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Original research article
Tumor necrosis factor alpha abolished the suppressive effect of insulin on hepatic glucose production and glycogenolysis stimulated by cAMP Aline Franco da Rocha a, Thaı´s Fernanda Liboni a, Mirian Ayumi Kurauti a, Camila Oliveira de Souza a, Daniele Romani Miksza a, Carolina Campos Lima Moreira a, Glaucia Regina Borba-Murad a, Roberto Barbosa Bazotte b, Helenir Medri de Souza a,* a b
Department of Physiological Sciences, State University of Londrina, Londrina, PR, Brazil Department of Pharmacology and Therapeutics, State University of Maringa´, Maringa´, PR, Brazil
A R T I C L E I N F O
Article history: Received 28 March 2013 Received in revised form 19 December 2013 Accepted 20 December 2013 Available online xxx Keywords: TNFa Insulin resistance Cyclic AMP Hepatic glucose production Glycogenolysis
A B S T R A C T
Background: Tumor necrosis factor alpha (TNFa) is implicated in the development of insulin resistance in obesity, type 2 diabetes and cancer. However, its ability to modulate the action of insulin on glycogen catabolism in the liver is controversial. The aim of the present study was to investigate whether TNFa acutely affects the suppression by insulin of hepatic glucose production (HGP) and glycogenolysis stimulated by cyclic adenosine monophosphate (cAMP). Methods: TNFa (10 mg/kg) was injected intravenously to rats and, 1 or 6 h later, their livers were subjected to in situ perfusion with cAMP (3 mM), in the presence or absence of physiological (20 mU/mL) or supraphysiological (500 mU/mL) concentrations of insulin. Results: The injection of TNFa, 1 or 6 h before liver perfusion, had no direct effect on the action of cAMP in stimulating HGP and glycogenolysis. However, when TNFa was injected 1 h, but not 6 h, before liver perfusion it completely abolished (p < 0.05) the suppressive effect of 20 mU/mL insulin on HGP and glycogenolysis stimulated by cAMP. Furthermore, the injection of TNFa 1 h or 6 h before liver perfusion did not influence the suppression of cAMP-stimulated HGP and glycogenolysis by 500 mU/mL insulin. Conclusion: TNFa acutely abolished the suppressive effect of physiological, but not supraphysiological, levels of insulin on HGP and glycogenolysis stimulated by cAMP, suggesting an important role of this mechanism to the increased HGP in several pathological states. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Tumor necrosis factor a (TNFa) is a multifunctional proinflammatory cytokine involved in many metabolic responses, which has been reported to act as a trigger of insulin resistance. The administration of TNFa was found to induce peripheral insulin resistance [1] in adipose tissue [2], muscle [3] and hepatocytes [4]. Consistently with this, the administration of anti-TNFa antibody or the genetic knockout of the TNFa receptor increased peripheral insulin sensitivity in the muscle and liver [5–10]. Furthermore, incubation of adipocytes, muscle cells or hepatocytes with TNFa inhibited essential steps in the insulin signal transduction pathway. In fact, TNFa increased serine phosphorylation of the insulin receptor (IR) and insulin receptor
* Corresponding author. E-mail address: [email protected] (H.M. de Souza).
substrate (IRS-1), probably by activating serine kinases [11], which reduced tyrosine phosphorylation of IR and IRS-1 stimulated by insulin [4,12–15] and prevented the activation of phosphatidylinositol 3-kinase (PI3K) [11,15,16] and protein kinase B (Akt/PKB) [3,4]. In addition, it has been shown that an increased production of TNFa is implicated in the pathogenesis of insulin resistance in type 2 diabetes and obesity and that enhanced expression of TNFa by adipose tissue plays an important role in the association between obesity and type 2 diabetes [8,14,17,18]. An increase in TNFa production seems also to be implicated in insulin resistance in cancer [19–21]. Interestingly, insulin resistance in cancer was associated with increased serum levels of TNFa [22] or increased expression of this cytokine in the muscle [19]. Despite the well established capacity of TNFa to induce insulin resistance in various pathological states, there are few studies assessing its influence on the action of insulin on glycogen catabolism in the liver, and the results are not clear. For example, there are reports
http://dx.doi.org/10.1016/j.pharep.2013.12.005 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Please cite this article in press as: da Rocha AF, et al. Tumor necrosis factor alpha abolished the suppressive effect of insulin on hepatic glucose production and glycogenolysis stimulated by cAMP. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2013.12.005
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that TNFa inhibits the suppressive effect of insulin on hepatic glucose production [1,6], whereas this inhibition was not observed in another study [8]. In view of these contradictory results, we investigated whether this cytokine acutely affects the suppressive action of insulin on HGP and glycogenolysis stimulated by cyclic adenosine monophosphate (cAMP) in rats. Materials and methods
TNFa (10 mg/kg) was administered to rats, 1 or 6 h before liver perfusion, by intravenous injection (penial vein) of 200 mL of a buffered solution (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.76 mM, KH2PO4 and 0.1% bovine serum albumin), pH 7.4, containing 2 mg TNFa. Control rats were injected with 200 mL of the buffered solution. All protocols were approved by the Ethics Committee for Animal Experimentation of the State University of Londrina.
