ATP: Adenosine Triphosphate; ANOVA: Analysis of Variance; CAT: Catalase; EDTA: Ethylenediamine Tetraacetic Acid; GLUT2: Glucose Transporter 2; GPx: Glutathione Peroxidase; GR: Glutathione Reductase; HSP70: Heat Shock Protein 70; NADPH: Nicotinamide Adenine Dinucleotide Phosphate; MDA: Malondialdehyde; ROS: Reactive Oxygen Species; SGLT1: Sodium- Glucose Trasporter 1; SOD: Superoxide Dismutase.

Creatine (Cr, N-[aminoiminomethyl]-N-methyl glycine) is an endogenous amino acid derived from glycine, methionine and arginine in the liver, kidney and pancreas [1]; in mammals, creatine is also obtained from diet by meat-containing products [2,3]. In normal conditions diet intake supplies about 50% of creatine requirement [2,3]. In both cases creatine is transported via the blood to tissues, where the uptake into the cells is carried out by a specific transporter (CT) that facilitates the entry of creatine against a very large concentration gradient [4-6]. Besides as an ergogenic aid to improve athletes exercise performance, creatine dietary supplement is increasingly used as a possible therapeutic agent in the treatment of a broad range of diseases, including myopathies, neurodegenerative disorders, cancer, rheumatic diseases, and type 2 diabetes [7-11]. The neuroprotective potential of creatine has been illustrated in numerous models of neurodegeneration as well as in animal and human models of traumatic brain injury and cerebral oxygen deprivation [12-15]. Moreover, creatine has been shown to maintain intestinal homeostasis and protects against colitis [16]; for instance, in mouse colitis models, creatine supplementation has been shown to attenuate the inflammatory response [17]. The putative benefits of creatine in these disorders have been generally attributed to the creatine-induced buffering of cellular ATP levels, which fall would stimulate the formation of reactive oxygen species (ROS) and tissue oxidative damage [3]. Indeed, most of these pathologies involve multiple etiological factors, among which the detrimental role of oxidative stress has been stably recognized. In fact, in vitro studies have revealed that creatine may have direct antioxidant properties by acting as a scavenger of free radicals [18,19]. In addition, studies show that the creatine prevents oxidative stress parameter, such as lipid peroxidation, in liver of mice treated with pervastatin [20].

Acidosis promotes lipid peroxidation or other manifestations of oxidant-mediated damage in various cell types [21-24]; moreover, a number of studies indicate acidosis to be involved in ROS-induced intestinal inflammatory diseases [25-27]. Acidosis associated with inflammatory conditions, in turn, produces oxidative stress and/or amplifies its effects [6,23,28-30]; in vivo and in vitro studies indicate that at an acidotic pH, the response of the gut to an insult is magnified [31].

One deleterious consequence of an altered cellular redox status in the intestine is the potential for genesis of gut pathologies. In fact, it has become increasingly apparent that oxidants, in addition to being agents of cytotoxicity, can play an important role in mediating specific cell responses and expression of genes involved in degenerative pathophysiologic states, such as inflammation and cancer. Reactive oxygen species are implicated in the pathogenesis of various gastro-intestinal diseases, including post-ischemic reperfusion injury of the small intestine, gastric ulcers [26,32], ulcerative colitis [33], Crohn’s disease [34], cancer and inflammation [35,36]. The aim of this study is therefore to show if creatine supplementation in vivo should ameliorate the antioxidant response of intestinal cells and prevent intestinal tissue injury induced by oxidative stress.

