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Neurogenic insulin resistance in guinea-pigs with cisplatin-induced neuropathy
European Journal of Pharmacology 531 (2006) 217 – 225 www.elsevier.com/locate/ejphar
Neurogenic insulin resistance in guinea-pigs with cisplatin-induced neuropathy Judit Szilvássy a , István Sziklai a , Réka Sári b , József Németh c , Barna Peitl b , Robert Porszasz b , János Lonovics d , Zoltán Szilvássy b,⁎ b
a Department of Oto-rhino-laryngology, Medical University of Debrecen H-4032 Nagyerdei krt. 98. Debrecen, Hungary Department of Pharmacology and Pharmacotherapy, Medical University of Debrecen H-4032 Nagyerdei krt. 98. Debrecen, Hungary c Neuropharmacology Research Group of The Hungarian Academy of Sciences, Pécs, Szigeti u. 12 H-7643, Pécs, Hungary d 1st Department Medicine, University of Szeged, Hungary, H-6720 Szeged, Korányi fasor 10, Hungary
Received 28 July 2005; received in revised form 29 November 2005; accepted 12 December 2005 Available online 24 January 2006
Abstract The aim of the present work was to study whether neurotoxicity produced by cisplatin modified tissue insulin sensitivity in guinea-pigs. One week after selective sensory denervation of the anterior hepatic plexus by means of perineurial 2% capsaicin treatment, hyperinsulinaemic euglycaemic glucose clamp were performed to estimate insulin sensitivity in male guinea-pigs. The guinea-pigs underwent regional sensory denervation of the anterior hepatic plexus exhibited insulin resistance, whereas systemic capsaicin desensitization increased insulin sensitivity. Intraportal administration of L-nitro-arginine methyl esther (L-NAME decreased, whereas capsaicin increased insulin sensitivity. Neither atropine nor acetylcholine produced any significant effect. In animals with preceding regional capsaicin desensitization, none of the pharmacological maneuvers modified the resulting insulin resistant state. Cisplatin pretreatment induced sensory neuropathy and decreased insulin sensitivity. Insulin sensitivity did not change after either regional or systemic capsaicin desensitization in the cisplatin-treated animals. CGRP8–37, a nonselective calcitonin gene-related peptide (CGRP) antagonist (50 μg/kg i.v.), significantly increased insulin sensitivity in normal animals but only a tendency to insulin sensitization was seen after cisplatin treatment. Cisplatin treatment, similar to regional capsaicin desensitization of the anterior hepatic plexus, produced a significant decrease in insulin-stimulated uptake of 2-deoxy-D [L-14C] glucose in cardiac and gastrocnemius muscle with no effect on percentage suppression of endogenous glucose production by hyperinsulinaemia. We conclude that the majority of cisplatin-induced insulin resistance is related to functional deterioration of the hepatic insulin sensitizing substance (HISS) mechanism. © 2005 Elsevier B.V. All rights reserved. Keywords: Cisplatin; Sensory neuropathy; Insulin resistance
1. Introduction Recent studies by Porszasz et al. (2002) have shown that capsaicin-sensitive sensory fibers in the anterior hepatic plexus underlie the release of somatostatin or a somatostatin-like substance from the liver that enters the circulation and sensitizes peripheral tissues to the hypoglycemic effect of insulin. Since the insulin sensitizing effect was mediated by a circulating peptide derived from a restricted population of capsaicin-
⁎ Corresponding author. Tel./fax: +36 52 427 899. E-mail address: [email protected] (Z. Szilvássy). 0014-2999/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2005.12.018
sensitive sensory fibers acting at remote sites, we termed this mechanism as “sensocrine insulin sensitization” (Porszasz et al., 2003). Furthermore, Porszasz et al. proposed that the sensocrine insulin sensitizing material was identical to HISS (hepatic insulin sensitizing substance) first described by Lautt (1999), a circulating undefined substance, the release of which from the liver was shown to result from the post-prandial parasympathetic reflex activation and attributed to be responsible for a significant part of post-prandial increase in insulin sensitivity in rats (Lautt, 2004). Since both the sensory hepatic insulin sensitization pathway first published by Porszasz et al. (2002) and the HISS mechanism described by Lautt (1999) were blocked either by nitric oxide synthase inhibition or surgical cut
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of the anterior hepatic plexus fibers, we considered possible that the two mechanisms were very closely related to each other or even they might be the same (Bajza et al., 2004; Porszasz et al., 2003; Sadri and Lautt, 1999). Nevertheless, since selective regional desensitization of the anterior hepatic plexus fibers by capsaicin, a substance acting on receptors exclusively expressed on sensory fibres (Caterina et al., 1997) produced the same effect as surgical partial hepatic denervation, there was no doubt about existence of a sensory nitrergic endogenous insulin sensitizing mechanism in both rabbits and rats (Bajza et al., 2004; Porszasz et al., 2002, 2003). Moreover, since this mechanism could be either activated, for example by nitrates (Bajza et al., 2004; Lautt, 2004) or blocked by atropine (Lautt, 2003), capsaicin desensitization (Porszasz et al., 2003), and nitrate tolerance (Bajza et al., 2004), it may serve as a subject of pharmacological exploitation to find new insulin sensitizer substances. However, it may also be a source of metabolic side effects in case of drugs deteriorating anterior hepatic plexus fiber function. Cisplatin is a chemotherapeutic agent used for the treatment of several types of cancer. Unfortunately, cisplatin's therapeutic potential is limited by diverse adverse effects such as myelosuppression, nephrotoxicity, ototoxicity and neurotoxicity (Fillastre and Raguenez-Viotte, 1989; Ozols and Young, 1985; Stewart et al., 1987). The drug-induced neurotoxicity is characterized by a decrease in sensory nerve conduction velocity and a deficiency in axonal transport of sensory neuropeptides such as that of CGRP, substance P, galanin and somatostatin (Barajon et al., 1996). However, to the best of our knowledge, no studies have been conducted as to whether a sensory-effector dysfunction occurring in cisplatin-neuropathy is reflected in changes in insulin sensitivity. The present work was therefore concerned with the possibility that a neurotoxic treatment schedule with cisplatin would modify tissue insulin sensitivity as determined by hyperinsulinaemic euglycaemic glucose clamping in anaesthetized guinea-pigs. 2. Materials and methods The experiments performed in the present work conform to European Community guiding principles for the care and use of laboratory animals. The experimental protocol applied has been approved by the local ethical board of the University of Debrecen, Hungary. 2.1. Treatment groups The study was carried out with hundred and twenty eight male guinea-pigs weighing 350–400 g. They were housed in an animal room (12-h light/dark periods a day, temperature of 22–25 °C, humidity of 50–70%) with two animals per pen fed commercial laboratory chow and tap water ad libitum. The animals were randomly divided into two main experimental groups (30 animals in each): One group of animals treated with the solvent for cisplatin (1 ml isotonic NaCl) with 75 mg/kg mannitol i.p. once a day over 9 days, and another group of guinea-pigs treated with 3 mg/kg cisplatin
with 75 mg/kg mannitol i.p. once a day over 9 days. Each main group was divided into three subgroups; one subgroup served as control (14 animals), a second subgroup of animals underwent regional capsaicin desensitization of plexus hepaticus anterior fibers (16 animals), and the guinea-pigs in the third subgroup underwent systemic capsaicin desensitization (16 animals). Six animals from the two control groups were devoted to nerve conduction velocity studies. An additional group (12 guinea-pigs) was used to test the effect of an intravenous dose of 50 μg/kg CGRP8–37, a nonselective calcitonin gene-related peptide (CGRP) receptor antagonist on insulin sensitivity in animals with a preceding 9-day treatment with cisplatin (6 animals) and/or its vehicle (6 animals). In a separate group of 60 animals, we studied whether sensory nitrergic components were involved in the HISS mechanism in this species, similar to that seen in rabbits (Porszasz et al., 2002) and rats (Porszasz et al., 2003), as follows: Thirty animals underwent perineurial capsaicin desensitization, and another thirty guinea-pigs served as controls. Each group was divided into five subgroups with 6 animals in each: the animals in one of these groups were given an intraportal injection of 10 mg/kg L-NAME, a nitric oxide (NO) synthase inhibitor. A second subgroup was devoted to study the effect of a single bolus of intraportal (l mg/kg) atropine. In a third subgroup of animals, we applied 0.3 mg/kg capsaicin into the portal vein over 1 min to activate hepatic sensory fibers. These injections were applied 10 min prior to commencement of glucose clamping. The fourth subgroup was used to study the effect of acetylcholine infusion (1, 3 and 10 μg/kg/min) on insulin sensitivity. The acetylcholine infusion was applied during the steady state period of glucose clamping. 2.2. Nerve conduction velocity studies This series of experiments was carried out to verify/ exclude sensory neuropathy involving unmyelinated slow conducting ‘C’ fibers previously shown to play an important role in the HISS mechanism. Left saphenous nerve conduction velocity was determined in animals from both groups as described (Nemeth et al., 1999a,b; Szilvassy et al., 2000). In brief, in artificially ventilated animals anaesthetized with sodium pentobarbital (30 mg/kg i.p.) the nerve was prepared, cleaned of fat and adhering connective tissues and strains of square-wave (500 μs) constant voltage stimuli were applied through pairs of platinum electrodes (Experimetria UK) placed as high as possible. Another pair of electrodes was applied 2 cm distal to the stimulating electrodes for recording the summation action potentials evoked by the proximal stimulation. The time lags between stimulation and the appearance of corresponding ‘A’ and ‘C’ signals were determined for calculation of average conduction velocity. The inter-electrode distance was divided by the interval between the end of the stimulatory impulse and the appearance of the corresponding ‘A’ and ‘C’ signals (Janig and Lisney, 1989). The cisplatin-induced ‘C’ signal delay was used for characterization of cisplatin-induced sensory neuropathy.
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The animals were anaesthetized with an intraperitoneal dose of 30 mg/kg sodium pentobarbital. Bundles of anterior hepatic plexus were prepared, cleaned of fat and adhering connective tissues. Surgical sponge slices of approximately 3 mm length impregnated with 2% capsaicin solution were applied around the plexus. The abdominal cavity was closed and the perineurial sponge slices were left in their places over three days. The abdomen was then re-opened and the sponge pieces were removed. The wounds were closed and a week period of recovery was allowed to each animal. After the period of convalescence, the animals were subjected to hyperinsulinaemic euglycaemic glucose clamp studies. For control to this series of guinea-pigs served those, which received matching sponge pieces impregnated with the solvent for capsaicin (ethanol + Tween 80).
