The AMP-activated protein kinase (AMPK) is believed to protect cells against environmental stress (e.g. heat shock) by switching off biosynthetic pathways, the key signal being elevation of AMP. Identification of novel targets for the kinase cascade would be facilitated by development of a specific agent for activating the kinase in intact cells. Incubation of rat hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) results in accumulation of the monophosphorylated derivative (5-aminoimidazole-4-carboxamide ribonucleoside; ZMP) within the cell. ZMP mimics both activating effects of AMP on AMPK, i.e. direct allosteric activation and promotion of phosphorylation by AMPK kinase. Unlike existing methods for activating AMPK in intact cells (e.g. fructose, heat shock), AICAR does not perturb the cellular contents of ATP, ADP or AMP. Incubation of hepatocytes with AICAR activates AMPK due to increased phosphorylation, causes phosphorylation and inactivation of a known target for AMPK (3-hydroxy-3-methylglutaryl-CoA reductase), and almost total cessation of two of the known target pathways, i.e. fatty acid and sterol synthesis. Incubation of isolated adipocytes with AICAR antagonizes isoprenaline-induced lipolysis. This provides direct evidence that the inhibition by AMPK of activation of hormone-sensitive lipase by cyclic-AMP-dependent protein kinase, previously demonstrated in cell-free assays, also operates in intact cells. AICAR should be a useful tool for identifying new target pathways and processes regulated by the protein kinase cascade.

5-aminoimidazole-4-carboxamide ribonucleoside. A specific method for activating AMP-activated protein kinase in intact cells?

The AMP-activated protein kinase (AMPK) is believed to protect cells against environmental stress (e.g. heat shock) by switching off biosynthetic pathways, the key signal being elevation of AMP. Identification of novel targets for the kinase cascade would be facilitated by development of a specific agent for activating the kinase in intact cells. Incubation of rat hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) results in accumulation of the monophosphorylated derivative (5-aminoimidaz-ole-4-carboxamide ribonucleoside; ZMP) within the cell. ZMP mimics both activating effects of AMP on AMPK, i.e. direct allosteric activation and promotion of phosphorylation by AMPK kinase. Unlike existing methods for activating AMPK in intact cells (e.g. fructose, heat shock), AICAR does not perturb the cellular contents of ATP, ADP or AMP. Incubation of hepatocytes with AICAR activates AMPK due to increased phosphorylation, causes phosphorylation and inactivation of a known target for AMPK (3-hydroxy-3-methylglutaryl-CoA reductase), and almost total cessation of two of the known target pathways, i.e. fatty acid and sterol synthesis. Incubation of isolated adipocytes with AICAR antagonizes isoprenaline-induced lipolysis. This provides direct evidence that the inhibition by AMPK of activation of hormone-sensitive lipase by cyclic-AMP-dependent protein kinase, previously demonstrated in cell-free assays, also operates in intact cells. AICAR should be a useful tool for identifying new target pathways and processes regulated by the protein kinase cascade.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1995.0558k.x

5‐Aminoimidazole‐4‐Carboxamide Ribonucleoside

The regulation of the matrix volume of mammalian mitochondria in vivo and in vitro and its role in the control of mitochondrial metabolism.

Status of the mitochondrial pool of glutathione in the isolated hepatocyte.

Alpha-ketoglutarate dehydrogenase (alpha-KGDH), a key enzyme in the Krebs' cycle, is a crucial early target of oxidative stress (Tretter and Adam-Vizi, 2000). The present study demonstrates that alpha-KGDH is able to generate H(2)O(2) and, thus, could also be a source of reactive oxygen species (ROS) in mitochondria. Isolated alpha-KGDH with coenzyme A (HS-CoA) and thiamine pyrophosphate started to produce H(2)O(2) after addition of alpha-ketoglutarate in the absence of nicotinamide adenine dinucleotide-oxidized (NAD(+)). NAD(+), which proved to be a powerful inhibitor of alpha-KGDH-mediated H(2)O(2) formation, switched the H(2)O(2) forming mode of the enzyme to the catalytic [nicotinamide adenine dinucleotide-reduced (NADH) forming] mode. In contrast, NADH stimulated H(2)O(2) formation by alpha-KGDH, and for this, neither alpha-ketoglutarate nor HS-CoA were required. When all of the substrates and cofactors of the enzyme were present, the NADH/NAD(+) ratio determined the rate of H(2)O(2) production. The higher the NADH/NAD(+) ratio the higher the rate of H(2)O(2) production. H(2)O(2) production as well as the catalytic function of the enzyme was activated by Ca(2+). In synaptosomes, using alpha-ketoglutarate as respiratory substrate, the rate of H(2)O(2) production increased by 2.5-fold, and aconitase activity decreased, indicating that alpha-KGDH can generate H(2)O(2) in in situ mitochondria. Given the NADH/NAD(+) ratio as a key regulator of H(2)O(2) production by alpha-KGDH, it is suggested that production of ROS could be significant not only in the respiratory chain but also in the Krebs' cycle when oxidation of NADH is impaired. Thus alpha-KGDH is not only a target of ROS but could significantly contribute to generation of oxidative stress in the mitochondria.