Chemicals
Liver perfusion
Recombinant rat TNFa was purchased from Peprotech Mexico (SA de CV). Aliquots of reconstituted TNFa were stored at 20 8C and thawed immediately before the experiments. Regular insulin was purchased from Eli Lilly (Brazil) and cAMP from Sigma Chemical Co. (St. Louis, USA). All other chemicals (98–99.8% purity) were purchased from Sigma Chemical Co. (St. Louis, USA), Acros Organics (New Jersey, USA), Reagen (Rio de Janeiro, Brazil) and Merck (Darmstadt, Germany).
For the surgical procedure, rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg) and subjected to in situ liver perfusion, as previously described [23,24]. The perfusion fluid, Krebs-Henseleit buffer (KHB), pH 7.4, at 37 8C and saturated with a 95%:5% O2:CO2 mixture, was introduced into the liver (4 mL/min per gram of liver) through a cannula inserted into the portal vein, while a second cannula in the inferior vena cava was used to collect the effluent perfusate. The composition of the KHB buffer was: 115 mM NaCl, 25 mM NaHCO3, 5.8 mM KCl, 1.2 mM Na2SO4, 1.18 mM MgCl2, 1.2 mM NaH2PO4 and 2.5 mM CaCl2. cAMP and insulin were dissolved in KHB. Livers were perfused with KHB for the first 10 min, with KHB + cAMP (3 mM) between 10 and 24 min and with KHB + cAMP (3 mM) or KHB + cAMP (3 mM) + insulin (20 mU/mL or 500 mU/mL) between
Animals and administration of TNFa Male Wistar rats (200–230 g), kept at a constant temperature (23 2 8C) in a light/dark cycle of 12:12 h and fed on a standard commercial laboratory diet (Nuvilab1), were used in all experiments.
Fig. 1. Effect of tumor necrosis factor alpha (TNFa) on the suppressive action of insulin on (A) hepatic glucose production and (C) glycogenolysis stimulated by cAMP and (B, D) the corresponding areas under curves (AUCs). Livers were perfused 1 h after the administration of 10 mg/kg TNFa (TNFa - 1 h) or buffered solution (Control), as described in Materials and Methods. cAMP (3 mM) was infused from 10 to 24 min. cAMP (3 mM) or cAMP (3 mM) + insulin (20 mU/mL) were infused from 24 to 50 min. Data are mean SEM of 6–9 experiments. *p < 0.05 vs. cAMP, #p < 0.05 vs. cAMP + Ins20 (one-way ANOVA followed by Newman–Keuls test).
Please cite this article in press as: da Rocha AF, et al. Tumor necrosis factor alpha abolished the suppressive effect of insulin on hepatic glucose production and glycogenolysis stimulated by cAMP. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2013.12.005
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24 and 50 min. The effluent perfusate was collected every 2 min to measure the production of glucose, lactate and pyruvate. At the end of the perfusion, the liver was removed and weighed, to allow precise metabolic calculations and the correction of flux rates. Glycogenolysis was calculated as the sum of glucose production plus half the sum of lactate and pyruvate production [glucose + 1/2 (lactate + pyruvate)]. The areas under curves (AUCs) of glucose production and glycogenolysis were calculated for the interval of 24–50 min. Analytical procedures The concentrations of glucose [25], pyruvate [26] and lactate [27] were assayed by enzymatic methods. Statistical procedures Normal distribution and variance homogeneity were tested and one-way ANOVA, followed by the Newman–Keuls test, was employed to analyze the results. Statistical analysis was carried out with the program GraphPad Prism 4.0 and significance was accepted when p < 0.05. Data are expressed as mean standard error of the mean (SEM).