[3-3H] glucose was added to the glucose infusion, to maintain specific activity within 25% of the baseline. Blood samples were taken simultaneously with those for plasma insulin immunoreactivity determination. For determination of insulinstimulated glucose uptake by the gastrocnemius muscle, tissue samples were estimated using the administration of an intravenous bolus of 2-deoxy-D [L-14C] glucose (NEN Life Science), a non-metabolizable glucose analogue and by determining the tissue content of 2-deoxyglucose-6-phosphate. A 10 μCi bolus of 2-deoxy-D [L-14C] glucose was given in the 10th min of the steady state of glucose clamping. For determination of tissue [L-14C] 2-deoxyglucose-6-phosphate, the heart and gastrocnemius muscles were removed from each animal within 3 min after the experiments. The tissue samples were homogenized and centrifuged. The supernatants were run on an ion exchange column to separate deoxyglucose from deoxyglucose phosphate.
2.4. Systemic capsaicin desensitization
2.7. Drugs and chemicals
Capsaicin was used to elicit a selective functional deterioration of sensory ‘C’ fibers. The guinea-pigs were given capsaicin/solvent subcutaneously in the sequence of 10, 30, and 50 mg/kg single daily doses over 3 days. Capsaicin (2% w/v) was dissolved in physiological saline containing 3% v/v ethanol and 4% v/v Tween 80. The animals pre-treated with capsaicin were used for further studies after a 7-day period of recovery to avoid aspecific effects of systemic capsaicin administration as described (Ferdinandy et al., 1997).
Beyond the isotopes used, all drugs and chemicals have been purchased from Sigma (St Louis, Mo) except capsaicin that was from Fluka (Buchs, Switzerland).
2.3. Perineurial capsaicin desensitization
2.5. Hyperinsulinaemic euglycaemic glucose clamp studies Human regular insulin was infused at a constant rate (3 mU/kg, NOVO Nordisk, Copenhagen) via one of the venous catheters over 120 min. This insulin infusion rate yielded plasma insulin immunoreactivity of 100 ± 5 μU/ml in the steady state (see below). Blood samples (0.05 ml) were taken from the arterial cannula for blood glucose concentration at 10 min intervals. Blood glucose concentration was maintained constant (5.5 ± 0.5 mmol/l) by a variable rate of glucose infusion via the second venous cannula. When blood glucose had stabilized for at least 20 min, we defined this condition as steady state. This occurred within 40 min succeeding commencement of the insulin infusion. In the steady state, additional blood samples (0.3 ml) were taken for plasma insulin determination three times at 10-min intervals. The glucose infusion rate (mg/kg/min) during steady state was used to characterize insulin sensitivity (Porszasz et al., 2002). Each clamp determination was done after a preceding 24-h period of fasting. 2.6. Tissue glucose flux A continuous infusion of high performance liquid chromatography-purified [3-3H]glucose (12 μCi bolus, followed by 10 μCi; DuPont-NEN) was commenced 60 min before determination of insulin sensitivity. Subsequently, 12 μCi
2.8. Statistics The results expressed as means ± S.D. were analyzed with one-way analysis of variance followed by a modified t-test for repeated measures according to Bonferroni's method (Wallenstein et al., 1980). Changes were considered significant at P = 0.05. 3. Results 3.1. Experimental protocol Fig. 1 shows the design of insulin sensitivity determination studies. In each group, insulin sensitivity determined by hyperinsulinaemic euglycaemic glucose clamping served as the end point. The first series of experiments was to estimate the involvement of capsaicin-sensitive hepatic sensory fiber in changes in insulin sensitivity produced by cisplatin. Since the hypothesis was based on that the putative deterioration of sensory nerve function achieved by cisplatin was of importance, subgroups of 6 animals in the cisplatin- and vehicletreated groups were anaesthetized for nerve conduction velocity studies. These experiments were to confirm the presence or absence of sensory neuropathy induced by cisplatin. Determinations were done 24 h after the last cisplatin/vehicle administration as described previously (Szilvassy et al., 2000). The next series of experiments was to confirm the involvement of capsaicin-sensitive sensory fibers of the anterior hepatic plexus in the HISS mechanism in anaesthetized guineapigs, similar to that found in conscious rabbits (Porszasz et al., 2002) and anaesthetized rats (Porszasz et al., 2003). This was
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Fig. 1. Schematic drawing of the insulin sensitivity study design. In each group, whole body insulin sensitivity was determined by means of hyperinsulinaemic euglycaemic glucose clamp (HEGC). The animals in the ‘Main Group 1’ were treated by cisplatin (3 mg/kg, i.p.) and mannitol (75 mg/kg, i.p.) once a day over 9 days. A group of animals (Main Group 2) were treated with the solvent for cisplatin (1 ml isotonic NaCl) with 75 mg/kg mannitol i.p. with the same schedule as described in the ‘Main Group 1’. Each main group was divided into three subgroups; one subgroup served as control, a second subgroup of animals underwent systemic capsaicin desensitization (the guinea-pigs were given capsaicin subcutaneously in the sequence of 10, 30, and 50 mg/kg single daily doses over 3 days), and the guinea-pigs in the third subgroup underwent regional capsaicin desensitization of the fibers of anterior hepatic plexus (surgical sponge slices impregnated with 2% capsaicin solution were applied around the plexus for three days). The animals from the two control groups were devoted to nerve conduction velocity studies. An additional group was used to test the effect of an intravenous dose of 50 μg/kg CGRP8–37, a non-selective calcitonin gene-related peptide (CGRP) receptor antagonist on insulin sensitivity. The animals of ‘Main Group 3’ and ‘Main Group 4’ were devoted to study the sensory nitrergic nature of HISS mechanism in guinea-pigs. The effect of capsaicin (0.3 mg/kg/min i.po.) (subgroup b), L-NAME (10 mg/kg i.po.) (subgroup c), Atropine (1 mg/kg i.po.) (subgroup d) and Acetylcholine (1, 3, 10 μg/kg/min) (subgroup e) were determined on insulin sensitivity by HEGC. The control animals were treated with perineurial capsaicin only (subgroup a). The animals of ‘Main Group 4’ without perineurial capsaicin treatment served as corresponding controls to animals of ‘Main Group 3’.
achieved by selective deterioration of sensory fibers of the anterior hepatic plexus by means of perineurial capsaicin treatment (group 1/a) as described above. For control to this series of guinea-pigs served those, who received matching
placebo sponge slices (group 2/a). Since the deterioration of sensory fibers of the anterior hepatic plexus yielded insulin resistance (Porszasz et al., 2002), we assumed that chemical stimulation of these fibers by intraportal capsaicin (group 1/b)
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would produce an increase in insulin sensitivity using group 2/b animals as controls. In another subgroup of guinea-pigs, we intended to confirm the sensitivity of the HISS mechanism to NO synthase inhibition (group 1/c) and atropine (group 1/d) as suggested by the original observation by the group of Lautt et al. (2001). Since the HISS mechanism has been shown to involve parasympathetic components in rats (Lautt et al., 2001), a group of 6 animals was devoted to try to activate the mechanism by intraportal acetylcholine infusion (group 1/e). Group 2/c–e animals served as controls. From the above experiments, we obtained evidence for the sensory nitrergic nature of the HISS mechanism (see Results) in anaesthetized guinea-pigs. As far as the influence of sensory denervation on tissue insulin sensitivity is concerned, previous studies by Koopmans et al. (1998) revealed an increased responsiveness of peripheral tissues to the hypoglycemic effect of insulin in animals with perinatal capsaicin desensitization. This latter intervention is known to cause generalized irreversible sensory nerve damage (Jancso et al., 1977; Szolcsanyi, 1996). Therefore, to elucidate the effect of regional (anterior hepatic plexus sensory denervation) vs. systemic capsaicin desensitization, we used a systemic capsaicin desensitization protocol (group 3) 7 days preceding insulin sensitivity determinations as described (Ferdinandy et al., 1997; Szilvassy et al., 2002). This protocol induces a longlasting neuropeptide loss in sensory fibers (Szilvassy et al., 2002). Guinea-pigs pre-treated with the solvent of capsaicin served as controls (group 4). When the effect of systemic or regional capsaicin desensitization on insulin sensitivity was studied, the desensitization protocol commenced 24 h after the last cisplatin dose. Since the activation or blockade of the HISS effect is suggested to be reflected in changes in skeletal muscle glucose uptake, supplementary sets of experiments were instituted to investigate the insulin-stimulated uptake of 2-deoxy-D [L-14C] glucose, a non-metabolizable glucose analogue in samples from the heart and gastrocnemius muscle (10 tissue samples from 5 animals in each group). In these experiments, determination of tissue 2-deoxy-D [L-14C] glucose-6 phosphate content was the end point. Rates of basal glucose turnover and whole body glucose uptake at the end of the steady state of the clamp period were calculated by the use of a modified form of Steele's equation, taking the extra tracer glucose into account. Endogenous glucose production rate was determined by subtracting the “during clamp” glucose infusion rate from the whole body glucose uptake.
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3.3. Body weight and rectal temperature Body weight decreased from pre-treatment value of 362 ± 33 to 312 ± 21 g (P ≤ 0.05) in the cisplatin-treated animals. The ‘control’ guinea-pigs did not exhibit any significant change in body weight during the treatment period. Neither regional, nor systemic capsaicin desensitization caused any further significant change in body weight in either group. Rectal temperature did not change in animals in either main group. However, a period of transient increase in rectal temperature occurred in animals with systemic capsaicin desensitization, the duration of which did not exceed four days. 3.4. Nerve conduction velocity Fig. 2 shows that cisplatin, at the treatment schedule applied, produced a significant decrease in nerve conduction velocity in slow conducting unmyelinated ‘C’ fibres. 3.5. Changes in insulin sensitivity The cisplatin treatment applied significantly decreased insulin sensitivity as determined by hyperinsulinaemic (100 μU/ml) euglycaemic (5.5 mmol/l) glucose clamping in anaesthetized guinea-pigs. Regional desensitization with 2% capsaicin solution of the sensory fibres of the anterior hepatic plexus, known to underlie the HISS mechanism, decreased; whereas systemic capsaicin desensitization increased insulin sensitivity. Nevertheless, the decreased insulin sensitivity changed neither after regional (anterior hepatic plexus) nor succeeding systemic desensitization by capsaicin in cisplatin treated animals (Fig. 3).
