Post-mortem muscle temperature affects the rate of decline in pH in a linear manner from 37.5 °C down near 0 °C, and this pH decline is correlated with the enzymatic degradation of glycogen to lactate. This transformation occurs in an anaerobic context that includes the metabolic splice between glycogenolysis and glycolysis; and both processes are strongly upregulated by AMPK enzyme. In this study we reported changes (0.5 h and 24 h post-mortem) in muscle glycogen concentration, lactate and AMPK activity from 12 samples of Longissimus dorsi from 38 steers that produced high pH (>5.9) and normal pH (<5.8) carcasses at 24 h post-mortem. Moreover, we evaluated changes in AMPK activity in samples from both categories incubated at 37, 25, 17 and 5 °C and supplemented with exogenous glycogen. Finally, we analysed if there were structural differences between polymers from both categories. Our analyses show that enzymatic AMPK activity was significantly higher at 17 °C than at 37 °C or 25 °C (p<0.0001 and p<0.05 in samples from normal and high pH categories, respectively), and was near zero at 5 °C. On the other hand, AMPK activity did not change in relation with excess glycogen and we did not detect structural differences in the polymers present in samples from both categories. We concluded that post-mortem AMPK activity level is highly sensitive to temperature and not at in vitro changes in glycogen concentration. Their results suggest that that normal levels of pre-mortem muscle glycogen and an adequate cooling managing of carcasses are relevant to let an efficient glycogenolytic/glycolytic flow required for lactate accumulation and pH decline, trough of post-mortem AMPK signalling pathway.

Diabetic nephropathy (DN) is a major complication of diabetic patients and the leading cause of end-stage renal disease. Glomerular dysfunction plays a critical role in DN, but deterioration of renal function also correlates with tubular alterations. Human DN is characterized by glycogen accumulation in tubules. Although this pathological feature has long been recognized, little information exists about the triggering mechanism. In this study, we detected over-expression of muscle glycogen synthase (MGS) in diabetic human kidney. This enhanced expression suggests the participation of MGS in renal metabolic changes associated with diabetes. HK2 human renal cell line exhibited an intrinsic ability to synthesize glycogen, which was enhanced after over-expression of protein targeting to glycogen. A correlation between increased glycogen amount and cell death was observed. Based on a previous transcriptome study on human diabetic kidney disease, significant differences in the expression of genes involved in glycogen metabolism were analyzed. We propose that glucose, but not insulin, is the main modulator of MGS activity in HK2 cells, suggesting that blood glucose control is the best approach to modulate renal glycogen-induced damage during long-term diabetes.

Understanding how glucose metabolism is finely regulated at molecular and cellular levels in the liver is critical for knowing its relationship to related pathologies, such as diabetes. In order to gain insight into the regulation of glucose metabolism, we studied the liver-expressed isoforms aldolase B and fructose-1,6-bisphosphatase-1 (FBPase-1), key enzymes in gluconeogenesis, analysing their cellular localization in hepatocytes under different metabolic conditions and their protein–protein interaction in vitro and in vivo. We observed that glucose, insulin, glucagon and adrenaline differentially modulate the intracellular distribution of aldolase B and FBPase-1. Interestingly, the in vitro protein–protein interaction analysis between aldolase B and FBPase-1 showed a specific and regulable interaction between them, whereas aldolase A (muscle isozyme) and FBPase-1 showed no interaction. The affinity of the aldolase B and FBPase-1 complex was modulated by intermediate metabolites, but only in the presence of K+. We observed a decreased association constant in the presence of adenosine monophosphate, fructose-2,6-bisphosphate, fructose-6-phosphate and inhibitory concentrations of fructose-1,6-bisphosphate. Conversely, the association constant of the complex increased in the presence of dihydroxyacetone phosphate (DHAP) and non-inhibitory concentrations of fructose-1,6-bisphosphate. Notably, in vivo FRET studies confirmed the interaction between aldolase B and FBPase-1. Also, the co-expression of aldolase B and FBPase-1 in cultured cells suggested that FBPase-1 guides the cellular localization of aldolase B. Our results provide further evidence that metabolic conditions modulate aldolase B and FBPase-1 activity at the cellular level through the regulation of their interaction, suggesting that their association confers a catalytic advantage for both enzymes.