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Results The infusion of cAMP (3 mM) in the liver stimulated (p < 0.05) HGP and glycogenolysis in all groups (Figs. 1–4). There were no differences in these variables between the TNFa and control groups (Figs. 1–4). The infusion of 20 mU/mL or 500 mU/mL insulin in the liver of control rats decreased (p < 0.05) the HGP and glycogenolysis stimulated by cAMP, as shown by the AUCs (Figs. 1–4). The intravenous injection of TNFa, 1 or 6 h before liver perfusion, did not influence the stimulatory effect of cAMP on glycogen catabolism, as observed by the similarity in the rates of HGP and glycogenolysis, from 10 to 24 min of perfusion, in comparison with control rats (Figs. 1–4). On the other hand, the administration of TNFa, 1 h before liver perfusion, completely abolished (p < 0.05) the inhibitory effect of 20 mU/mL insulin on HGP (Fig. 1A and B) and glycogenolysis (Fig. 1C and D) stimulated by cAMP. However, when the TNFa was injected 6 h before liver perfusion, it did not change significantly the suppressive effect of 20 mU/mL insulin on HGP (Fig. 2A and B) or glycogenolysis (Fig. 2C and D) stimulated by cAMP. Moreover, TNFa administered 1 h before liver perfusion did not influence the suppressive effect of 500 mU/mL insulin on HGP
Fig. 2. Effect of tumor necrosis factor alpha (TNFa) on the suppressive action of insulin on (A) hepatic glucose production and (C) glycogenolysis stimulated by cAMP and (B, D) the corresponding areas under curves (AUCs). Livers were perfused 6 h after the administration of 10 mg/kg TNFa (TNFa – 6 h) or buffered solution (Control), as described in Materials and Methods Section. cAMP (3 mM) was infused from 10 to 24 min. cAMP (3 mM) or cAMP (3 mM) + insulin (20 mU/mL) were infused from 24 to 50 min. Data are mean SEM of 6–7 experiments. *p < 0.05 vs. cAMP, **p < 0.01 vs. cAMP (one-way ANOVA followed by Newman–Keuls test).
Please cite this article in press as: da Rocha AF, et al. Tumor necrosis factor alpha abolished the suppressive effect of insulin on hepatic glucose production and glycogenolysis stimulated by cAMP. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2013.12.005
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Fig. 3. Effect of tumor necrosis factor alpha (TNFa) on the suppressive action of insulin on (A) hepatic glucose production and (C) glycogenolysis stimulated by cAMP and (B, D) the corresponding areas under curves (AUCs). Livers were perfused 1 h after the administration of 10 mg/kg TNFa (TNFa – 1 h) or buffered solution (Control), as described in Materials and Methods. cAMP (3 mM) was infused from 10 to 24 min. cAMP (3 mM) or cAMP (3 mM) + insulin (500 mU/mL) were infused from 24 to 50 min. Data are mean SEM of 6–7 experiments. *p < 0.05 and **p < 0.01 vs. cAMP (one-way ANOVA followed by Newman–Keuls test).
(Fig. 3A and B) or glycogenolysis (Fig. 3C and D) stimulated by cAMP; nor did TNFa injected 6 h before liver perfusion have any effect on the inhibitory action of 500 mU/mL insulin on HGP (Fig. 4A and B) and glycogenolysis (Fig. 4C and D) stimulated by cAMP. Discussion The main question raised in this study was whether TNFa acutely affects the action of insulin on the liver glycogen catabolism stimulated by cAMP. To answer this question, the livers of fed rats were perfused in situ with cAMP in the presence or absence of physiological (20 mU/mL) or supraphysiological (500 mU/mL) insulin levels, 1 or 6 h after an intravenous injection of TNFa. This cytokine was administered intravenously because its direct infusion into the liver did not alter the HGP from alanine, while intravenous injection of TNFa decreased the HGP from this amino acid in isolated perfused livers [28]. The administration of TNFa did not influence the stimulatory effect of cAMP on glycogen catabolism (Figs. 1–4). In contrast to our study, the TNFa suppressed the dibutyryl cAMP/dexamethasone-induced increase in glucose-6-phosphatase mRNA levels by activation of nuclear factor kappaB (NFkB) [29]. The stimulation of HGP and glycogenolysis by cAMP was probably mediated by
activation of cAMP-dependent protein kinase (PKA) and subsequent phosphorylation and activation of glycogen phosphorylase [30]. Similarly, incubation of isolated rat hepatocytes with TNFa also did not influence glycogenolysis [20]. In rats that did not receive TNFa (controls), the activation of glycogen catabolism promoted by cAMP was reduced in the presence of 20 mU/mL or 500 mU/mL insulin (Figs. 1–4), confirming the direct inhibitory effect of insulin in liver perfused in situ [24,31,32]. This effect can be attributed to a reduction in the intracellular concentration of cAMP, due to the activation of phosphodiesterase-3B (PDE3B), an enzyme that promotes the hydrolysis of cAMP [33,34]. PDE3B is activated by Akt/PKB, via stimulation of PI3K associated with IRS-1 [35]. However, the administration of TNFa, 1 h before liver perfusion, completely abolished the inhibitory effect of 20 mU/mL insulin on cAMP-stimulated HGP and glycogenolysis (Fig. 1). These results reveal an in vivo acute inhibitory effect of TNFa on the hepatic response to insulin; i.e., TNFa reduced the ability of insulin to inhibit glycogen catabolism activated by cAMP. In corroboration, it has been reported that the infusion of TNFa in rats inhibited insulin-induced suppression of HGP [1]. Furthermore, in obese rats, in which the activity of TNFa was inhibited, HGP was completely suppressed during hyperinsulinemic glucose clamp [6]. However, in another