3.2. Exclusions Seven cisplatin-treated animals had to be excluded from the experiments, four of them as cisplatin failed to produce any decrease in nerve conduction velocity in either ‘A’ or ‘C’ fibers, one because of respiratory insufficiency due to pneumonia and two because of the development of extended skin lesions. (Exclusions are not involved in the number of animals consumed by the study).
Fig. 2. Cisplatin-induced decrease in nerve conduction velocity in slow conducting unmyelinated C fibers of the femoral nerve. Measurements were done 24 h after a series of 9 intraperitoneal injections of cisplatin (3 mg/kg) and/ or its vehicle once a day. The data are means? S.D. obtained with 6 animals per group. ⁎: significantly different from control at P ≤ 0.05.
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Fig. 3. Effects of systemic and regional capsaicin desensitization of the anterior hepatic plexus (AHP capsaicin) on insulin sensitivity in anaesthetized guineapigs with a preceding 9-day treatment with cisplatin (3 mg/kg/day) and/or its vehicle. Systemic desensitization (Systemic capsaicin) was induced by subcutaneous injection of 10, 30 and 50 mg/kg capsaicin in three consecutive days. Regional desensitization was induced by wrapping anterior hepatic plexus fibers up with “Gelaspon” sponges impregnated with 2% capsaicin solution. The data are expressed as means ± S.D. obtained with 8 animals per group. ⁎ P ≤ 0.05 vs. control; + : P ≤ 0.05 vs. vehicle.
Fig. 5. Effects of perineurial treatment of the anterior hepatic plexus with capsaicin/its solvent on insulin sensitivity in anaesthetized rats and the modifying effects of L-NAME (hatched column), atropine (crosshatched column), or infusion of capsaicin (black column), acetylcholine (grey columns) into the portal vein. Capsaicin was applied as perineurial sponges impregnated with 2% capsaicin solution. The data are expressed as means ± S.D. obtained with 6 animals per group.⁎ P ≤ 0.05.
pharmacological maneuvers modified the resulting insulin resistant state (Fig. 5). CGRP8–37, a non-selective CGRP receptor antagonist at an intravenous dose of 50 μg/kg, significantly increased insulin sensitivity in normal animals but it failed to attain a significant change in this parameter after the 9-day treatment with cisplatin (Fig. 4). 3.6. Effect of intraportal L-NAME, capsaicin, atropine and acetylcholine on insulin sensitivity with and without preceding regional capsaicin desensitization Intraportal administration of L-NAME (10 mg/kg) decreased, whereas capsaicin (0.3 mg/kg over 1 min) increased insulin sensitivity. Neither atropine (1 mg/kg) nor acetylcholine (1–10 μg/kg/min) produced any significant effect. In animals with preceding regional capsaicin desensitization, none of the
Fig. 4. Effect of an intravenous dose of CGRP8–37 (50 μ g/kg) on insulin sensitivity in anaesthetized guinea-pigs with a preceding 9-day treatment with cisplatin (3 mg/kg/day) and/or its vehicle. The data are expressed as means ± S.D. obtained with 8 animals per group. ⁎ P ≤ 0.05 vs. control; + : P ≤ 0.05 vs. vehicle.
3.7. Effect of cisplatin neuropathy, partial hepatic sensory denervation, intraportal L-NAME or atropine on percentage suppression from basal of endogenous glucose production at hyperinsulinaemic euglycaemic glucose clamping The applied insulin plus glucose infusion as shown by Fig. 6, induced complete inhibition of endogenous glucose production during the steady state. Neither cisplatin neuropathy nor partial hepatic sensory denervation affected this inhibition. However, both intraportal L-NAME and atropine significantly attenuated the ‘steady state’ reduction of endogenous glucose production.
Fig. 6. Suppression from baseline of endogenous glucose production (EGP) by cisplatin neuropathy, perineurial capsaicin desensitization of the AHP fibres, intraportal L-NAME and atropine during hyperinsulinaemic euglycaemic glucose clamping in anaesthetized guinea-pigs. The data are means ± standard deviation. ⁎ significantly different from basal values at P ≤ 0.05.
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Fig. 7. 2-deoxy-D [L-14C] glucose uptake by cardiac and gastrocnemius muscle tissues during hyperinsulinaemic euglycaemic glucose clamping. In anaesthetized guinea-pigs. Cisplatin neuropathy, perineurial capsaicin desensitization of the AHP fibers, intraportal L-NAME and atropine. The data are means ± standard deviation. ⁎ significantly different from basal values at P ≤ 0.05.
3.8. Effect of cisplatin neuropathy, partial hepatic sensory denervation, intraportal L-NAME or atropine on cardiac and skeletal muscle 2-deoxy-D [L-14C] glucose uptake Insulin-stimulated glucose uptake was more than four times higher in cardiac tissue than in samples from the gastrocnemius muscle. It was significantly decreased by cisplatin neuropathy, partial hepatic sensory denervation and intraportal L-NAME in samples from either tissue. Atropine at the dose applied was without effect (Fig. 7). 4. Discussion These results confirm our previous finding that capsaicinsensitive sensory fibers in the anterior hepatic plexus are involved in endogenous insulin sensitization. This is suggested by the data that selective deterioration of these fibers by perineurial capsaicin yields insulin resistance in guinea-pigs, similar to that found in rabbits (Porszasz et al., 2002). The results also show that systemic capsaicin desensitization causes a significant increase in sensitivity to the hypoglycemic effect of insulin, an effect similar to that found by Koopmans's group (Koopmans et al., 1998) in conscious rats with neonatal deafferentation of capsaicin-sensitive sensory nerves. Nevertheless, the major original finding of this paper is that subacute treatment with cisplatin at a dose producing deterioration of ‘C’ fiber function, results in insulin resistance. Moreover, since neither regional nor systemic capsaicin desensitization could modify insulin sensitivity in cisplatin-treated animals, it is suggested that the predominant mechanism of cisplatin-induced insulin resistance is an impairment of the sensory effector function of fibers of the anterior hepatic plexus that underlie the HISS mechanism (Porszasz et al., 2002). This work in part is an extension of our previous results obtained in rabbits and rats that sensory fibers in the anterior hepatic play a crucial role in the regulation of insulin sensitivity
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(Porszasz et al., 2002). Based on the observation that blockade of NO synthesis by low-dose intraportal L-NAME significantly attenuated the HISS effect, the sensory nitrergic nature of the HISS mechanism has now been confirmed in three species i.e. in rabbits (Porszasz et al., 2002), rats (Porszasz et al., 2003) and, according to the present results, in guinea-pigs. That nerves in the anterior hepatic plexus play a crucial role in the regulation of post-prandial increase in insulin sensitivity in rats was first described in the pioneer works by Lautt's group (Sadri et al., 1997; Xie and Lautt, 1995, 1996) This discovery was then principally confirmed by Moore et al., (Moore et al., 2002) who found that chronic partial hepatic denervation induced insulin resistance in dogs (Moore et al., 2002). However, the latter group carried out insulin sensitivity tests 16 days after hepatic denervation, whereas Lautt et al. found acute hepatic denervation to produce insulin resistance in rats (Lautt et al., 2001). According to the original concept by Lautt's group, the insulin sensitizing mechanism linked to functional integrity of the anterior hepatic plexus fibers worked as follows: An insulin sensitizing parasympathetic reflex was activated in the liver in response to post-prandial hyperinsulinaemia, the atypical efferent pathway of which involved the release of HISS, an unidentified hormone-like substance that increased the sensitivity of peripheral tissue (predominantly the skeletal muscle) to the hypoglycemic effect of insulin (Lautt et al., 2001; Sadri et al., 1997; Szolcsanyi et al., 1998; Xie et al., 1993; Xie and Lautt, 1994, 1996). This mechanism was shown to substantially involve nitrergic components, since various NO synthase inhibitors apparently induced insulin resistance, and intraportal administration of NO donors restored insulin sensitivity in such animals (Sadri and Lautt, 1999). As shown from the results in Fig. 2, neither atropine nor intraportal acetylcholine affected insulin sensitivity in guinea-pigs, similar to that recently described by Porszasz et al. (Porszasz et al., 2003) in rats. Theoretically, the discrepancy may in part be explained by a difference in the way of determining insulin sensitivity, as Lautt's group used the rapid insulin sensitivity test termed RIST (Lautt et al., 1998) to characterize changes in insulin sensitivity, whereas we remained with using the hyperinsulinaemic euglycaemic glucose clamping, the gold standard of determining insulin sensitivity in whole animals (DeFronzo et al., 1979). The RIST method utilizes hyperinsulinaemia produced by a short term insulin infusion (usually 5 min), the hypoglycemic effect of which is compensated by a succeeding longer lasting glucose infusion (Lautt et al., 1998). During the hyperinsulinaemic clamp method, however, plasma insulin immunoreactivity is maintained at a level approximately 10 times higher (100 μU/ml) than corresponding fasting values confirmed by succeeding radioimmunoassay determinations and the hypoglycemic effect of continuous hyperinsulinaemia is opposed by continuous glucose infusion. Assuming that the HISS mechanism is activated by hyperinsulinaemia, it is possible that a much higher degree of the HISS mechanism activation is achieved by hyperinsulinaemic glucose clamping (because of the long-lasting stable hyperinsulinaemia) than that attained at the RIST method by preceding exposure to food. This may serve as an explanation for the lack of effect of either