Previous experiments have shown that in vitro treatment with creatine had positive effects on rat jejunal epithelium under conditions of oxidative stress induced by an ischemia and reperfusion model [37]. In the present study the effect of creatine was studied in vivo, by a subministration of creatine lasted 7 days, while oxidative stress was induced by acidosis. We measured various oxidant and antioxidant parameters on cells extracted from rats jejunum after administration of creatine in control condition and in condition of chronic acidosis. In particular we investigated whether creatine administration had effects on the activities of the main antioxidant enzymes of the cell, and whether creatine could affect parameters associated with oxidative stress, such as the level of lipid peroxidation. Furthermore, to investigate the effects of the molecule on intestinal function, the expression of (Na⁺/K⁺)- ATPase, SGLT1, and GLUT2 was examined. Finally, to assess whether the presence of creatine may have any cytoprotective effects in relation to stress conditions, we measured the expression of heat shock proteins (HSP70), which are known to play a protective role in the intestinal epithelial cells against thermal and oxidative stress.

The experiments were performed according to national ethical guidelines and approved by “Comune di Milano – Uff. Diritti degli animali”, “Regione Lombardia” and “Ministero della Salute” on male albino rats (Wistar strain, Charles River Italiana) weighing 250-300 g (about two months age).

Experiments were performed on 16 rats, maintained on standard chow and with ad libitum access to drinking water. To induce metabolic acidosis, rats were given 0.28 M NH₄Cl in drinking water for 7 days. Four different experimental conditions were set up: 1. control; 2. creatine; 3. NH₄Cl; 4. creatine + NH₄Cl. For each condition rats were watered with 75 ml of the respective solutions, all prepared using tap water. At the first group was administered net water, at the second a 20 mM creatine monohydrate solution, at the third a 280 mM NH₄Cl solution, while at the fourth a solution of NH₄Cl 280 mM and 20 mM creatine. For the first two conditions, the animals were treated for a total of 11 days. For the last two conditions the treatment above described lasted a total of 11 days and was preceded by a four-day pre-treatment in which rats were given only water (third condition) or a 20 mM solution of creatine monohydrate (fourth condition). Body weight of the animals was recorded the first and the last day. After treatment, animals were killed under anaesthesia, always between 9:00 and 10:00 a.m. to avoid any possible cyclic daily variations in antioxidant levels. To confirm acidosis, blood pH was measured immediately before death directly from blood in the left ventricle. The intestinal tissues were dissected, the jejunum resected, and the mucosa was scraped, weighed, rapidly freeze-clamped at liquid nitrogen temperature and stored at -80°C until use.

Enzyme activities

The jejunal scraped material was homogenized in 50 mM potassium phosphate buffer, pH 7.4, containing EDTA 1 mM. The samples were centrifuged for 10 min at 12000xg, 4°C, and the supernatant was used for activity assays of enzymes.

All enzyme activities were expressed as mU/mg proteins and reported in percent over the control.

Catalase (CAT) activity was measured according to the method of Aebi [38] by following the decrease in absorbance of H₂O₂ at 240 nm for 5 min.

Superoxide dismutase (SOD) activity was measured by the inhibition of pyrogallol autoxidation at 420 nm according to Guzik et al. [39].

Glutathione peroxidase (GPx) activity was measured by following the oxidation of NADPH at 340 nm according to Anwer et al. [40].

Glutathione reductase (GR) activity was measured as a decrease in absorbance of NADPH for 5 min at 340 nm according to Ojano-Dirain et al. [41].

Lipid peroxidation

The jejunal scraped material was homogenized in KCl 1.15% and centrifuged for 10 min at 12000xg, 4°C. Malondialdehyde (MDA) production, expressed as nmol/mg proteins, was assessed spectrophotometrically on the supernatant with the method defined by Ohkawa et al. [42].