Please cite this article in press as: da Rocha AF, et al. Tumor necrosis factor alpha abolished the suppressive effect of insulin on hepatic glucose production and glycogenolysis stimulated by cAMP. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2013.12.005
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Fig. 4. Effect of tumor necrosis factor alpha (TNFa) on the suppressive action of insulin on (A) hepatic glucose production and (C) glycogenolysis stimulated by cAMP and (B, D) the corresponding areas under curves (AUCs). Livers were perfused 6 h after administration of 10 mg/kg TNFa (TNFa – 6 h) or buffered solution (Control), as described in Materials and Methods Section. cAMP (3 mM) was infused from 10 to 24 min. cAMP (3 mM) or cAMP (3 mM) + insulin (500 mU/mL) were infused from 24 to 50 min. Data are mean SEM of 6–7 experiments. *p < 0.05 and **p < 0.01 vs. cAMP (one-way ANOVA followed by Newman–Keuls test).
study with obese rats, the inhibition of TNFa did not affect the suppression of HGP mediated by insulin [8]. Also in agreement with our results, several studies have shown that TNFa inhibits the insulin signaling cascade in hepatocytes, probably via the activation of serine kinases [11]. In fact, portal infusion of TNFa increased serine phosphorylation of IRS-1 and inhibited the insulin signaling in the liver [4]. Similarly, incubation of hepatoma cells with TNFa induced serine phosphorylation of IRS-1 [13]. Further, incubation of hepatoma cells [12] or adipocytes [15] with TNFa, for 1 h, decreased tyrosine phosphorylation of IR and IRS-1 stimulated by insulin, indicating that the period of 1 h is enough for the induction of the effects of TNFa on the insulin signaling pathway. A reduction of the suppressive effect of insulin in the liver, as promoted by TNFa in the present study, results in increased HGP. Thus, increased production of TNFa may be one of the factors that contribute to the increased HGP in type 2 diabetes, obesity and cancer. Although the intravenous injection of TNFa, 1 h before the liver perfusion, completely abolished the inhibitory effect of 20 mU/mL insulin on cAMP-stimulated catabolism of glycogen, when the TNFa was administered 6 h before liver perfusion, it did not change significantly this suppressive effect of insulin on HGP or
glycogenolysis (Fig. 2), demonstrating that the effect of TNFa declines over time. In contrast to the results obtained with physiological concentrations of insulin, the administration of TNFa, either 1 h (Fig. 3) or 6 h (Fig. 4) before the liver was perfused with supraphysiological concentrations of insulin (500 mU/mL), had no effect on the insulin-mediated suppression of HGP and glycogenolysis stimulated by cAMP. Therefore, high levels of insulin, mimicking the compensatory hyperinsulinemia that occurs during insulin resistance associated with type 2 diabetes [36] and obesity [9], were necessary to overcome the inhibitory effect of TNFa on hepatic actions of insulin. These results suggest that supraphysiological concentrations of insulin can attenuate the reduction of insulin sensitivity induced by TNFa. In agreement with these results, it was found that increasing concentration of insulin was able to reduce the inhibition of the insulin signaling cascade by TNFa [14]. It was also shown that a compensatory hyperinsulinemia, resulting from hypertrophy of pancreatic islets, maintained normoglycemia in an animal model of insulin resistance induced by obesity [9]. In addition, hepatic ketogenesis, which is inhibited by insulin in the normal state, remained suppressed by compensatory hyperinsulinemia in a state of insulin resistance [37].
Please cite this article in press as: da Rocha AF, et al. Tumor necrosis factor alpha abolished the suppressive effect of insulin on hepatic glucose production and glycogenolysis stimulated by cAMP. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2013.12.005
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It can be concluded that intravenous injection of TNFa completely abolished the suppressive action of insulin on cAMPstimulated glycogen catabolism in the perfused liver, suggesting an important role of this mechanism to the increased HGP observed in several pathological states. Conflict of interest statement The authors declare that there are no conflicts of interest.