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acetylcholine infusion or electrical stimulation of peripheral anterior hepatic plexus fibers subsequent to an acute nerve cut to further increase insulin sensitivity in rats (Porszasz et al., 2003). The present results clearly show that neither regional capsaicin nor cisplatin neuropathy influenced insulin-induced suppression of endogenous glucose production with a strong inhibitory effect on insulin-stimulated 2-deoxy-D [L- 14 C] glucose uptake. Therefore, assuming that the HISS mechanism is activated in the liver and acts on skeletal muscle (Lautt, 2003), the insulin resistant state associated with cisplatininduced sensory neuropathy seems to be very similar to that suggested for HISS deficiency. The cisplatin-treated guinea-pigs exhibited characteristic neurophysiological features of sensory neuropathy at level of unmyelinated “C” fibers, reflected in a significant decrease in conduction velocity in slow conducting sensory nerve fibers. However, it has been found that the dorsal root ganglia cells serve as the primary targets for cisplatin to induce peripheral neuropathy (Barajon et al., 1996; Cavaletti et al., 1998; Gispen et al., 1992). It is therefore not surprising that sensory neuropathy highlights neurotoxicity produced by cisplatin (Gispen et al., 1992; Tredici et al., 1994). Neuromorphologic studies by Barajon and his co-workers (Barajon et al., 1996) on cisplatin-induced changes revealed an accumulation of sensory neuropeptides (CGRP, substance P, galanin and somatostatin) in dorsal root ganglia with much more severe histological alterations in ganglionic cells than those seen in peripheral fibers. These studies also suggested an impaired axonal transport of sensory neuropeptides by cisplatin. Previous studies elucidated the sensory effector nature of the HISS mechanism with the involvement of nitrergic pathways (Lautt, 2003; Porszasz et al., 2002). It has also been shown that the anterior hepatic plexus contains a significant amount of capsaicinsensitive peptidergic sensory nerve fibers, a significant part of which derives from dorsal root ganglia, reaching the liver with vagal efferents (Miao et al., 1997; Erin et al., 2000). If this latter were true, the neurotoxic doses of cisplatin through causing a deficiency in axonal transport of sensory neuropeptides, might have caused changes in insulin sensitivity similar to that characteristic of a transient neuropeptide loss produced by repetitive capsaicin doses (Ferdinandy et al., 1997; Szolcsanyi, 1996). However, the effect of systemic capsaicin desensitization on insulin sensitivity was opposite to that attained by cisplatin, whereas sensory neuropathy induced by the subacute intraperitoneal cisplatin treatment schedule, produced changes similar to that seen after regional sensory denervation of the anterior hepatic plexus by capsaicin. This might suggest the dominant toxic influence of intraperitoneal cisplatin doses on sensory nerves supplying the liver, however, the same cisplatin treatment schedule has been shown to cause severe alterations in bronchomotility as well (Szilvassy et al., 2000). That systemic capsaicin desensitization yields an increase in insulin sensitivity through elimination of CGRP, an insulin antagonist from sensory nerve terminals was elegantly demonstrated by Koopmans et al. (Koopmans et al., 1998). Indeed, CGRP8–37, a non-selective CGRP receptor antagonist, at the dose applied (Peitl et al., 1999) was found to increase insulin
sensitivity in our model as well. Nevertheless, the CGRP receptor antagonist failed to amend insulin resistance attained by cisplatin, similarly to systemic capsaicin desensitization applied after the cisplatin treatment period. Taken these findings together, it is suggested that insulin resistance deriving from cisplatin-induced sensory neuropathy is predominantly underlain by an impairment of the HISS mechanism. Whatever the precise mechanism, the results call attention to the possibility that insulin resistance seen in patients with several types of cancer (Yoshikawa et al., 2001) may be accentuated by anticancer therapy per se producing sensory neuropathy. Since insulin resistance significantly contributes to cancer cachexia (Pisters et al., 1992), insulin sensitization might be an important adjunct in cancer chemotherapy (Benko et al., 2003). Acknowledgement This work was supported by grants from the Hungarian Ministry of Education (OTKA T032002, TT042266, D45992 and T043467; ETT 6003/1/200, 03597/2003 and 585/2003), the National Research and Development Fund (NRDP 1/007/ 2001), the Hungarian–German academic grant MÖB-DAAD 1627/2002, a grant from a Higher Education Fund (FKFP 2000–2003), OMFB ALK-00190/2002 and the Association of Innovative Pharmaceutical Manufacturers. The technical assistance by Miss Susanna Koszorus is greatly acknowledged. Robert Porszasz MD, PhD is supported by the Hungarian Academy of Sciences (Janos Bolyai Postdoctoral Fellowship). The work by Barna Peitl MD, PhD was supported by a postdoctoral OTKA fellowship. References Bajza, A., Peitl, B., Nemeth, J., Porszasz, R., Rabloczky, G., Literati-Nagy, P., Szilvassy, J., Szilvassy, Z., 2004. Development of insulin resistance by nitrate tolerance in conscious rabbits. J. Cardiovasc. Pharmacol. 43, 471–476. Barajon, I., Bersani, M., Quartu, M., Del Fiacco, M., Cavaletti, G., Holst, J.J., Tredici, G., 1996. Neuropeptides and morphological changes in cisplatin-induced dorsal root ganglion neuronopathy. Exp. Neurol. 138, 93–104. Benko, I., Djazayeri, K., Abraham, C., Zsuga, J., Szilvassy, Z., 2003. Rosiglitazone-induced protection against myelotoxicity produced by 5fluorouracil. Eur. J. Pharmacol. 477, 179–182. Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., Levine, J.D., Julius, D., 1997. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389, 816–824. Cavaletti, G., Fabbrica, D., Minoia, C., Frattola, L., Tredici, G., 1998. Carboplatin toxic effects on the peripheral nervous system of the rat. Ann. Oncol. 9, 443–447. DeFronzo, R.A., Tobin, J.D., Andres, R., 1979. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am. J. Physiol. 237, E214–E223. Erin, N., Ercan, F., Yegen, B.C., Arbak, S., Okar, I., Oktay, S., 2000. Role of capsaicin-sensitive nerves in gastric and hepatic injury induced by coldrestraint stress. Dig. Dis. Sci. 45, 1889–1899. Ferdinandy, P., Csont, T., Csonka, C., Torok, M., Dux, M., Nemeth, J., Horvath, L.I., Dux, L., Szilvassy, Z., Jancso, G., 1997. Capsaicin-sensitive local sensory innervation is involved in pacing-induced preconditioning in rat hearts: role of nitric oxide and CGRP? Naunyn-Schmiedeberg's Arch. Pharmacol. 356, 356–363.
J. Szilvássy et al. / European Journal of Pharmacology 531 (2006) 217–225 Fillastre, J.P., Raguenez-Viotte, G., 1989. Cisplatin nephrotoxicity. Toxicol. Lett. 46, 163–175. Gispen, W.H., Hamers, F.P., Vecht, C.J., Jennekens, F.G., Neyt, J.P., 1992. ACTH/MSH like peptides in the treatment of cisplatin neuropathy. J. Steroid Biochem. Mol. Biol. 43, 179–183. Jancso, G., Kiraly, E., Jancso-Gabor, A., 1977. Pharmacologically induced selective degeneration of chemosensitive primary sensory neurones. Nature 270, 741–743. Janig, W., Lisney, S.J., 1989. Small diameter myelinated afferents produce vasodilatation but not plasma extravasation in rat skin. J. Physiol. 415, 477–486. Koopmans, S.J., Leighton, B., DeFronzo, R.A., 1998. Neonatal de-afferentation of capsaicin-sensitive sensory nerves increases in vivo insulin sensitivity in conscious adult rats. Diabetologia 41, 813–820. Lautt, W.W., 1999. The HISS story overview: a novel hepatic neurohumoral regulation of peripheral insulin sensitivity in health and diabetes. Can. J. Physiol. Pharm. 77, 553–562. Lautt, W.W., 2003. Practice and principles of pharmacodynamic determination of HISS-dependent and HISS-independent insulin action: methods to quantitate mechanisms of insulin resistance. Med. Res. Rev. 23, 1–14. Lautt, W.W., 2004. A new paradigm for diabetes and obesity: the hepatic insulin sensitizing substance (HISS) hypothesis. J. Pharmacol. Sci. 95, 9–17. Lautt, W.W., Wang, X., Sadri, P., Legare, D.J., Macedo, M.P., 1998. Rapid insulin sensitivity test (RIST). Can. J. Physiol. Pharm. 76, 1080–1086. Lautt, W.W., Macedo, M.P., Sadri, P., Takayama, S., Duarte, R.F., Legare, D.J., 2001. Hepatic parasympathetic (HISS) control of insulin sensitivity determined by feeding and fasting. Am. J. Physiol. Gasterointest. Liver Physiol. 281, G29–G36. Miao, F.J., Janig, W., Levine, J.D., 1997. Vagal branches involved in inhibition of bradykinin-induced synovial plasma extravasation by intrathecal nicotine and noxious stimulation in the rat. J. Physiol. 498 (Pt 2), 473–481. Moore, M.C., Satake, S., Baranowski, B., Hsieh, P.S., Neal, D.W., Cherrington, A.D., 2002. Effect of hepatic denervation on peripheral insulin sensitivity in conscious dogs. Am. J. Physiol. Endocrinol. Metab. 282, E286–E296. Nemeth, J., Szilvassy, Z., Than, M., Oroszi, G., Sari, R., Szolcsanyi, J., 1999a. Decreased sensory neuropeptide release from trachea of rats with streptozotocin-induced diabetes. Eur. J. Pharmacol. 369, 221–224. Nemeth, J., Than, M., Sari, R., Peitl, B., Oroszi, G., Farkas, B., Szolcsanyi, J., Szilvassy, Z., 1999b. Impairment of neurogenic inflammatory and antiinflammatory responses in diabetic rats. Eur. J. Pharmacol. 386, 83–88. Ozols, R.F., Young, R.C., 1985. High-dose cisplatin therapy in ovarian cancer. Semin. Oncol. 12, 21–30. Peitl, B., Petho, G., Porszasz, R., Nemeth, J., Szolcsanyi, J., 1999. Capsaicininsensitive sensory-efferent meningeal vasodilatation evoked by electrical stimulation of trigeminal nerve fibres in the rat. Br. J. Pharmacol. 127, 457–467. Pisters, P.W., Cersosimo, E., Rogatko, A., Brennan, M.F., 1992. Insulin action on glucose and branched-chain amino acid metabolism in cancer cachexia: differential effects of insulin. Surgery 111, 301–310. Porszasz, R., Legvari, G., Nemeth, J., Literati, P.N., Szolcsanyi, J., Szilvassy, Z., 2002. The sensory nitrergic nature of the hepatic insulin sensitizing substance mechanism in conscious rabbits. Eur. J. Pharmacol. 443, 211–212.
