FEBS Letters 585 (2011) 92–98 journal homepage: www.FEBSLetters.org Lactate downregulates the glycolytic enzymes hexokinase and phosphofructokinase in diverse tissues from mice Tiago C. Leite a,b, Raquel G. Coelho a,b, Daniel Da Silva a, Wagner S. Coelho a, Monica M. Marinho-Carvalho a, ⇑ Mauro Sola-Penna a, a Laboratório de Enzimologia e Controle do Metabolismo (LabECoM), Departamento de Fármacos, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil b Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil article info abstract Article history: We examined the effects of lactate on the enzymatic activity of hexokinase (HK), phosphofructoki- Received 11 August 2010 nase (PFK) and pyruvate kinase (PK) in various mouse tissues. Our results showed that lactate inhib- Revised 4 November 2010 ited PFK activity in all the analyzed tissues. This inhibitory effect was observed in skeletal muscle Accepted 6 November 2010 even in the presence of insulin. Lactate directly inhibited the phosphorylation of PFK tyrosine res- Available online 11 November 2010 idues in skeletal muscle, an important mechanism of the enzyme activation. Moreover, lactate indi- Edited by Judit Ovádi rectly inhibited HK activity, which resulted from its cellular redistribution, here attributed to alterations of HK structure. PK activity was not affected by lactate. The activity of HK and PFK is directly related to glucose metabolism. Thus, it is conceivable that lactate exposure can induce inhi- Keywords: Hexokinase bition of glucose consumption in tissues. Phosphofructokinase Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. Glycolysis Open access under the Elsevier OA license. Insulin resistance Lactate 1. Introduction demonstrated that lactate could inhibit 6-phosphofructo-1-kinase (PFK, phosphofructokinase), a regulatory enzyme of glycolytic flux, In the last century, lactate had been considered as the end prod- by dissociating the active enzyme tetramers into the less active di- uct of glycolytic flux with no major metabolic functions other than mers [9]. inducing metabolic acidosis and tissue damage [1,2]. However, in This study aimed to contribute to, and to expand the knowledge recent years, lactate has been studied based on its ability to serve regarding, the action mechanism of lactate in skeletal muscle, liver, as an energy source and a cell-signaling and tissue-repairing mol- kidney and heart. Our results demonstrate that lactate can inhibit ecule [1,3]. Chronic hyperlactatemia has been described as an inde- both hexokinase (HK) and PFK, but not pyruvate kinase (PK) in a pendent risk factor for diabetes development, with lactate being an variety of tissues, supporting the hypothesis of negative regulation important factor for maintaining insulin resistance [4,5]. To date, of glucose consumption by glycolytic flux downregulation. only a few metabolic explanations have been provided for this ef- fect of lactate. Depré et al. reported decreased tissue glucose con- 2. Materials and methods sumption in the presence of lactate [6]. Lombardi et al. proposed that hyperlactatemia could decrease the GLUT-4 level and glucose 2.1. Materials uptake by skeletal muscle [7], a rate-limiting step of glucose metabolism in skeletal muscle. In addition, Choi et al. demon- ATP, fructose-6-phosphate, fructose-2,6-biphosphate (F2,6BP), strated that lactate could induce insulin resistance in skeletal mus- hexokinase, insulin and glucose were obtained from Sigma Chem- cle by inhibiting glycolytic flux through suppressing insulin ical Co. (St. Louis, MO, USA). 32Pi was obtained from the Instituto de signaling [8]. However, they did not specify which step(s) of the Pesquisas Energéticas e Nucleares (São Paulo, Brazil). [c-32P]ATP glycolytic flux was (were) inhibited. In a previous study, we was prepared according to Maia et al. [10]. ⇑ Corresponding author. Address: Laboratório de Enzimologia e Controle do 2.2. Mouse tissue homogenates Metabolismo (LabECoM), Departamento de Fármacos, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Rio de Janeiro – RJ 21941- 590, Brazil. Fax: +55 21 2260 9192x231. All mouse Experiments were performed according to the animal E-mail address: [email protected] (M. Sola-Penna). experimental protocols. Male Swiss mice (20–25 g) fed ad libitum 0014-5793 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. Open access under the Elsevier OA license. doi:10.1016/j.febslet.2010.11.009 T.C. Leite et al. / FEBS Letters 585 (2011) 92–98 93 were sacrificed by cervical dislocation. The heart, liver, kidney and 3. Results and discussion quadriceps were promptly removed, stripped of fat and connective tissue and incubated in the homogenization buffer consisting of 3.1. Effects of lactate on PFK activity 50 mM Tris–HCl (pH 7.4), 250 mM sucrose, 20 mM KF, 0.2 mM