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Funding [20]
Research supported by Fundac¸a˜o Arauca´ria. References
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Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Tumor necrosis factor alpha abolished the suppressive effect of insulin on hepatic glucose production and glycogenolysis stimulated by cAMP Aline Franco da Rocha a, Thaı´s Fernanda Liboni a, Mirian Ayumi Kurauti a, Camila Oliveira de Souza a, Daniele Romani Miksza a, Carolina Campos Lima Moreira a, Glaucia Regina Borba-Murad a, Roberto Barbosa Bazotte b, Helenir Medri de Souza a,* a b
Department of Physiological Sciences, State University of Londrina, Londrina, PR, Brazil Department of Pharmacology and Therapeutics, State University of Maringa´, Maringa´, PR, Brazil
A R T I C L E I N F O
Article history: Received 28 March 2013 Received in revised form 19 December 2013 Accepted 20 December 2013 Available online xxx Keywords: TNFa Insulin resistance Cyclic AMP Hepatic glucose production Glycogenolysis
A B S T R A C T
Background: Tumor necrosis factor alpha (TNFa) is implicated in the development of insulin resistance in obesity, type 2 diabetes and cancer. However, its ability to modulate the action of insulin on glycogen catabolism in the liver is controversial. The aim of the present study was to investigate whether TNFa acutely affects the suppression by insulin of hepatic glucose production (HGP) and glycogenolysis stimulated by cyclic adenosine monophosphate (cAMP). Methods: TNFa (10 mg/kg) was injected intravenously to rats and, 1 or 6 h later, their livers were subjected to in situ perfusion with cAMP (3 mM), in the presence or absence of physiological (20 mU/mL) or supraphysiological (500 mU/mL) concentrations of insulin. Results: The injection of TNFa, 1 or 6 h before liver perfusion, had no direct effect on the action of cAMP in stimulating HGP and glycogenolysis. However, when TNFa was injected 1 h, but not 6 h, before liver perfusion it completely abolished (p < 0.05) the suppressive effect of 20 mU/mL insulin on HGP and glycogenolysis stimulated by cAMP. Furthermore, the injection of TNFa 1 h or 6 h before liver perfusion did not influence the suppression of cAMP-stimulated HGP and glycogenolysis by 500 mU/mL insulin. Conclusion: TNFa acutely abolished the suppressive effect of physiological, but not supraphysiological, levels of insulin on HGP and glycogenolysis stimulated by cAMP, suggesting an important role of this mechanism to the increased HGP in several pathological states. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Tumor necrosis factor a (TNFa) is a multifunctional proinflammatory cytokine involved in many metabolic responses, which has been reported to act as a trigger of insulin resistance. The administration of TNFa was found to induce peripheral insulin resistance [1] in adipose tissue [2], muscle [3] and hepatocytes [4]. Consistently with this, the administration of anti-TNFa antibody or the genetic knockout of the TNFa receptor increased peripheral insulin sensitivity in the muscle and liver [5–10]. Furthermore, incubation of adipocytes, muscle cells or hepatocytes with TNFa inhibited essential steps in the insulin signal transduction pathway. In fact, TNFa increased serine phosphorylation of the insulin receptor (IR) and insulin receptor
* Corresponding author. E-mail address: [email protected] (H.M. de Souza).
substrate (IRS-1), probably by activating serine kinases [11], which reduced tyrosine phosphorylation of IR and IRS-1 stimulated by insulin [4,12–15] and prevented the activation of phosphatidylinositol 3-kinase (PI3K) [11,15,16] and protein kinase B (Akt/PKB) [3,4]. In addition, it has been shown that an increased production of TNFa is implicated in the pathogenesis of insulin resistance in type 2 diabetes and obesity and that enhanced expression of TNFa by adipose tissue plays an important role in the association between obesity and type 2 diabetes [8,14,17,18]. An increase in TNFa production seems also to be implicated in insulin resistance in cancer [19–21]. Interestingly, insulin resistance in cancer was associated with increased serum levels of TNFa [22] or increased expression of this cytokine in the muscle [19]. Despite the well established capacity of TNFa to induce insulin resistance in various pathological states, there are few studies assessing its influence on the action of insulin on glycogen catabolism in the liver, and the results are not clear. For example, there are reports
http://dx.doi.org/10.1016/j.pharep.2013.12.005 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Please cite this article in press as: da Rocha AF, et al. Tumor necrosis factor alpha abolished the suppressive effect of insulin on hepatic glucose production and glycogenolysis stimulated by cAMP. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2013.12.005
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that TNFa inhibits the suppressive effect of insulin on hepatic glucose production [1,6], whereas this inhibition was not observed in another study [8]. In view of these contradictory results, we investigated whether this cytokine acutely affects the suppressive action of insulin on HGP and glycogenolysis stimulated by cyclic adenosine monophosphate (cAMP) in rats. Materials and methods
TNFa (10 mg/kg) was administered to rats, 1 or 6 h before liver perfusion, by intravenous injection (penial vein) of 200 mL of a buffered solution (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.76 mM, KH2PO4 and 0.1% bovine serum albumin), pH 7.4, containing 2 mg TNFa. Control rats were injected with 200 mL of the buffered solution. All protocols were approved by the Ethics Committee for Animal Experimentation of the State University of Londrina.