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Porszasz, R., Legvari, G., Pataki, T., Szilvassy, J., Nemeth, J., Kovacs, P., Paragh, G., Szolcsanyi, J., Szilvassy, Z., 2003. Hepatic insulin sensitizing substance: a novel ‘sensocrine’ mechanism to increase insulin sensitivity in anaesthetized rats. Br. J. Pharmacol. 139, 1171–1179. Sadri, P., Lautt, W.W., 1999. Blockade of hepatic nitric oxide synthase causes insulin resistance. Am. J. Physiol. 277, G101–G108. Sadri, P., Legare, D.J., Lautt, W.W., 1997. Insulin resistance caused by nitric oxide synthase inhibition. Proc. West. Pharmacol. Soc. 40, 19–20. Stewart, D.J., Verma, S., Maroun, J.A., 1987. Phase I study of the combination of disulfiram with cisplatin. Am. J. Clin. Oncol. 10, 517–519. Szilvassy, J., Sziklai, I., Racz, T., Horvath, P., Rabloczky, G., Szilvassy, Z., 2000. Impaired bronchomotor responses to field stimulation in guinea-pigs with cisplatin-induced neuropathy. Eur. J. Pharmacol. 403, 259–265. Szilvassy, J., Sziklai, I., Horvath, P., Szilasi, M., Nemeth, J., Kovacs, P., Szilvassy, Z., 2002. Feeble bronchomotor responses in diabetic rats in association with decreased sensory neuropeptide release. Am. J. Physiol., Lung Cell. Mol. Physiol. 282, L1023–L1030. Szolcsanyi, J., 1996. Capsaicin-sensitive sensory nerve terminals with local and systemic efferent functions: facts and scopes of an unorthodox neuroregulatory mechanism. Prog. Brain Res. 113, 343–359. Szolcsanyi, J., Helyes, Z., Oroszi, G., Nemeth, J., Pinter, E., 1998. Release of somatostatin and its role in the mediation of the anti-inflammatory effect induced by antidromic stimulation of sensory fibres of rat sciatic nerve. Br. J. Pharmacol. 123, 936–942. Tredici, G., Cavaletti, G., Petruccioli, M.G., Fabbrica, D., Tedeschi, M., Venturino, P., 1994. Low-dose glutathione administration in the prevention of cisplatin-induced peripheral neuropathy in rats. Neurotoxicology 15, 701–704. Wallenstein, S., Zucker, C.L., Fleiss, J.L., 1980. Some statistical methods useful in circulation research. Circ. Res. 47, 1–9. Xie, H., Lautt, W.W., 1994. Insulin resistance produced by hepatic denervation or muscarinic cholinergic blockade. Proc. West. Pharmacol. Soc. 37, 39–40. Xie, H., Lautt, W.W., 1995. Induction of insulin resistance by cholinergic blockade with atropine in the cat. J. Auton. Pharm. 15, 361–369. Xie, H., Lautt, W.W., 1996. Insulin resistance caused by hepatic cholinergic interruption and reversed by acetylcholine administration. Am. J. Physiol. 271, E587–E592. Xie, H., Tsybenko, V.A., Johnson, M.V., Lautt, W.W., 1993. Insulin resistance of glucose response produced by hepatic denervations. Can. J. Physiol. Pharm. 71, 175–178. Yoshikawa, T., Noguchi, Y., Doi, C., Makino, T., Nomura, K., 2001. Insulin resistance in patients with cancer: relationships with tumor site, tumor stage, body-weight loss, acute-phase response, and energy expenditure. Nutrition 17, 590–593.
Further reading Guimaraes, A.P., Guimaraes, F.S., Prado, W.A., 2000. Modulation of carbachol-induced antinociception from the rat periaqueductal gray. Brain Res. Bull. 518 471–478. Migliore, M., Giuliano, R., Aziz, T., Saad, R.A., Sgalambro, F., 2002. Fourstep local anesthesia and sedation for thoracoscopic diagnosis and management of pleural diseases. Chest 1218 2032–2035.
Neurogenic insulin resistance in guinea-pigs with cisplatin-induced neuropathy Judit Szilvássy a , István Sziklai a , Réka Sári b , József Németh c , Barna Peitl b , Robert Porszasz b , János Lonovics d , Zoltán Szilvássy b,⁎ b
a Department of Oto-rhino-laryngology, Medical University of Debrecen H-4032 Nagyerdei krt. 98. Debrecen, Hungary Department of Pharmacology and Pharmacotherapy, Medical University of Debrecen H-4032 Nagyerdei krt. 98. Debrecen, Hungary c Neuropharmacology Research Group of The Hungarian Academy of Sciences, Pécs, Szigeti u. 12 H-7643, Pécs, Hungary d 1st Department Medicine, University of Szeged, Hungary, H-6720 Szeged, Korányi fasor 10, Hungary
Received 28 July 2005; received in revised form 29 November 2005; accepted 12 December 2005 Available online 24 January 2006
Abstract The aim of the present work was to study whether neurotoxicity produced by cisplatin modified tissue insulin sensitivity in guinea-pigs. One week after selective sensory denervation of the anterior hepatic plexus by means of perineurial 2% capsaicin treatment, hyperinsulinaemic euglycaemic glucose clamp were performed to estimate insulin sensitivity in male guinea-pigs. The guinea-pigs underwent regional sensory denervation of the anterior hepatic plexus exhibited insulin resistance, whereas systemic capsaicin desensitization increased insulin sensitivity. Intraportal administration of L-nitro-arginine methyl esther (L-NAME decreased, whereas capsaicin increased insulin sensitivity. Neither atropine nor acetylcholine produced any significant effect. In animals with preceding regional capsaicin desensitization, none of the pharmacological maneuvers modified the resulting insulin resistant state. Cisplatin pretreatment induced sensory neuropathy and decreased insulin sensitivity. Insulin sensitivity did not change after either regional or systemic capsaicin desensitization in the cisplatin-treated animals. CGRP8–37, a nonselective calcitonin gene-related peptide (CGRP) antagonist (50 μg/kg i.v.), significantly increased insulin sensitivity in normal animals but only a tendency to insulin sensitization was seen after cisplatin treatment. Cisplatin treatment, similar to regional capsaicin desensitization of the anterior hepatic plexus, produced a significant decrease in insulin-stimulated uptake of 2-deoxy-D [L-14C] glucose in cardiac and gastrocnemius muscle with no effect on percentage suppression of endogenous glucose production by hyperinsulinaemia. We conclude that the majority of cisplatin-induced insulin resistance is related to functional deterioration of the hepatic insulin sensitizing substance (HISS) mechanism. © 2005 Elsevier B.V. All rights reserved. Keywords: Cisplatin; Sensory neuropathy; Insulin resistance
1. Introduction Recent studies by Porszasz et al. (2002) have shown that capsaicin-sensitive sensory fibers in the anterior hepatic plexus underlie the release of somatostatin or a somatostatin-like substance from the liver that enters the circulation and sensitizes peripheral tissues to the hypoglycemic effect of insulin. Since the insulin sensitizing effect was mediated by a circulating peptide derived from a restricted population of capsaicin-
⁎ Corresponding author. Tel./fax: +36 52 427 899. E-mail address: [email protected] (Z. Szilvássy). 0014-2999/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2005.12.018
sensitive sensory fibers acting at remote sites, we termed this mechanism as “sensocrine insulin sensitization” (Porszasz et al., 2003). Furthermore, Porszasz et al. proposed that the sensocrine insulin sensitizing material was identical to HISS (hepatic insulin sensitizing substance) first described by Lautt (1999), a circulating undefined substance, the release of which from the liver was shown to result from the post-prandial parasympathetic reflex activation and attributed to be responsible for a significant part of post-prandial increase in insulin sensitivity in rats (Lautt, 2004). Since both the sensory hepatic insulin sensitization pathway first published by Porszasz et al. (2002) and the HISS mechanism described by Lautt (1999) were blocked either by nitric oxide synthase inhibition or surgical cut
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of the anterior hepatic plexus fibers, we considered possible that the two mechanisms were very closely related to each other or even they might be the same (Bajza et al., 2004; Porszasz et al., 2003; Sadri and Lautt, 1999). Nevertheless, since selective regional desensitization of the anterior hepatic plexus fibers by capsaicin, a substance acting on receptors exclusively expressed on sensory fibres (Caterina et al., 1997) produced the same effect as surgical partial hepatic denervation, there was no doubt about existence of a sensory nitrergic endogenous insulin sensitizing mechanism in both rabbits and rats (Bajza et al., 2004; Porszasz et al., 2002, 2003). Moreover, since this mechanism could be either activated, for example by nitrates (Bajza et al., 2004; Lautt, 2004) or blocked by atropine (Lautt, 2003), capsaicin desensitization (Porszasz et al., 2003), and nitrate tolerance (Bajza et al., 2004), it may serve as a subject of pharmacological exploitation to find new insulin sensitizer substances. However, it may also be a source of metabolic side effects in case of drugs deteriorating anterior hepatic plexus fiber function. Cisplatin is a chemotherapeutic agent used for the treatment of several types of cancer. Unfortunately, cisplatin's therapeutic potential is limited by diverse adverse effects such as myelosuppression, nephrotoxicity, ototoxicity and neurotoxicity (Fillastre and Raguenez-Viotte, 1989; Ozols and Young, 1985; Stewart et al., 1987). The drug-induced neurotoxicity is characterized by a decrease in sensory nerve conduction velocity and a deficiency in axonal transport of sensory neuropeptides such as that of CGRP, substance P, galanin and somatostatin (Barajon et al., 1996). However, to the best of our knowledge, no studies have been conducted as to whether a sensory-effector dysfunction occurring in cisplatin-neuropathy is reflected in changes in insulin sensitivity. The present work was therefore concerned with the possibility that a neurotoxic treatment schedule with cisplatin would modify tissue insulin sensitivity as determined by hyperinsulinaemic euglycaemic glucose clamping in anaesthetized guinea-pigs. 2. Materials and methods The experiments performed in the present work conform to European Community guiding principles for the care and use of laboratory animals. The experimental protocol applied has been approved by the local ethical board of the University of Debrecen, Hungary. 2.1. Treatment groups The study was carried out with hundred and twenty eight male guinea-pigs weighing 350–400 g. They were housed in an animal room (12-h light/dark periods a day, temperature of 22–25 °C, humidity of 50–70%) with two animals per pen fed commercial laboratory chow and tap water ad libitum. The animals were randomly divided into two main experimental groups (30 animals in each): One group of animals treated with the solvent for cisplatin (1 ml isotonic NaCl) with 75 mg/kg mannitol i.p. once a day over 9 days, and another group of guinea-pigs treated with 3 mg/kg cisplatin