https://www.jneurosci.org/content/jneuro/24/36/7771.full.pdf

Generation of reactive oxygen species in the reaction catalyzed by α-ketoglutarate dehydrogenase

1 The subcellular distribution of adenine nucleotides, acetyl-CoA, CoA, glutamate, 2-oxoglutarate, malate, oxaloacetate, pyruvate, phosphoenolpyruvate, 3-phosphoglycerate, glucose 6-phosphate, aspartate and citrate was studied in isolated hepatocytes in the absence and presence of glucagon by using a modified digitonin procedure for cell fractionation 2 In the absence of glucagon, the cytosol contains about two-thirds of cellular ATP, some 40–50% of ADP, acetyl-CoA, citrate and phosphoenolpyruvate, more than 75% of total 2-oxoglutarate, glutamate, malate, oxaloacetate, pyruvate, 3-phosphoglycerate and aspartate, and all of glucose 6-phosphate 3 In the presence of glucagon the cytosolic space shows an increase in the content of malate, phosphoenolpyruvate and 3-phosphoglycerate by more than 60%, and those of aspartate and glucose 6-phosphate rise by about 25% Other metabolites remain unchanged After glucagon treatment, cytosolic pyruvate is decreased by 37%, whereas glutamate and 2-oxoglutarate decrease by 70% The [NAD+]/[NADH] ratios calculated from the cytosolic concentrations of the reactants of lactate dehydrogenase and malate dehydrogenase were the same Glucagon shifts this ratio and also that of the [NADP+]/[NADPH] couple towards a more reduced state 4 In the mitochondrial space glucagon causes an increase in the acetyl-CoA and ATP contents by 25%, and an increase in [phosphoenolpyruvate] by 50% Other metabolites are not changed by glucagon Oxaloacetate in the matrix is only slightly decreased after glucagon, yet glutamate and 2-oxoglutarate fall to about 25% of the respective control values The [NAD+]/[NADH] ratios as calculated from the [3-hydroxybutyrate]/[acetoacetate] ratio and from the matrix [malate]/[oxaloacetate] couple are lowered by glucagon, yet in the latter case the values are about tenfold higher than in the former 5 Glucagon and oleate stimulate gluconeogenesis from lactate to nearly the same extent Oleate, however, does not produce the changes in cellular 2-oxoglutarate and glutamate as observed with glucagon 6 The changes of the subcellular metabolite distribution after glucagon are compatible with the proposal that the stimulation of gluconeogenesis results from as yet unknown action(s) of the hormone at the mitochondrial level in concert with its established effects on proteolysis and lipolysis

Effect of glucagon on metabolite compartmentation in isolated rat liver cells during gluconeogenesis from lactate.

In isolated hepatocytes from normal fed rats, the subcellular distribution of malate, citrate, 2-oxoglutarate, glutamate, aspartate, oxaloacetate, acetyl-CoA and CoASH has been determined by a modified digitonin method. Incubation with various substrates (lactate, pyruvate, alanine, oleate, oleate plus lactate, ethanol and aspartate) markedly changed the total cellular amounts of metabolites, but their distribution between the cytosolic and mitochondrial compartments was kept fairly constant. In the presence of lactate, pyruvate or alanine, about 90% of cellular aspartate, malate and oxaloacetate, and 50% of citrate was located in the cytosol. The changes in acetyl-CoA in the cytosol were opposite to those in the mitochondrial space, the sum of both remaining nearly constant. The mitochondrial acetyl-CoA/CoASH ratio ranged from 0.3-0.9 and was positively correlated with the rate of ketone body formation. The mitochondrial/cytosolic (m/c) concentration gradients for malate, citrate, 2-oxoglutarate, glutamate, aspartate, oxaloacetate, acetyl-CoA and CoASH averaged from hepatocytes under different substrate conditions were determined to be 1.0, 8.8, 1.6, 2.2, 0.5, 0.7, 13 and 40, respectively. From the distribution of citrate, a pH difference of 0.3 across the inner mitochondrial membrane was calculated, yet lower values resulted from the m/c gradients of 2-oxoglutarate, glutamate and malate. The mass action ratios for citrate synthase and mitochondrial aspartate aminotransferase have been calculated from the metabolite concentrations measured in the mitochondrial pellet fraction. A comparison with the respective equilibrium constants indicates that in intact hepatocytes, neither enzyme maintains its reactants at equilibrium. On the assumption that mitochondrial malate dehydrogenase and 3-hydroxybutyrate dehydrogenase operate near equilibrium, the concentration of free oxaloacetate appears to be 0.3-2 micron, depending on the substrate used. Plotting the calculated free mitochondrial oxaloacetate concentration against the citrate concentration measured in the mitochondrial pellet yielded a hyperbolic saturation curve, from which an apparent Km of citrate synthase for oxaloacetate in the intact cells of 2 micron can be derived, which is comparable to the value determined with purified rat liver citrate synthase. The results are discussed with respect to the supply of substrates and effectors of anion carriers and of key enzymes of the tricarboxylic acid cycle and fatty acid biosynthesis.