Protein extraction and western blot

The jejunal scraped material from each rat was resuspended in cold buffer sucrose-histidine (IS) containing 0.3 M sucrose, 25 mM Histidine, 1 mM EDTA, supplemented with protease inhibitors (Roche), homogenized and then centrifuged at 4°C for 15 min at 5000 × g. Supernatant was recovered; protein concentration was measured [43] and equal amounts of proteins (5 mg for Na⁺/K⁺-ATPase and 60 mg for GLUT2 and HSP70) were analysed by 7% SDS/PAGE electrophoresis. The proteins transferred to a polyvinylidene difluoride membrane (Miniprotean 3, Biorad), were probed overnight at 4°C with the specific primary antibodies. In particular, the antibodies used were: anti-ATPase alpha 1 (Na⁺/K⁺) (Novus Biologicals) diluted 1:5000, polyclonal anti-GLUT2 (Chemicon) diluted 1:1000, and anti HSPA1A-Heat shock protein 70 kDa protein 1A (Aviva) diluted 1:5000. The primary antibody for GLUT2 was detected with a goat anti-rabbit IgG conjugated to horseradish peroxidase (Chemicon) diluted 1:40000, the anti-ATPase alpha 1 (Na⁺, K⁺) was detected with a goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotech) diluted 1:3000, and the primary antibody for HSP70 was detected with a goat anti-rabbit IgG conjugated to horseradish peroxidase (Chemicon) diluted 1:40000. All primary and secondary antibodies were diluted in 5% non-fat dry milk TBST buffer (Tris-buffered saline with Tween). Site of antibody-antigen reaction was visualized by using Amersham ECL Plusfollowed by autoradiography.

Statistics

Statistical analysis was performed by Student’s t test or by analysis of variance (ANOVA) followed by post-hoc Tukey’s limitation. Values are reported as means ± S.E. For all determinations, in each experimental condition, we carried out 4 replications. The level of significance was taken at p ≤ 0.05.

Blood pH and body weight

The measurements of blood pH immediately before death of treated animals (Figure 1) confirmed the data previous published [6]: in rats treated with NH₄Cl there was a statistically significant decrease in the pH value with respect to the control, confirming that acidosis condition was actually induced. As in the previous research [6] the acidotic condition was associated with a reduced animal growth (Figure 2), also in the rats treated with creatine.

Values are means ± S.E.
** p = 0.05 vs. control.

Figure 1: Blood pH values after induction of metabolic acidosis by administration of 0.28 M NH₄Cl in drinking water for 7 days.

Values are means ± S.E.
*** p = 0.05 vs control ** P = 0.05 vs. creatine.

Figure 2: Rat weight (g) gain after 11 days of treatment.

Antioxidant enzyme activities, MDA production, and HSP70 expression

Figure 3 shows the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and catalase (CAT), respectively, in the different conditions studied. As shown in a previous research [6], the activity of SOD, GPx, and GR does not vary significantly in chronic acidosis with respect to the control condition. Creatine does not have effects nor in control condition, neither in acidosis. Evidence was given that in murine neurons, chronic acidosis reduces the activities of GPx and GR [44], while in renal tubular cells, GPx activity is increased [45]. In rats, a 6 days creatine oral supplementation decreased ROS content in slow and fast-twitch skeletal muscles but did not change expression and activities of antioxidant enzymes [46]. Our data show that none of these effects occurs in the jejunal portion of the intestine undergoing chronic acidosis and that creatine does not act on these antioxidant activities under basic conditions.

Values are means ± S.E. Number of experiments = 4 with duplicate estimation.
*** p = 0.001 vs. control and acidosis. ** p = 0.05 vs. control and acidosis.

Figure 3: Effects of 11 days administration of 20 mM creatine on superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and catalase (CAT) activities of jejunal mucosal homogenate in control condition and in chronic acidosis.

As previously shown [6], CAT activity (Figure 3) is not influenced by chronic acidosis. Anyway, an inhibiting effect of creatine on the activity of the enzyme is evident, both in control and in acidosis. It is known that the CAT cell level is substrate-dependent [47]: since it has been suggested that the antioxidant effect of creatine is due to a direct scavenging action on ROS [48], it could be hypothesized that the interaction of creatine administered with ROS (H₂O₂) produced by normal cellular metabolism, determines the decrease in CAT activity we observed. The reduction of CAT activity in the presence of creatine could also be explained by taking into account mitochondrial metabolism. In fact, ROS production in the mitochondria depends strongly on the mitochondrial transmembrane potential. When mitochondrial ADP levels decrease, membrane potential increases as ROS formation does [49]. One of the enzymes involved in the recycling mechanism of ADP is mitochondrial creatine kinase (mt- CK), which is located in the transmembrane space of mitochondria. This enzyme catalyzes the reaction: MgATP + Cr ↔ PCr + MgADP + H⁺. Phosphocreatine produced at mitochondrial level is exported to the cytosol, while ADP produced at cytosolic level is pumped into the mitochondrion. This causes an increase in ADP levels of in the mitochondria and therefore reduces the production of ROS and H₂O₂. Thus the administration of creatine could have an antioxidant role acting through this mechanism [49].