with 75 mg/kg mannitol i.p. once a day over 9 days. Each main group was divided into three subgroups; one subgroup served as control (14 animals), a second subgroup of animals underwent regional capsaicin desensitization of plexus hepaticus anterior fibers (16 animals), and the guinea-pigs in the third subgroup underwent systemic capsaicin desensitization (16 animals). Six animals from the two control groups were devoted to nerve conduction velocity studies. An additional group (12 guinea-pigs) was used to test the effect of an intravenous dose of 50 μg/kg CGRP8–37, a nonselective calcitonin gene-related peptide (CGRP) receptor antagonist on insulin sensitivity in animals with a preceding 9-day treatment with cisplatin (6 animals) and/or its vehicle (6 animals). In a separate group of 60 animals, we studied whether sensory nitrergic components were involved in the HISS mechanism in this species, similar to that seen in rabbits (Porszasz et al., 2002) and rats (Porszasz et al., 2003), as follows: Thirty animals underwent perineurial capsaicin desensitization, and another thirty guinea-pigs served as controls. Each group was divided into five subgroups with 6 animals in each: the animals in one of these groups were given an intraportal injection of 10 mg/kg L-NAME, a nitric oxide (NO) synthase inhibitor. A second subgroup was devoted to study the effect of a single bolus of intraportal (l mg/kg) atropine. In a third subgroup of animals, we applied 0.3 mg/kg capsaicin into the portal vein over 1 min to activate hepatic sensory fibers. These injections were applied 10 min prior to commencement of glucose clamping. The fourth subgroup was used to study the effect of acetylcholine infusion (1, 3 and 10 μg/kg/min) on insulin sensitivity. The acetylcholine infusion was applied during the steady state period of glucose clamping. 2.2. Nerve conduction velocity studies This series of experiments was carried out to verify/ exclude sensory neuropathy involving unmyelinated slow conducting ‘C’ fibers previously shown to play an important role in the HISS mechanism. Left saphenous nerve conduction velocity was determined in animals from both groups as described (Nemeth et al., 1999a,b; Szilvassy et al., 2000). In brief, in artificially ventilated animals anaesthetized with sodium pentobarbital (30 mg/kg i.p.) the nerve was prepared, cleaned of fat and adhering connective tissues and strains of square-wave (500 μs) constant voltage stimuli were applied through pairs of platinum electrodes (Experimetria UK) placed as high as possible. Another pair of electrodes was applied 2 cm distal to the stimulating electrodes for recording the summation action potentials evoked by the proximal stimulation. The time lags between stimulation and the appearance of corresponding ‘A’ and ‘C’ signals were determined for calculation of average conduction velocity. The inter-electrode distance was divided by the interval between the end of the stimulatory impulse and the appearance of the corresponding ‘A’ and ‘C’ signals (Janig and Lisney, 1989). The cisplatin-induced ‘C’ signal delay was used for characterization of cisplatin-induced sensory neuropathy.
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The animals were anaesthetized with an intraperitoneal dose of 30 mg/kg sodium pentobarbital. Bundles of anterior hepatic plexus were prepared, cleaned of fat and adhering connective tissues. Surgical sponge slices of approximately 3 mm length impregnated with 2% capsaicin solution were applied around the plexus. The abdominal cavity was closed and the perineurial sponge slices were left in their places over three days. The abdomen was then re-opened and the sponge pieces were removed. The wounds were closed and a week period of recovery was allowed to each animal. After the period of convalescence, the animals were subjected to hyperinsulinaemic euglycaemic glucose clamp studies. For control to this series of guinea-pigs served those, which received matching sponge pieces impregnated with the solvent for capsaicin (ethanol + Tween 80).
[3-3H] glucose was added to the glucose infusion, to maintain specific activity within 25% of the baseline. Blood samples were taken simultaneously with those for plasma insulin immunoreactivity determination. For determination of insulinstimulated glucose uptake by the gastrocnemius muscle, tissue samples were estimated using the administration of an intravenous bolus of 2-deoxy-D [L-14C] glucose (NEN Life Science), a non-metabolizable glucose analogue and by determining the tissue content of 2-deoxyglucose-6-phosphate. A 10 μCi bolus of 2-deoxy-D [L-14C] glucose was given in the 10th min of the steady state of glucose clamping. For determination of tissue [L-14C] 2-deoxyglucose-6-phosphate, the heart and gastrocnemius muscles were removed from each animal within 3 min after the experiments. The tissue samples were homogenized and centrifuged. The supernatants were run on an ion exchange column to separate deoxyglucose from deoxyglucose phosphate.
2.4. Systemic capsaicin desensitization
2.7. Drugs and chemicals
Capsaicin was used to elicit a selective functional deterioration of sensory ‘C’ fibers. The guinea-pigs were given capsaicin/solvent subcutaneously in the sequence of 10, 30, and 50 mg/kg single daily doses over 3 days. Capsaicin (2% w/v) was dissolved in physiological saline containing 3% v/v ethanol and 4% v/v Tween 80. The animals pre-treated with capsaicin were used for further studies after a 7-day period of recovery to avoid aspecific effects of systemic capsaicin administration as described (Ferdinandy et al., 1997).
Beyond the isotopes used, all drugs and chemicals have been purchased from Sigma (St Louis, Mo) except capsaicin that was from Fluka (Buchs, Switzerland).
2.3. Perineurial capsaicin desensitization
2.5. Hyperinsulinaemic euglycaemic glucose clamp studies Human regular insulin was infused at a constant rate (3 mU/kg, NOVO Nordisk, Copenhagen) via one of the venous catheters over 120 min. This insulin infusion rate yielded plasma insulin immunoreactivity of 100 ± 5 μU/ml in the steady state (see below). Blood samples (0.05 ml) were taken from the arterial cannula for blood glucose concentration at 10 min intervals. Blood glucose concentration was maintained constant (5.5 ± 0.5 mmol/l) by a variable rate of glucose infusion via the second venous cannula. When blood glucose had stabilized for at least 20 min, we defined this condition as steady state. This occurred within 40 min succeeding commencement of the insulin infusion. In the steady state, additional blood samples (0.3 ml) were taken for plasma insulin determination three times at 10-min intervals. The glucose infusion rate (mg/kg/min) during steady state was used to characterize insulin sensitivity (Porszasz et al., 2002). Each clamp determination was done after a preceding 24-h period of fasting. 2.6. Tissue glucose flux A continuous infusion of high performance liquid chromatography-purified [3-3H]glucose (12 μCi bolus, followed by 10 μCi; DuPont-NEN) was commenced 60 min before determination of insulin sensitivity. Subsequently, 12 μCi
2.8. Statistics The results expressed as means ± S.D. were analyzed with one-way analysis of variance followed by a modified t-test for repeated measures according to Bonferroni's method (Wallenstein et al., 1980). Changes were considered significant at P = 0.05. 3. Results 3.1. Experimental protocol Fig. 1 shows the design of insulin sensitivity determination studies. In each group, insulin sensitivity determined by hyperinsulinaemic euglycaemic glucose clamping served as the end point. The first series of experiments was to estimate the involvement of capsaicin-sensitive hepatic sensory fiber in changes in insulin sensitivity produced by cisplatin. Since the hypothesis was based on that the putative deterioration of sensory nerve function achieved by cisplatin was of importance, subgroups of 6 animals in the cisplatin- and vehicletreated groups were anaesthetized for nerve conduction velocity studies. These experiments were to confirm the presence or absence of sensory neuropathy induced by cisplatin. Determinations were done 24 h after the last cisplatin/vehicle administration as described previously (Szilvassy et al., 2000). The next series of experiments was to confirm the involvement of capsaicin-sensitive sensory fibers of the anterior hepatic plexus in the HISS mechanism in anaesthetized guineapigs, similar to that found in conscious rabbits (Porszasz et al., 2002) and anaesthetized rats (Porszasz et al., 2003). This was
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Fig. 1. Schematic drawing of the insulin sensitivity study design. In each group, whole body insulin sensitivity was determined by means of hyperinsulinaemic euglycaemic glucose clamp (HEGC). The animals in the ‘Main Group 1’ were treated by cisplatin (3 mg/kg, i.p.) and mannitol (75 mg/kg, i.p.) once a day over 9 days. A group of animals (Main Group 2) were treated with the solvent for cisplatin (1 ml isotonic NaCl) with 75 mg/kg mannitol i.p. with the same schedule as described in the ‘Main Group 1’. Each main group was divided into three subgroups; one subgroup served as control, a second subgroup of animals underwent systemic capsaicin desensitization (the guinea-pigs were given capsaicin subcutaneously in the sequence of 10, 30, and 50 mg/kg single daily doses over 3 days), and the guinea-pigs in the third subgroup underwent regional capsaicin desensitization of the fibers of anterior hepatic plexus (surgical sponge slices impregnated with 2% capsaicin solution were applied around the plexus for three days). The animals from the two control groups were devoted to nerve conduction velocity studies. An additional group was used to test the effect of an intravenous dose of 50 μg/kg CGRP8–37, a non-selective calcitonin gene-related peptide (CGRP) receptor antagonist on insulin sensitivity. The animals of ‘Main Group 3’ and ‘Main Group 4’ were devoted to study the sensory nitrergic nature of HISS mechanism in guinea-pigs. The effect of capsaicin (0.3 mg/kg/min i.po.) (subgroup b), L-NAME (10 mg/kg i.po.) (subgroup c), Atropine (1 mg/kg i.po.) (subgroup d) and Acetylcholine (1, 3, 10 μg/kg/min) (subgroup e) were determined on insulin sensitivity by HEGC. The control animals were treated with perineurial capsaicin only (subgroup a). The animals of ‘Main Group 4’ without perineurial capsaicin treatment served as corresponding controls to animals of ‘Main Group 3’.