Distribution of metabolites between the cytosolic and mitochondrial compartments of hepatocytes isolated from fed rats.

1. A modification of the digitonin method of Zuurendonk & Tager (1974) (Biochim. Biophys. Acta 333, 393-399) (i.e. the 'convaentional' method) was developed that allows the fractionation of isolated hepatocytes at -5 degrees C (i.e. 'low-temperature' method). 2. With respect to compartmentation of adenine nucleotides, glutamate and citrate, the two methods yielded very similar results. 3. In contrast, the mitochondrial amounts of aspartate and malate, as revealed by the low-temperature method, were about twice as high as those found by the conventional procedure. No change in the total cellular content occurred. 4. With n-butylmalonate and glisoxepid present in the conventional digitonin medium, significantly higher amounts of malate and aspartate respectively were found in the mitochondrial pellets. The results obtained by the low-temperature method, however, were not influenced by the these inhibitors. 5. It is concluded that under the conventional conditions of cell fractionation no appreciable redistribution of adenine nucleotides, glutamate and citrate occurs.

/pdf/validity-of-the-digitonin-method-for-metabolite-pvlfq805om.pdf

Validity of the digitonin method for metabolite compartmentation in isolated hepatocytes.

Gluconeogenesis from lactate in isolated hepatocytes is activated by a variety of hormonal stimuli. As to the mechanism(s) of action, it is widely accepted that the effect of glucagon is mediated by cyclic AMP (Exton et al., 1970, 1971), and adrenaline and phenylephrine have been shown to activate gluconeogenesis without involvement of cyclic AMP (Tolbert et al., 1973; Tolbert & Fain, 1974; Kneer et al., 1974; Exton & Harper, 1975; Hutson et al., 1976; Cherrington et al., 1976). Moreover, vasopressin has been demonstrated to increase glucose production in the perfused liver (Hems & Whitton, 1973) independently of changes in the hepatic cyclic AMP content (Kirk & Hems, 1974). Recently we have reported on the subcellular distridtion of metabolites in isolated hepatocytes after incubation with glucagon, which is known to cause a large increase in cyclic AMP (Johnson et al., 1972; Ingebretsen et al., 1972; Kirk & Hems, 1974;Christoffersen &Berg, 1974; Exton &Harper, 1975; Hutsonet al., 1976; Cherrington et al., 1976; Hems et al., 1978). The present study is concerned with the question of what changes in metabolite compartmentation might occur when liver cells are incubated with dibutyryl cyclic AMP or with glucogenic agents that cause either a relatively small increase or no change in cellular cyclic AMP, such as adrenaline and isoproterenol (Tolbert et al., 1973; Exton & Harper, 1975; Hutson et al., 1976; Chan & Exton, 1977) or phenylephrine (Exton & Harper, 1975; Hutson et al., 1976; Cherrington et al., 1976; Chan & Exton, 1977) and vasopressin (Kirk & Hems, 1974; Hems et al., 1978) respectively. Surprisingly, the effects of dibutyryl cyclic AMP on metabolite distribution proved to be different from those produced by glucagon.

Comparative Studies on the Influence of Hormones on Metabolite Compartmentation in Isolated Liver Cells during Gluconeogenesis from Lactate

1
The effect of oleate on the subcellular distribution of ATP, 2-oxoglutarate, glutamate, citrate, malate and phosphoenolpyruvate was studied in hepatocytes from rats starved for 48 h by applying a modified digitonin method. The results markedly differ from those observed after glucagon [Siess, E. A., Brocks, D. G., Lattke, H. K., and Wieland, O. H. (1977) Biochem. J. 166, 225–235]. Total cellular amounts and the distribution of ATP and 2-oxoglutarate remained unchanged. In the mitochondrial matrix glutamate was increased, while mitochondrial phosphoenolpyruvate was decreased. Citrate and malate were increased both in the mitochondrial and cytosolic space.

2
In contrast to the effect of glucagon, gluconeogenesis from dihydroxyacetone, fructose or glutamine was not stimulated by oleate. Gluconeogenesis from propionate was even inhibited by the fatty acid.

3
The stimulation by glucagon of glucose production from dihydroxyacetone or fructose was undiminished in biotin-deficient hepatocytes. Glucose formation from lactate, however, was stimulated only in biotin-substituted hepatocytes.

4
The results indicate that oleate stimulates gluconeogenesis by increasing pyruvate carboxylase activity (EC 6.4.1.1), whereas glucagon displays a more complex mode of action.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1980.tb06136.x

Dietrich G. Brocks

Papers

Effect of glucagon on metabolite compartmentation in isolated rat liver cells during gluconeogenesis from lactate.

Distribution of metabolites between the cytosolic and mitochondrial compartments of hepatocytes isolated from fed rats.

Validity of the digitonin method for metabolite compartmentation in isolated hepatocytes.

Comparative Studies on the Influence of Hormones on Metabolite Compartmentation in Isolated Liver Cells during Gluconeogenesis from Lactate

Distinctive Roles of Oleate and Glucagen in Gluconeogenesis