Chemicals
Liver perfusion
Recombinant rat TNFa was purchased from Peprotech Mexico (SA de CV). Aliquots of reconstituted TNFa were stored at 20 8C and thawed immediately before the experiments. Regular insulin was purchased from Eli Lilly (Brazil) and cAMP from Sigma Chemical Co. (St. Louis, USA). All other chemicals (98–99.8% purity) were purchased from Sigma Chemical Co. (St. Louis, USA), Acros Organics (New Jersey, USA), Reagen (Rio de Janeiro, Brazil) and Merck (Darmstadt, Germany).
For the surgical procedure, rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg) and subjected to in situ liver perfusion, as previously described [23,24]. The perfusion fluid, Krebs-Henseleit buffer (KHB), pH 7.4, at 37 8C and saturated with a 95%:5% O2:CO2 mixture, was introduced into the liver (4 mL/min per gram of liver) through a cannula inserted into the portal vein, while a second cannula in the inferior vena cava was used to collect the effluent perfusate. The composition of the KHB buffer was: 115 mM NaCl, 25 mM NaHCO3, 5.8 mM KCl, 1.2 mM Na2SO4, 1.18 mM MgCl2, 1.2 mM NaH2PO4 and 2.5 mM CaCl2. cAMP and insulin were dissolved in KHB. Livers were perfused with KHB for the first 10 min, with KHB + cAMP (3 mM) between 10 and 24 min and with KHB + cAMP (3 mM) or KHB + cAMP (3 mM) + insulin (20 mU/mL or 500 mU/mL) between
Animals and administration of TNFa Male Wistar rats (200–230 g), kept at a constant temperature (23 2 8C) in a light/dark cycle of 12:12 h and fed on a standard commercial laboratory diet (Nuvilab1), were used in all experiments.
Fig. 1. Effect of tumor necrosis factor alpha (TNFa) on the suppressive action of insulin on (A) hepatic glucose production and (C) glycogenolysis stimulated by cAMP and (B, D) the corresponding areas under curves (AUCs). Livers were perfused 1 h after the administration of 10 mg/kg TNFa (TNFa - 1 h) or buffered solution (Control), as described in Materials and Methods. cAMP (3 mM) was infused from 10 to 24 min. cAMP (3 mM) or cAMP (3 mM) + insulin (20 mU/mL) were infused from 24 to 50 min. Data are mean SEM of 6–9 experiments. *p < 0.05 vs. cAMP, #p < 0.05 vs. cAMP + Ins20 (one-way ANOVA followed by Newman–Keuls test).
Please cite this article in press as: da Rocha AF, et al. Tumor necrosis factor alpha abolished the suppressive effect of insulin on hepatic glucose production and glycogenolysis stimulated by cAMP. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2013.12.005
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24 and 50 min. The effluent perfusate was collected every 2 min to measure the production of glucose, lactate and pyruvate. At the end of the perfusion, the liver was removed and weighed, to allow precise metabolic calculations and the correction of flux rates. Glycogenolysis was calculated as the sum of glucose production plus half the sum of lactate and pyruvate production [glucose + 1/2 (lactate + pyruvate)]. The areas under curves (AUCs) of glucose production and glycogenolysis were calculated for the interval of 24–50 min. Analytical procedures The concentrations of glucose [25], pyruvate [26] and lactate [27] were assayed by enzymatic methods. Statistical procedures Normal distribution and variance homogeneity were tested and one-way ANOVA, followed by the Newman–Keuls test, was employed to analyze the results. Statistical analysis was carried out with the program GraphPad Prism 4.0 and significance was accepted when p < 0.05. Data are expressed as mean standard error of the mean (SEM).