b- mercaptoethanol and 0.5 mM PMSF (1:3). The tissues were then We assessed the ability of lactate to modulate PFK and found treated with or without 5 or 10 mM lactate, depending on the that lactate inhibited PFK activity in the analyzed tissues under sev- requirements of the experiments. eral conditions (Fig. 1). Acute exposure to 10 mM lactate exerted no effect on PFK activity in skeletal and cardiac muscle. However, we 2.3. Tissue fractionation observed a 20% and 33% reduction in PFK activity in the liver and kidney, respectively. Unlike the acute exposure to 10 mM lactate, Tissue fractionation was performed according to a modification lactate preincubation for three hours inhibited PFK activity in skel- of the Lilling and Beitner protocol [11] proposed by Alves and Sola- etal and cardiac muscle. However, in the liver and kidney, lactate Penna [12]. The pH of all tissues homogenate and fractions were preincubation did not cause a further decrease in PFK activity. To controlled before and after the addition of lactate to assure that evaluate if the ability of lactate to inhibit PFK activity remains in the pH was controlled. the presence of hormones that stimulate glycolytic flux, isolated mouse tissues were incubated in a buffer containing 4.5 mM glu- 2.4. Enzymatic activity assays cose and stimulated with 100 nM insulin (Fig. 1). We observed that after insulin exposure, the ability of lactate to inhibit PFK was lost, HK and PFK enzymatic activities were assessed by the independent of the analyzed tissue type and time frame. The only radiometric method described by Sola-Penna et al. [13] with exception was found in skeletal muscle, where we observed a 52% the modifications proposed by Zancan and Sola-Penna [14,15]. reduction in PFK activity in the tissue preincubated with 10 mM This assay was performed at 37 °C in a 0.4-ml reaction system lactate, even in the presence of 100 nM insulin (Fig. 1). Insulin containing 50 mM Tris–HCl (pH 7.4), 5 mM MgCl2 and can promote an increase in intracellular synthesis of F2,6BP, a [c-32P]ATP (4 lCi/lmol). PK was evaluated in a basic medium known positive allosteric factor of PFK [18,19]. Therefore, it is pos- containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2,5mM sible that the increased F2,6BP synthesis triggered by insulin could phospho(enol)pyruvate (PEP), 5 mM ADP and 120 mM KCl. The counterbalance the inhibitory effect of lactate on PFK activity. reaction was initiated by the addition of enzyme preparation. To evaluate whether F2,6BP is capable of reversing the inhibi- Aliquots were withdrawn 2, 4, 6, 8 and 10 min after the reaction tory effect of lactate on PFK activity, isolated mouse tissues were was initiated and the reaction was stopped by the addition of preincubated with or without 10 mM lactate for three hours and 0.1 N HCl. The medium was neutralized with NaOH and the subsequently treated with 100 nM F2,6BP (Fig. 2). As expected, ATP content was evaluated using the commercial kit ATPlite 1 PFK was activated when tissues were treated with 100 nM step (PerkinElmer, MA, USA). Blanks were performed in parallel F2,6BP. As shown in Fig. 2, PFK activity in skeletal muscle, heart, li- in the absence of PEP. ver and kidney was increased by approximately 148%, 156%, 132% and 134%, respectively. However, the stimulatory effect of 2.5. Intrinsic fluorescence spectroscopy F2,6BPF2, 6BP on PFK activity was decreased by preincubating the tissues with lactate for three hours. Under these conditions, Intrinsic fluorescence analysis was perfumed on a spectrofluo- PFK activity in skeletal muscle, heart, liver and kidney was approx- rimeter (Jasco Ò) in media consisting of 100 mM Tris–HCl (pH imately 70%, 121%, 104% and 96%, respectively, of the levels ob- 7.4), 5 mM (NH4)2SO4 and purified hexokinase (5 lg/ml) in the served in control samples. Only in skeletal muscle was the PFK presence or absence of 5 or 10 mM lactate. Respective spectra were activity significantly lower than that in the control samples. subtracted for background and interference correction. The excita- It has been shown that PFK is regulated by different protein ki- tion wavelength used was 280 nm, and fluorescence emission was nases capable of phosphorylating its serine, threonine and/or tyro- scanned from 300 to 400 nm (0.5 nm intervals at a rate of 100 nm/ sine residues [20–22]. Therefore, we proceeded to examine if min). The center of mass of the intrinsic fluorescence spectrum was lactate is capable of regulating the phosphorylation levels of these calculated according to Leite et al. [9] using the SigmaPlot 10.0 (Sy- residues in PFK. For this purpose, skeletal and cardiac muscle, as stat) software. well as liver and kidney tissues, was incubated for three hours with 10 mM lactate or 100 mM insulin (positive control) for evaluating 2.6.