achieved by selective deterioration of sensory fibers of the anterior hepatic plexus by means of perineurial capsaicin treatment (group 1/a) as described above. For control to this series of guinea-pigs served those, who received matching
placebo sponge slices (group 2/a). Since the deterioration of sensory fibers of the anterior hepatic plexus yielded insulin resistance (Porszasz et al., 2002), we assumed that chemical stimulation of these fibers by intraportal capsaicin (group 1/b)
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would produce an increase in insulin sensitivity using group 2/b animals as controls. In another subgroup of guinea-pigs, we intended to confirm the sensitivity of the HISS mechanism to NO synthase inhibition (group 1/c) and atropine (group 1/d) as suggested by the original observation by the group of Lautt et al. (2001). Since the HISS mechanism has been shown to involve parasympathetic components in rats (Lautt et al., 2001), a group of 6 animals was devoted to try to activate the mechanism by intraportal acetylcholine infusion (group 1/e). Group 2/c–e animals served as controls. From the above experiments, we obtained evidence for the sensory nitrergic nature of the HISS mechanism (see Results) in anaesthetized guinea-pigs. As far as the influence of sensory denervation on tissue insulin sensitivity is concerned, previous studies by Koopmans et al. (1998) revealed an increased responsiveness of peripheral tissues to the hypoglycemic effect of insulin in animals with perinatal capsaicin desensitization. This latter intervention is known to cause generalized irreversible sensory nerve damage (Jancso et al., 1977; Szolcsanyi, 1996). Therefore, to elucidate the effect of regional (anterior hepatic plexus sensory denervation) vs. systemic capsaicin desensitization, we used a systemic capsaicin desensitization protocol (group 3) 7 days preceding insulin sensitivity determinations as described (Ferdinandy et al., 1997; Szilvassy et al., 2002). This protocol induces a longlasting neuropeptide loss in sensory fibers (Szilvassy et al., 2002). Guinea-pigs pre-treated with the solvent of capsaicin served as controls (group 4). When the effect of systemic or regional capsaicin desensitization on insulin sensitivity was studied, the desensitization protocol commenced 24 h after the last cisplatin dose. Since the activation or blockade of the HISS effect is suggested to be reflected in changes in skeletal muscle glucose uptake, supplementary sets of experiments were instituted to investigate the insulin-stimulated uptake of 2-deoxy-D [L-14C] glucose, a non-metabolizable glucose analogue in samples from the heart and gastrocnemius muscle (10 tissue samples from 5 animals in each group). In these experiments, determination of tissue 2-deoxy-D [L-14C] glucose-6 phosphate content was the end point. Rates of basal glucose turnover and whole body glucose uptake at the end of the steady state of the clamp period were calculated by the use of a modified form of Steele's equation, taking the extra tracer glucose into account. Endogenous glucose production rate was determined by subtracting the “during clamp” glucose infusion rate from the whole body glucose uptake.
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3.3. Body weight and rectal temperature Body weight decreased from pre-treatment value of 362 ± 33 to 312 ± 21 g (P ≤ 0.05) in the cisplatin-treated animals. The ‘control’ guinea-pigs did not exhibit any significant change in body weight during the treatment period. Neither regional, nor systemic capsaicin desensitization caused any further significant change in body weight in either group. Rectal temperature did not change in animals in either main group. However, a period of transient increase in rectal temperature occurred in animals with systemic capsaicin desensitization, the duration of which did not exceed four days. 3.4. Nerve conduction velocity Fig. 2 shows that cisplatin, at the treatment schedule applied, produced a significant decrease in nerve conduction velocity in slow conducting unmyelinated ‘C’ fibres. 3.5. Changes in insulin sensitivity The cisplatin treatment applied significantly decreased insulin sensitivity as determined by hyperinsulinaemic (100 μU/ml) euglycaemic (5.5 mmol/l) glucose clamping in anaesthetized guinea-pigs. Regional desensitization with 2% capsaicin solution of the sensory fibres of the anterior hepatic plexus, known to underlie the HISS mechanism, decreased; whereas systemic capsaicin desensitization increased insulin sensitivity. Nevertheless, the decreased insulin sensitivity changed neither after regional (anterior hepatic plexus) nor succeeding systemic desensitization by capsaicin in cisplatin treated animals (Fig. 3).
3.2. Exclusions Seven cisplatin-treated animals had to be excluded from the experiments, four of them as cisplatin failed to produce any decrease in nerve conduction velocity in either ‘A’ or ‘C’ fibers, one because of respiratory insufficiency due to pneumonia and two because of the development of extended skin lesions. (Exclusions are not involved in the number of animals consumed by the study).
Fig. 2. Cisplatin-induced decrease in nerve conduction velocity in slow conducting unmyelinated C fibers of the femoral nerve. Measurements were done 24 h after a series of 9 intraperitoneal injections of cisplatin (3 mg/kg) and/ or its vehicle once a day. The data are means? S.D. obtained with 6 animals per group. ⁎: significantly different from control at P ≤ 0.05.
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Fig. 3. Effects of systemic and regional capsaicin desensitization of the anterior hepatic plexus (AHP capsaicin) on insulin sensitivity in anaesthetized guineapigs with a preceding 9-day treatment with cisplatin (3 mg/kg/day) and/or its vehicle. Systemic desensitization (Systemic capsaicin) was induced by subcutaneous injection of 10, 30 and 50 mg/kg capsaicin in three consecutive days. Regional desensitization was induced by wrapping anterior hepatic plexus fibers up with “Gelaspon” sponges impregnated with 2% capsaicin solution. The data are expressed as means ± S.D. obtained with 8 animals per group. ⁎ P ≤ 0.05 vs. control; + : P ≤ 0.05 vs. vehicle.
Fig. 5. Effects of perineurial treatment of the anterior hepatic plexus with capsaicin/its solvent on insulin sensitivity in anaesthetized rats and the modifying effects of L-NAME (hatched column), atropine (crosshatched column), or infusion of capsaicin (black column), acetylcholine (grey columns) into the portal vein. Capsaicin was applied as perineurial sponges impregnated with 2% capsaicin solution. The data are expressed as means ± S.D. obtained with 6 animals per group.⁎ P ≤ 0.05.
pharmacological maneuvers modified the resulting insulin resistant state (Fig. 5). CGRP8–37, a non-selective CGRP receptor antagonist at an intravenous dose of 50 μg/kg, significantly increased insulin sensitivity in normal animals but it failed to attain a significant change in this parameter after the 9-day treatment with cisplatin (Fig. 4). 3.6. Effect of intraportal L-NAME, capsaicin, atropine and acetylcholine on insulin sensitivity with and without preceding regional capsaicin desensitization Intraportal administration of L-NAME (10 mg/kg) decreased, whereas capsaicin (0.3 mg/kg over 1 min) increased insulin sensitivity. Neither atropine (1 mg/kg) nor acetylcholine (1–10 μg/kg/min) produced any significant effect. In animals with preceding regional capsaicin desensitization, none of the
Fig. 4. Effect of an intravenous dose of CGRP8–37 (50 μ g/kg) on insulin sensitivity in anaesthetized guinea-pigs with a preceding 9-day treatment with cisplatin (3 mg/kg/day) and/or its vehicle. The data are expressed as means ± S.D. obtained with 8 animals per group. ⁎ P ≤ 0.05 vs. control; + : P ≤ 0.05 vs. vehicle.
3.7. Effect of cisplatin neuropathy, partial hepatic sensory denervation, intraportal L-NAME or atropine on percentage suppression from basal of endogenous glucose production at hyperinsulinaemic euglycaemic glucose clamping The applied insulin plus glucose infusion as shown by Fig. 6, induced complete inhibition of endogenous glucose production during the steady state. Neither cisplatin neuropathy nor partial hepatic sensory denervation affected this inhibition. However, both intraportal L-NAME and atropine significantly attenuated the ‘steady state’ reduction of endogenous glucose production.
Fig. 6. Suppression from baseline of endogenous glucose production (EGP) by cisplatin neuropathy, perineurial capsaicin desensitization of the AHP fibres, intraportal L-NAME and atropine during hyperinsulinaemic euglycaemic glucose clamping in anaesthetized guinea-pigs. The data are means ± standard deviation. ⁎ significantly different from basal values at P ≤ 0.05.
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Fig. 7. 2-deoxy-D [L-14C] glucose uptake by cardiac and gastrocnemius muscle tissues during hyperinsulinaemic euglycaemic glucose clamping. In anaesthetized guinea-pigs. Cisplatin neuropathy, perineurial capsaicin desensitization of the AHP fibers, intraportal L-NAME and atropine. The data are means ± standard deviation. ⁎ significantly different from basal values at P ≤ 0.05.
