the present work was to investigate the distribution of the response to extracellular NAD+ along the hepatic acinus. The bivascularly perfused rat liver was used. Various combinations of perfusion directions (antegrade and retrograde) and infusion routes (portal vein, hepatic vein and hepatic artery) were used in order to supply NAD+ to different regions of the liver parenchyma, also taking advantage of the fact that its extracellular transformation generates steep concentration gradients. Oxygen uptake was stimulated by NAD+ in retrograde perfusion (irrespective of the infusion route) and transiently inhibited in antegrade perfusion. This indicates that the signal causing oxygen uptake inhibition is generated in the periportal area. The signal responsible for oxygen uptake stimulation is homogenously distributed. Stimulation of glucose release was more intense when NAD+ was infused into the portal vein or into the hepatic artery, indicating that stimulation of glycogenolysis predominates in the periportal area. The increases in perfusion pressure were more pronounced when the periportal area was supplied with NAD+ suggesting that the vasoconstrictive elements responding to NAD+ predominate in this region. The response to extracellular NAD+ is thus unequally distributed in the liver. As a paracrine agent, NAD+ is likely to be released locally. It can be concluded that its effects will be different depending on the area where it is released.     

Glycogen phosphorylase sequences from the amitochondriate protists, Trichomonas vaginalis, Mastigamoeba balamuthi, Entamoeba histolytica and Giardia intestinalis

Glycogen phosphorylase genes or messages from four amitochondriate eukaryotes, Trichomonas vaginalis, Mastigamoeba balamuthi, Entamoeba histolytica (two genes) and Giardia intestinalis, have been isolated and sequenced. The sequences of the amitochondriate protist enzymes appear to share a most recent common ancestor. The clade containing these sequences is closest to that of another protist, the slime mold (Dictyostelium discoideum), and is more closely related to fungal and plant phosphorylases than to mammalian and eubacterial homologs. Structure-based amino acid alignment shows conservation of the residues and domains involved in catalysis and allosteric regulation by glucose 6-phosphate but high divergence at domains involved in phosphorylation-dependent regulation and AMP binding in fungi and animals. Protist phosphorylases, as their prokaryotic and plant counterparts, are probably not regulated by phosphorylation.     

Hepatic heterogeneity in the response to ATP studied in the bivascularly perfused rat liver

The zonation of the purinergic action of ATP in the hepatic parenchyma was investigated in the bivascularly perfused rat liver by means of anterograde and retrograde perfusion. Livers from fed rats were used, and ATP was infused according to four different experimental protocols: (A) anterograde perfusion and ATP infusion via the portal vein; (B) anterograde perfusion and ATP via the hepatic artery; (C) retrograde perfusion and ATP via the hepatic vein; (D) retrograde perfusion and ATP via the hepatic artery. The following metabolic parameters were measured: glucose release, lactate production and oxygen consumption. The hemodynamic effects were evaluated by measuring the sinusoidal mean transit times by means of the indicator-dilution technique. ATP was infused during 20 min at four different rates (between 0.06-0.77 µmol min_-1 g liver_-1; 20-200 µM) in each of the four experimental protocols.The results that were obtained allow several conclusions with respect to the localization of the effects of ATP along the hepatic acini: (1) In retrograde perfusion the sinusoidal mean transit times were approximately twice those observed in anterograde perfusion. ATP increased the sinusoidal mean transit times only in retrograde perfusion (protocols C and D). The effect was more pronounced with protocol D. These results allow the conclusion that the responsive vasoconstrictive elements are localized in a pre-sinusoidal region; (2) All hepatic cells, periportal as well as perivenous, were able to metabolize ATP, so that concentration gradients were generated with all experimental protocols. Extraction of ATP was more pronounced in retrograde perfusion, an observation that can be attributed, partly at least, to the longer sinusoidal transit times. In anterograde perfusion, the extraction of ATP was time-dependent, a phenomenon that cannot be satisfactorily explained with the available data; (3) ATP produced a transient initial inhibition of oxygen uptake when protocols A and B were employed. These protocols are the only ones in which the cells situated shortly after the intrasinusoidal confluence of the portal vein and the hepatic artery were effectively supplied with ATP. The decrease in oxygen consumption was more pronounced at low ATP infusions when protocol B was employed. These observations allow the conclusion that the former phenomenon is localized mainly in cells situated shortly after the intrasinusoidal confluence of the portal vein and hepatic artery. Oxygen consumption in all other cells, especially the proximal periportal ones, is increased by ATP; (4) In agreement with previous data found in the literature, glycogenolysis stimulation by ATP was more pronounced in the periportal region. The cells that respond more intensively are not the proximal periportal ones, but those situated in the region of the intrasinusoidal confluence of the portal vein and the hepatic artery.     