The development and survival of male germ cells depend on the antioxidant capacity of the seminiferous tubule. Glutathione (GSH) plays an important role in the antioxidant defenses of the spermatogenic epithelium. Autophagy can act as a pro-survival response during oxidative stress or nutrient deficiency. In this work, we evaluated whether autophagy is involved in spermatogonia-type germ cell survival during severe GSH deficiency. We showed that the disruption of GSH metabolism with l-buthionine-(S,R)-sulfoximine (BSO) decreased reduced (GSH), oxidized (GSSG) glutathione content, and GSH/GSSG ratio in germ cells, without altering reactive oxygen species production and cell viability, evaluated by 2',7'-dichlorodihydrofluorescein (DCF) fluorescence and exclusion of propidium iodide assays, respectively. Autophagy was assessed by processing the endogenous protein LC3I and observing its sub-cellular distribution. Immunoblot and immunofluorescence analysis showed a consistent increase in LC3II and accumulation of autophagic vesicles under GSH-depletion conditions. This condition did not show changes in the level of phosphorylation of AMP-activated protein kinase (AMPK) or the ATP content. A loss in S-glutathionylated protein pattern was also observed. However, inhibition of autophagy resulted in decreased ATP content and increased caspase-3/7 activity in GSH-depleted germ cells. These findings suggest that GSH deficiency triggers an AMPK-independent induction of autophagy in germ cells as an adaptive stress response.

Background: Fructose-1,6-bisphosphatase, a major enzyme of gluconeogenesis, is inhibited by AMP, Fru-2,6-P-2 and by high concentrations of its substrate Fru-1,6-P-2. The mechanism thaf produces substrate inhibition continues to be obscure.Methods: Four types of experiments were used to shed light on this: (1) kinetic measurements over a very wide range of substrate concentrations, subjected to detailed statistical analysis; (2) fluorescence studies of mutants in which phenylalanine residues were replaced by tryptophan; (3) effect of Fru-2,6-P-2 and Fru-1,6-P-2 on the exchange of subunits between wild-type and Glu-tagged oligomers; and (4) kinetic studies of hybrid forms of the enzyme containing subunits mutated at the active site residue tyrosine-244.Results: The kinetic experiments with the wild-type enzyme indicate that the binding of Fru-1,6-P-2 induces the appearance of catalytic sites with lower affinity for substrate and lower catalytic activity. Binding of substrate to the high-affinity sites, but not to the low-affinity sites, enhances the fluorescence emission of the Phe219Trp mutant; the inhibitor, Fru-2,6-P-2, competes with the substrate for the high-affinity sites. Binding of substrate to the low-affinity sites acts as a "stapler" that prevents dissociation of the tetramer and hence exchange of subunits, and results in substrate inhibition.Conclusions: Binding of the first substrate molecule, in one dimer of the enzyme, produces a conformational change at the other dimer, reducing the substrate affinity and catalytic activity of its subunits.General significance: Mimics of the substrate inhibition of fructose-1,6-bisphosphatase may provide a future option for combatting both postprandial and fasting hyperglycemia. (C) 2013 Elsevier B.V. All rights reserved.