3.8. Effect of cisplatin neuropathy, partial hepatic sensory denervation, intraportal L-NAME or atropine on cardiac and skeletal muscle 2-deoxy-D [L-14C] glucose uptake Insulin-stimulated glucose uptake was more than four times higher in cardiac tissue than in samples from the gastrocnemius muscle. It was significantly decreased by cisplatin neuropathy, partial hepatic sensory denervation and intraportal L-NAME in samples from either tissue. Atropine at the dose applied was without effect (Fig. 7). 4. Discussion These results confirm our previous finding that capsaicinsensitive sensory fibers in the anterior hepatic plexus are involved in endogenous insulin sensitization. This is suggested by the data that selective deterioration of these fibers by perineurial capsaicin yields insulin resistance in guinea-pigs, similar to that found in rabbits (Porszasz et al., 2002). The results also show that systemic capsaicin desensitization causes a significant increase in sensitivity to the hypoglycemic effect of insulin, an effect similar to that found by Koopmans's group (Koopmans et al., 1998) in conscious rats with neonatal deafferentation of capsaicin-sensitive sensory nerves. Nevertheless, the major original finding of this paper is that subacute treatment with cisplatin at a dose producing deterioration of ‘C’ fiber function, results in insulin resistance. Moreover, since neither regional nor systemic capsaicin desensitization could modify insulin sensitivity in cisplatin-treated animals, it is suggested that the predominant mechanism of cisplatin-induced insulin resistance is an impairment of the sensory effector function of fibers of the anterior hepatic plexus that underlie the HISS mechanism (Porszasz et al., 2002). This work in part is an extension of our previous results obtained in rabbits and rats that sensory fibers in the anterior hepatic play a crucial role in the regulation of insulin sensitivity
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(Porszasz et al., 2002). Based on the observation that blockade of NO synthesis by low-dose intraportal L-NAME significantly attenuated the HISS effect, the sensory nitrergic nature of the HISS mechanism has now been confirmed in three species i.e. in rabbits (Porszasz et al., 2002), rats (Porszasz et al., 2003) and, according to the present results, in guinea-pigs. That nerves in the anterior hepatic plexus play a crucial role in the regulation of post-prandial increase in insulin sensitivity in rats was first described in the pioneer works by Lautt's group (Sadri et al., 1997; Xie and Lautt, 1995, 1996) This discovery was then principally confirmed by Moore et al., (Moore et al., 2002) who found that chronic partial hepatic denervation induced insulin resistance in dogs (Moore et al., 2002). However, the latter group carried out insulin sensitivity tests 16 days after hepatic denervation, whereas Lautt et al. found acute hepatic denervation to produce insulin resistance in rats (Lautt et al., 2001). According to the original concept by Lautt's group, the insulin sensitizing mechanism linked to functional integrity of the anterior hepatic plexus fibers worked as follows: An insulin sensitizing parasympathetic reflex was activated in the liver in response to post-prandial hyperinsulinaemia, the atypical efferent pathway of which involved the release of HISS, an unidentified hormone-like substance that increased the sensitivity of peripheral tissue (predominantly the skeletal muscle) to the hypoglycemic effect of insulin (Lautt et al., 2001; Sadri et al., 1997; Szolcsanyi et al., 1998; Xie et al., 1993; Xie and Lautt, 1994, 1996). This mechanism was shown to substantially involve nitrergic components, since various NO synthase inhibitors apparently induced insulin resistance, and intraportal administration of NO donors restored insulin sensitivity in such animals (Sadri and Lautt, 1999). As shown from the results in Fig. 2, neither atropine nor intraportal acetylcholine affected insulin sensitivity in guinea-pigs, similar to that recently described by Porszasz et al. (Porszasz et al., 2003) in rats. Theoretically, the discrepancy may in part be explained by a difference in the way of determining insulin sensitivity, as Lautt's group used the rapid insulin sensitivity test termed RIST (Lautt et al., 1998) to characterize changes in insulin sensitivity, whereas we remained with using the hyperinsulinaemic euglycaemic glucose clamping, the gold standard of determining insulin sensitivity in whole animals (DeFronzo et al., 1979). The RIST method utilizes hyperinsulinaemia produced by a short term insulin infusion (usually 5 min), the hypoglycemic effect of which is compensated by a succeeding longer lasting glucose infusion (Lautt et al., 1998). During the hyperinsulinaemic clamp method, however, plasma insulin immunoreactivity is maintained at a level approximately 10 times higher (100 μU/ml) than corresponding fasting values confirmed by succeeding radioimmunoassay determinations and the hypoglycemic effect of continuous hyperinsulinaemia is opposed by continuous glucose infusion. Assuming that the HISS mechanism is activated by hyperinsulinaemia, it is possible that a much higher degree of the HISS mechanism activation is achieved by hyperinsulinaemic glucose clamping (because of the long-lasting stable hyperinsulinaemia) than that attained at the RIST method by preceding exposure to food. This may serve as an explanation for the lack of effect of either
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acetylcholine infusion or electrical stimulation of peripheral anterior hepatic plexus fibers subsequent to an acute nerve cut to further increase insulin sensitivity in rats (Porszasz et al., 2003). The present results clearly show that neither regional capsaicin nor cisplatin neuropathy influenced insulin-induced suppression of endogenous glucose production with a strong inhibitory effect on insulin-stimulated 2-deoxy-D [L- 14 C] glucose uptake. Therefore, assuming that the HISS mechanism is activated in the liver and acts on skeletal muscle (Lautt, 2003), the insulin resistant state associated with cisplatininduced sensory neuropathy seems to be very similar to that suggested for HISS deficiency. The cisplatin-treated guinea-pigs exhibited characteristic neurophysiological features of sensory neuropathy at level of unmyelinated “C” fibers, reflected in a significant decrease in conduction velocity in slow conducting sensory nerve fibers. However, it has been found that the dorsal root ganglia cells serve as the primary targets for cisplatin to induce peripheral neuropathy (Barajon et al., 1996; Cavaletti et al., 1998; Gispen et al., 1992). It is therefore not surprising that sensory neuropathy highlights neurotoxicity produced by cisplatin (Gispen et al., 1992; Tredici et al., 1994). Neuromorphologic studies by Barajon and his co-workers (Barajon et al., 1996) on cisplatin-induced changes revealed an accumulation of sensory neuropeptides (CGRP, substance P, galanin and somatostatin) in dorsal root ganglia with much more severe histological alterations in ganglionic cells than those seen in peripheral fibers. These studies also suggested an impaired axonal transport of sensory neuropeptides by cisplatin. Previous studies elucidated the sensory effector nature of the HISS mechanism with the involvement of nitrergic pathways (Lautt, 2003; Porszasz et al., 2002). It has also been shown that the anterior hepatic plexus contains a significant amount of capsaicinsensitive peptidergic sensory nerve fibers, a significant part of which derives from dorsal root ganglia, reaching the liver with vagal efferents (Miao et al., 1997; Erin et al., 2000). If this latter were true, the neurotoxic doses of cisplatin through causing a deficiency in axonal transport of sensory neuropeptides, might have caused changes in insulin sensitivity similar to that characteristic of a transient neuropeptide loss produced by repetitive capsaicin doses (Ferdinandy et al., 1997; Szolcsanyi, 1996). However, the effect of systemic capsaicin desensitization on insulin sensitivity was opposite to that attained by cisplatin, whereas sensory neuropathy induced by the subacute intraperitoneal cisplatin treatment schedule, produced changes similar to that seen after regional sensory denervation of the anterior hepatic plexus by capsaicin. This might suggest the dominant toxic influence of intraperitoneal cisplatin doses on sensory nerves supplying the liver, however, the same cisplatin treatment schedule has been shown to cause severe alterations in bronchomotility as well (Szilvassy et al., 2000). That systemic capsaicin desensitization yields an increase in insulin sensitivity through elimination of CGRP, an insulin antagonist from sensory nerve terminals was elegantly demonstrated by Koopmans et al. (Koopmans et al., 1998). Indeed, CGRP8–37, a non-selective CGRP receptor antagonist, at the dose applied (Peitl et al., 1999) was found to increase insulin
sensitivity in our model as well. Nevertheless, the CGRP receptor antagonist failed to amend insulin resistance attained by cisplatin, similarly to systemic capsaicin desensitization applied after the cisplatin treatment period. Taken these findings together, it is suggested that insulin resistance deriving from cisplatin-induced sensory neuropathy is predominantly underlain by an impairment of the HISS mechanism. Whatever the precise mechanism, the results call attention to the possibility that insulin resistance seen in patients with several types of cancer (Yoshikawa et al., 2001) may be accentuated by anticancer therapy per se producing sensory neuropathy. Since insulin resistance significantly contributes to cancer cachexia (Pisters et al., 1992), insulin sensitization might be an important adjunct in cancer chemotherapy (Benko et al., 2003). Acknowledgement This work was supported by grants from the Hungarian Ministry of Education (OTKA T032002, TT042266, D45992 and T043467; ETT 6003/1/200, 03597/2003 and 585/2003), the National Research and Development Fund (NRDP 1/007/ 2001), the Hungarian–German academic grant MÖB-DAAD 1627/2002, a grant from a Higher Education Fund (FKFP 2000–2003), OMFB ALK-00190/2002 and the Association of Innovative Pharmaceutical Manufacturers. The technical assistance by Miss Susanna Koszorus is greatly acknowledged. Robert Porszasz MD, PhD is supported by the Hungarian Academy of Sciences (Janos Bolyai Postdoctoral Fellowship). The work by Barna Peitl MD, PhD was supported by a postdoctoral OTKA fellowship. References Bajza, A., Peitl, B., Nemeth, J., Porszasz, R., Rabloczky, G., Literati-Nagy, P., Szilvassy, J., Szilvassy, Z., 2004. Development of insulin resistance by nitrate tolerance in conscious rabbits. J. Cardiovasc. Pharmacol. 43, 471–476. Barajon, I., Bersani, M., Quartu, M., Del Fiacco, M., Cavaletti, G., Holst, J.J., Tredici, G., 1996. Neuropeptides and morphological changes in cisplatin-induced dorsal root ganglion neuronopathy. Exp. Neurol. 138, 93–104. Benko, I., Djazayeri, K., Abraham, C., Zsuga, J., Szilvassy, Z., 2003. Rosiglitazone-induced protection against myelotoxicity produced by 5fluorouracil. Eur. J. Pharmacol. 477, 179–182. Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., Levine, J.D., Julius, D., 1997. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389, 816–824. Cavaletti, G., Fabbrica, D., Minoia, C., Frattola, L., Tredici, G., 1998. Carboplatin toxic effects on the peripheral nervous system of the rat. Ann. Oncol. 9, 443–447. DeFronzo, R.A., Tobin, J.D., Andres, R., 1979. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am. J. Physiol. 237, E214–E223. Erin, N., Ercan, F., Yegen, B.C., Arbak, S., Okar, I., Oktay, S., 2000. Role of capsaicin-sensitive nerves in gastric and hepatic injury induced by coldrestraint stress. Dig. Dis. Sci. 45, 1889–1899. Ferdinandy, P., Csont, T., Csonka, C., Torok, M., Dux, M., Nemeth, J., Horvath, L.I., Dux, L., Szilvassy, Z., Jancso, G., 1997. Capsaicin-sensitive local sensory innervation is involved in pacing-induced preconditioning in rat hearts: role of nitric oxide and CGRP? Naunyn-Schmiedeberg's Arch. Pharmacol. 356, 356–363.
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Further reading Guimaraes, A.P., Guimaraes, F.S., Prado, W.A., 2000. Modulation of carbachol-induced antinociception from the rat periaqueductal gray. Brain Res. Bull. 518 471–478. Migliore, M., Giuliano, R., Aziz, T., Saad, R.A., Sgalambro, F., 2002. Fourstep local anesthesia and sedation for thoracoscopic diagnosis and management of pleural diseases. Chest 1218 2032–2035.