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Results The infusion of cAMP (3 mM) in the liver stimulated (p < 0.05) HGP and glycogenolysis in all groups (Figs. 1–4). There were no differences in these variables between the TNFa and control groups (Figs. 1–4). The infusion of 20 mU/mL or 500 mU/mL insulin in the liver of control rats decreased (p < 0.05) the HGP and glycogenolysis stimulated by cAMP, as shown by the AUCs (Figs. 1–4). The intravenous injection of TNFa, 1 or 6 h before liver perfusion, did not influence the stimulatory effect of cAMP on glycogen catabolism, as observed by the similarity in the rates of HGP and glycogenolysis, from 10 to 24 min of perfusion, in comparison with control rats (Figs. 1–4). On the other hand, the administration of TNFa, 1 h before liver perfusion, completely abolished (p < 0.05) the inhibitory effect of 20 mU/mL insulin on HGP (Fig. 1A and B) and glycogenolysis (Fig. 1C and D) stimulated by cAMP. However, when the TNFa was injected 6 h before liver perfusion, it did not change significantly the suppressive effect of 20 mU/mL insulin on HGP (Fig. 2A and B) or glycogenolysis (Fig. 2C and D) stimulated by cAMP. Moreover, TNFa administered 1 h before liver perfusion did not influence the suppressive effect of 500 mU/mL insulin on HGP
Fig. 2. Effect of tumor necrosis factor alpha (TNFa) on the suppressive action of insulin on (A) hepatic glucose production and (C) glycogenolysis stimulated by cAMP and (B, D) the corresponding areas under curves (AUCs). Livers were perfused 6 h after the administration of 10 mg/kg TNFa (TNFa – 6 h) or buffered solution (Control), as described in Materials and Methods Section. cAMP (3 mM) was infused from 10 to 24 min. cAMP (3 mM) or cAMP (3 mM) + insulin (20 mU/mL) were infused from 24 to 50 min. Data are mean SEM of 6–7 experiments. *p < 0.05 vs. cAMP, **p < 0.01 vs. cAMP (one-way ANOVA followed by Newman–Keuls test).
Please cite this article in press as: da Rocha AF, et al. Tumor necrosis factor alpha abolished the suppressive effect of insulin on hepatic glucose production and glycogenolysis stimulated by cAMP. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2013.12.005
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Fig. 3. Effect of tumor necrosis factor alpha (TNFa) on the suppressive action of insulin on (A) hepatic glucose production and (C) glycogenolysis stimulated by cAMP and (B, D) the corresponding areas under curves (AUCs). Livers were perfused 1 h after the administration of 10 mg/kg TNFa (TNFa – 1 h) or buffered solution (Control), as described in Materials and Methods. cAMP (3 mM) was infused from 10 to 24 min. cAMP (3 mM) or cAMP (3 mM) + insulin (500 mU/mL) were infused from 24 to 50 min. Data are mean SEM of 6–7 experiments. *p < 0.05 and **p < 0.01 vs. cAMP (one-way ANOVA followed by Newman–Keuls test).
(Fig. 3A and B) or glycogenolysis (Fig. 3C and D) stimulated by cAMP; nor did TNFa injected 6 h before liver perfusion have any effect on the inhibitory action of 500 mU/mL insulin on HGP (Fig. 4A and B) and glycogenolysis (Fig. 4C and D) stimulated by cAMP. Discussion The main question raised in this study was whether TNFa acutely affects the action of insulin on the liver glycogen catabolism stimulated by cAMP. To answer this question, the livers of fed rats were perfused in situ with cAMP in the presence or absence of physiological (20 mU/mL) or supraphysiological (500 mU/mL) insulin levels, 1 or 6 h after an intravenous injection of TNFa. This cytokine was administered intravenously because its direct infusion into the liver did not alter the HGP from alanine, while intravenous injection of TNFa decreased the HGP from this amino acid in isolated perfused livers [28]. The administration of TNFa did not influence the stimulatory effect of cAMP on glycogen catabolism (Figs. 1–4). In contrast to our study, the TNFa suppressed the dibutyryl cAMP/dexamethasone-induced increase in glucose-6-phosphatase mRNA levels by activation of nuclear factor kappaB (NFkB) [29]. The stimulation of HGP and glycogenolysis by cAMP was probably mediated by
activation of cAMP-dependent protein kinase (PKA) and subsequent phosphorylation and activation of glycogen phosphorylase [30]. Similarly, incubation of isolated rat hepatocytes with TNFa also did not influence glycogenolysis [20]. In rats that did not receive TNFa (controls), the activation of glycogen catabolism promoted by cAMP was reduced in the presence of 20 mU/mL or 500 mU/mL insulin (Figs. 1–4), confirming the direct inhibitory effect of insulin in liver perfused in situ [24,31,32]. This effect can be attributed to a reduction in the intracellular concentration of cAMP, due to the activation of phosphodiesterase-3B (PDE3B), an enzyme that promotes the hydrolysis of cAMP [33,34]. PDE3B is activated by Akt/PKB, via stimulation of PI3K associated with IRS-1 [35]. However, the administration of TNFa, 1 h before liver perfusion, completely abolished the inhibitory effect of 20 mU/mL insulin on cAMP-stimulated HGP and glycogenolysis (Fig. 1). These results reveal an in vivo acute inhibitory effect of TNFa on the hepatic response to insulin; i.e., TNFa reduced the ability of insulin to inhibit glycogen catabolism activated by cAMP. In corroboration, it has been reported that the infusion of TNFa in rats inhibited insulin-induced suppression of HGP [1]. Furthermore, in obese rats, in which the activity of TNFa was inhibited, HGP was completely suppressed during hyperinsulinemic glucose clamp [6]. However, in another
Please cite this article in press as: da Rocha AF, et al. Tumor necrosis factor alpha abolished the suppressive effect of insulin on hepatic glucose production and glycogenolysis stimulated by cAMP. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2013.12.005
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Fig. 4. Effect of tumor necrosis factor alpha (TNFa) on the suppressive action of insulin on (A) hepatic glucose production and (C) glycogenolysis stimulated by cAMP and (B, D) the corresponding areas under curves (AUCs). Livers were perfused 6 h after administration of 10 mg/kg TNFa (TNFa – 6 h) or buffered solution (Control), as described in Materials and Methods Section. cAMP (3 mM) was infused from 10 to 24 min. cAMP (3 mM) or cAMP (3 mM) + insulin (500 mU/mL) were infused from 24 to 50 min. Data are mean SEM of 6–7 experiments. *p < 0.05 and **p < 0.01 vs. cAMP (one-way ANOVA followed by Newman–Keuls test).