Using a streptozotocin‐induced type 1 diabetic rat model, we analyzed and separated the effects of hyperglycemia and hyperinsulinemia over the in vivo expression and subcellular localization of hepatic fructose 1,6‐bisphosphatase (FBPase) in the multicellular context of the liver. Our data showed that FBPase subcellular localization was modulated by the nutritional state in normal but not in diabetic rats. By contrast, the liver zonation was not affected in any condition. In healthy starved rats, FBPase was localized in the cytoplasm of hepatocytes, whereas in healthy re‐fed rats it was concentrated in the nucleus and the cell periphery. Interestingly, despite the hyperglycemia, FBPase was unable to accumulate in the nucleus in hepatocytes from streptozotocin‐induced diabetic rats, suggesting that insulin is a critical in vivo modulator. This idea was confirmed by exogenous insulin supplementation to diabetic rats, where insulin was able to induce the rapid accumulation of FBPase within the hepatocyte nucleus. Besides, hepatic FBPase was found phosphorylated only in the cytoplasm, suggesting that the phosphorylation state is involved in the nuclear translocation. In conclusion, insulin and not hyperglycemia plays a crucial role in the nuclear accumulation of FBPase in vivo and may be an important regulatory mechanism that could account for the increased endogenous glucose production of liver of diabetic rodents. J. Cell. Biochem. 113: 848–856, 2012. © 2011 Wiley Periodicals, Inc.

A substrate cycle composed of phosphofructo 1‐kinase I (PFK) and fructose 1,6 bisphosphatase I (FBPase) has been proposed in rat spermatids. This substrate cycle can explain the ability of glucose to induce a decrease in intracellular ATP, a phenomenon that was related to regulation of [Ca2+]i in these cells. In spite of the importance of this metabolic cycle, the expression and activities of the enzymes that compose such cycle have not been systematically studied in spermatogenic cells. Here, we show that PFK and FBPase activities were present in pachytene spermatocytes and round spermatids extracts. Expression of PFK at the mRNA and protein levels showed a relatively similar expression in spermatogenic cells, but a stronger expression in Sertoli cells. Instead, expression of FBPase at the mRNA and protein levels was stronger in round and elongating spermatids as compared to other spermatogenic cells. A similar pattern was observed when evidencing FBPase activity by a NADPH‐nitroblue tetrazolium‐linked cytochemical assay in isolated pachytene spermatocytes and round spermatids. Rat spermatids also showed the ability to convert lactate to fructose‐ and glucose‐6‐P, indicating that both glycolytic and gluconeogenic fluxes are present in these cells. Our results indicate that a coordinated expression of key substrate cycle enzymes, at the level of PFK/FBPase, appear in the last stages of spermatogenic cell differentiation, suggesting that the co‐regulation of these enzymes are required for the ability of these cells to respond to glucose and induce metabolic and Ca2+ signals that can be important for sperm development and function. J. Cell. Physiol. 212:807–816, 2007. © 2007 Wiley‐Liss, Inc.

Pig kidney fructose‐1,6‐bisphosphatase is a homotetrameric enzyme which does not contain tryptophan. In a previous report the guanidine hydrochloride‐induced unfolding of the enzyme has been described as a multistate process [Reyes, A. M., Ludwig, H. C., Yañez, A. J., Rodriguez, P. H and Slebe, J. C. (2003) Biochemistry 42, 6956–6964]. To monitor spectroscopically the unfolding transitions, four mutants were constructed containing a single tryptophan residue either near the C1–C2 or the C1–C4 intersubunit interface of the tetramer. The mutants were shown to retain essentially all of the structural and kinetic properties of the enzyme isolated from pig kidney. The enzymatic activity, intrinsic fluorescence, size‐exclusion chromatographic profiles and 1‐anilinonaphthalene‐8‐sulfonate binding by the mutants were studied under unfolding equilibrium conditions. The unfolding profiles were multisteps, and formation of hydrophobic structures was detected. The enzymatic activity of wild‐type and mutant FBPases as a function of guanidine hydrochloride concentration showed an initial enhancement (maximum ∼ 30%) followed by a biphasic decay. The activity and fluorescence results indicate that these transitions involve conformational changes in the fructose‐1,6‐bisphosphate and AMP domains. The representation of intrinsic fluorescence data as a ‘phase diagram’ reveals the existence of five intermediates, including two catalytically active intermediates that have not been previously described, and provides the first spectroscopic evidence for the formation of dimers. The intrinsic fluorescence unfolding profiles indicate that the dimers are formed by selective disruption of the C1–C2 interface.