Up-Regulation of 5-HT2B Receptor Density and Receptor-Mediated Glycogenolysis in Mouse Astrocytes by Long-Term Fluoxetine Administration

The effects were studied of short-term (1 week) versus long-term (2-3 weeks) fluoxetine treatment of primary cultures of mouse astrocytes, differentiated by treatment with dibutyryl cyclic AMP. From previous experiments it is known that acute treatment with fluoxetine stimulates glycogenolysis and increases free cytosolic Ca²⁺ concentration ([Ca²⁺]_i]) in these cultures, whereas short-term (one week) treatment with 10 M down-regulates the effects on glycogen and [Ca²⁺]_i, when fluoxetine administration is renewed (or when serotonin is administered). Moreover, antagonist studies have shown that these responses are evoked by activation of a 5-HT₂ receptor that is different from the 5-HT_2A receptor and therefore at that time tentatively were interpreted as being exerted on 5-HT_2C receptors. In the present study the cultures were found by RT-PCR to express mRNA for 5-HT_2A and 5-HT_2B receptors, but not for the 5-HT_2C receptor, identifying the 5-HT₂ receptor activated by fluoxetine as the 5-HT_2B receptor, the most recently cloned 5-HT₂ receptor and a 5-HT receptor known to be more abundant in human, than in rodent, brain. Both short-term and long-term treatment with fluoxetine increased the specific binding of [³H]mesulergine, a ligand for all three 5-HT₂ receptors. Long-term treatment with fluoxetine caused an agonist-induced up-regulation of the glycogenolytic response to renewed administration of fluoxetine, whereas short-term treatment abolished the fluoxetine-induced hydrolysis of glycogen. Thus, during a treatment period similar to that required for fluoxetine's clinical response to occur, 5-HT_2B-mediated effects are initially down-regulated and subsequently up-regulated.     

Dual-action hypoglycemic and hypocholesterolemic agents that inhibit glycogen phosphorylase and lanosterol demethylase

Diabetic dyslipidemia requires simultaneous treatment with hypoglycemic agents and lipid-modulating drugs. We recently described glycogen phosphorylase inhibitors that reduce glycogenolysis in cells and lower plasma glucose in ob/ob mice (J. Med. Chem., 41: 2934, 1998). In evaluating the series prototype, CP-320626, in dogs, up to 90% reduction in plasma cholesterol was noted after 2 week treatment. Cholesterol reductions were also noted in ob/ob mice and in rats. In HepG2 cells, CP-320626 acutely and dose-dependently inhibited cholesterolgenesis without affecting fatty acid synthesis. Inhibition occurred together with a dose-dependent increase in the cholesterol precursor, lanosterol, suggesting that cholesterolgenesis inhibition was due to lanosterol 14alpha-demethylase (CYP51) inhibition. In ob/ob mice, acute treatment with CP-320626 resulted in a decrease in hepatic cholesterolgenesis with concomitant lanosterol accumulation, further implicating CYP51 inhibition as the mechanism of cholesterol lowering in these animals. CP-320626 and analogs directly inhibited rhCYP51, and this inhibition was highly correlated with HepG2 cell cholesterolgenesis inhibition (R2 = 0.77). These observations indicate that CP-320626 inhibits cholesterolgenesis via direct inhibition of CYP51, and that this is the mechanism whereby CP-320626 lowers plasma cholesterol in experimental animals. Dual-action glycogenolysis and cholesterolgenesis inhibitors therefore have the potential to favorably affect both the hyperglycemia and the dyslipidemia of type 2 diabetes.     

Systems biology approaches to metabolic and cardiovascular disorders: network perspectives of cardiovascular metabolism

In this review, we examine cardiovascular metabolism from three different, but highly complementary, perspectives. First, from the abstract perspective of a metabolite network, composed of nodes and links. We present fundamental concepts in network theory, including emergence, to illustrate how nature has designed metabolism with a hierarchal modular scale-free topology to provide a robust system of energy delivery. Second, from the physical perspective of a modular spatially compartmentalized network. We review evidence that cardiovascular metabolism is functionally compartmentalized, such that oxidative phosphorylation, glycolysis, and glycogenolysis preferentially channel ATP to ATPases in different cellular compartments, using creatine kinase and adenylate kinase to maximize efficient energy delivery. Third, from the dynamics perspective, as a network of dynamically interactive metabolic modules capable of self-oscillation. Whereas normally, cardiac metabolism exists in a regime in which excitation-metabolism coupling closely matches energy supply and demand, we describe how under stressful conditions, the network can be pushed into a qualitatively new dynamic regime, manifested as cell-wide oscillations in ATP levels, in which the coordination between energy supply and demand is lost. We speculate how this state of "metabolic fibrillation" leads to cell death if not corrected and discuss the implications for cardioprotection.