study with obese rats, the inhibition of TNFa did not affect the suppression of HGP mediated by insulin [8]. Also in agreement with our results, several studies have shown that TNFa inhibits the insulin signaling cascade in hepatocytes, probably via the activation of serine kinases [11]. In fact, portal infusion of TNFa increased serine phosphorylation of IRS-1 and inhibited the insulin signaling in the liver [4]. Similarly, incubation of hepatoma cells with TNFa induced serine phosphorylation of IRS-1 [13]. Further, incubation of hepatoma cells [12] or adipocytes [15] with TNFa, for 1 h, decreased tyrosine phosphorylation of IR and IRS-1 stimulated by insulin, indicating that the period of 1 h is enough for the induction of the effects of TNFa on the insulin signaling pathway. A reduction of the suppressive effect of insulin in the liver, as promoted by TNFa in the present study, results in increased HGP. Thus, increased production of TNFa may be one of the factors that contribute to the increased HGP in type 2 diabetes, obesity and cancer. Although the intravenous injection of TNFa, 1 h before the liver perfusion, completely abolished the inhibitory effect of 20 mU/mL insulin on cAMP-stimulated catabolism of glycogen, when the TNFa was administered 6 h before liver perfusion, it did not change significantly this suppressive effect of insulin on HGP or
glycogenolysis (Fig. 2), demonstrating that the effect of TNFa declines over time. In contrast to the results obtained with physiological concentrations of insulin, the administration of TNFa, either 1 h (Fig. 3) or 6 h (Fig. 4) before the liver was perfused with supraphysiological concentrations of insulin (500 mU/mL), had no effect on the insulin-mediated suppression of HGP and glycogenolysis stimulated by cAMP. Therefore, high levels of insulin, mimicking the compensatory hyperinsulinemia that occurs during insulin resistance associated with type 2 diabetes [36] and obesity [9], were necessary to overcome the inhibitory effect of TNFa on hepatic actions of insulin. These results suggest that supraphysiological concentrations of insulin can attenuate the reduction of insulin sensitivity induced by TNFa. In agreement with these results, it was found that increasing concentration of insulin was able to reduce the inhibition of the insulin signaling cascade by TNFa [14]. It was also shown that a compensatory hyperinsulinemia, resulting from hypertrophy of pancreatic islets, maintained normoglycemia in an animal model of insulin resistance induced by obesity [9]. In addition, hepatic ketogenesis, which is inhibited by insulin in the normal state, remained suppressed by compensatory hyperinsulinemia in a state of insulin resistance [37].
Please cite this article in press as: da Rocha AF, et al. Tumor necrosis factor alpha abolished the suppressive effect of insulin on hepatic glucose production and glycogenolysis stimulated by cAMP. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2013.12.005
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It can be concluded that intravenous injection of TNFa completely abolished the suppressive action of insulin on cAMPstimulated glycogen catabolism in the perfused liver, suggesting an important role of this mechanism to the increased HGP observed in several pathological states. Conflict of interest statement The authors declare that there are no conflicts of interest.
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Funding [20]
Research supported by Fundac¸a˜o Arauca´ria. References
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Please cite this article in press as: da Rocha AF, et al. Tumor necrosis factor alpha abolished the suppressive effect of insulin on hepatic glucose production and glycogenolysis stimulated by cAMP. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2013.12.005