Selective treatment of pig kidney fructose 1,6-bisphosphatase with cyanate leads to the formation of an active carbamoylated derivative that shows no cooperative interaction between the AMP-binding sites, but completely retains the sensitivity to the inhibitor. By an exhaustive carbamoylation of the enzyme a derivative is formed that has a complete loss of cooperativity and a decrease of sensitivity to AMP. It was proposed that the observed changes of allosteric properties were due to the chemical modification of two lysine residues per enzyme subunit [Slebe et al. (1983), J. Protein Chem.2, 437–443]. Studies of the temperature dependence of AMP sensitivity and the interaction with Cibacron Blue Sepharose of carbamoylated fructose 1,6-bisphosphatase derivatives indicate that the lysine residue involved in AMP sensitivity is located at the allosteric AMP site, while the lysine residue involved in AMP cooperativity is at a distinct location. Using [14C]cyanate, we identified both lysine residues in the primary structure of the enzyme; Lys50 is essential for AMP cooperativity and Lys112 appears to be the reactive residue involved in the AMP sensitivity. According to the fructose 1,6-bisphosphatase crystal structure, Lys50 is strategically positioned at the C1–C2 interface, near the molecular center of the tetramer, and Lys112 is in the AMP-binding site. The results reported here, combined with the structural data of the enzyme, strongly suggest that the C1ndash;C2 interface is critical for the propagation of the allosteric signal among the AMP sites on different subunits.

Treatment of fructose 1,6-bisphosphatase with N-ethylmaleimide was shown to abolish the inhibition by fructose 2,6-bisphosphate, which also protected the enzyme against this chemical modification [Reyes, A., Burgos, M. E., Hubert, E., and Slebe, J. C. (1987),J. Biol. Chem.262, 8451–8454]. On the basis of these results, it was suggested that a single reactive sulfhydryl group was essential for the inhibition. We have isolated a peptide bearing the N-ethylmaleimide target site and the modified residue has been identified as cysteine-128. We have further examined the reactivity of this group and demonstrated that when reagents with bulky groups are used to modify the protein at the reactive sulfhydryl [e.g., N-ethylmaleimide or 5,5′-dithiobis-(2-nitrobenzoate)], most of the fructose 2,6-bisphosphate inhibition potential is lost. However, there is only partial or no loss of inhibition when smaller groups (e.g., cyanate or cyanide) are introduced. Kinetic and ultraviolet difference spectroscopy-binding studies show that the treatment of fructose 1,6-bisphosphatase with N-ethylmaleimide causes a considerable reduction in the affinity of the enzyme for fructose 2,6-bisphosphate while affinity for fructose 1,6-bisphosphate does not change. We can conclude that modification of this reactive sulfhydryl affects the enzyme sensitivity to fructose 2,6-bisphosphate inhibition by sterically interfering with the binding of this sugar bisphosphate, although this residue does not seem to be essential for the inhibition to occur. The results also suggest that fructose 1,6-bisphosphate and fructose 2,6-bisphosphate may interact with the enzyme in a different way.

Selective treatment of pig kidney fructose 1,6-bisphosphatase with potassium cyanate leads to the formation of an active carbamylated enzyme that has lost the cooperative interactions among AMP sites, but retains sensitivity to inhibition of catalytic activity by the regulator AMP. Incorporation data on [14C]KNCO indicate that the loss of enzyme cooperativity at the AMP sites is related to selective carbamylation of four lysine residues per mole of tetrameric enzyme. Exhaustive carbamylation suggests that a second lysine residue per subunit is essential for AMP inhibition.