One of the deleterious consequences of oxidative damage is lipid peroxidation, which produces structural and functional damage to membranes as well as several secondary products, among which malondialdehyde (MDA). From Figure 4 it can be observed that MDA levels of jejunal mucosa do not undergo modifications in the different experimental cases, suggesting that the condition of chronic acidosis does not induce oxidative damage and that creatine does not act on the degree of lipid peroxidation nor in control condition, neither in acidosis. Discordant data are reported in literature on this subject. At plasma level, for example, creatine administration is associated with a significant reduction of lipid peroxidation biomarkers [50]. Also in skeletal muscle of rats subjected to hyperhomocysteinemia creatine reduces lipid peroxidation [51]. However, the antioxidant effect of creatine found in plasma has not been observed in the liver [52], suggesting that the action of the molecule may be different in different tissues.

Values are means ± S.E. Number of experiments = 4 with duplicate estimation.

Figure 4: Effects of 11 days administration of 20 mM creatine on malondialdehyde (MDA) production of jejunal mucosal homogenate in control condition and in chronic acidosis.

HSP70 confers stress tolerance and cytoprotection against several environmental-induced injury conditions [53,54], thus the possible induction of HSP70 by creatine was investigated. HSP70 protein expression was measured using Western blot analysis (Figure 5). In all experimental conditions the presence of a band at 45 kDa, corresponding to the molecular weight of the 1A subunit of the tested protein, was observed. The related densitometric analysis shows that there are no significant variations in all considered cases. We cannot however exclude that there is a temporal dependence in the expression of HSP70, whose levels are notoriously modulated in a transient manner [55].

** p = 0.05 vs. control.
Western blot analysis for HSP 70 was performed on jejunum total proteins and was carried out on 4 experiments. Densitometric analysis of the bands did not reveal significant statistical difference in the intensity of the signals.

Figure 5: Effects of 11 days administration of 20 mM creatine on HSP70 expression in jejunal mucosal homogenates in control condition and in chronic acidosis.

Expression of Na⁺/K⁺-ATPase, GLUT2, and SGLT1

To evaluate the effects of creatine treatment on intestinal function in control and in chronic acidosis conditions, we investigated the expression of some important transport proteins: Na⁺/K⁺-ATPase, GLUT2, and SGLT1.

Figure 6 shows the results of western blot assays on Na⁺/K⁺- ATPase expression. In all the experimental conditions considered, the immunoblots show the presence of a 110 kDa band, corresponding to the molecular weight of the α1 subunit of the Na⁺/K⁺-ATPase. A significant increase in the signal obtained is observed both after treatment with creatine and in conditions of chronic acidosis (about +200% in both conditions), as previously observed in other tissues. In fact, it has been reported that chronic treatment with creatine induces an increase in the expression of Na⁺/K⁺-ATPase in the cerebral cortex [13], probably as a result of a functional enhancement due to the ergogenic properties of this molecule. As previously observed [6], and similarly to what was already proposed in duodenum [56], the increase in the signal observed in conditions of chronic acidosis could be interpreted as a long-term adaptive response, able to compensate for the reduction of Na⁺/K⁺- ATPase expression (and the consequent functional alterations of the reabsorption of ions and glucose) induced by acidosis in a short time. Moreover, this compensatory effect would allow to counteract the acidosis itself, since it can be hypothesized that the increase in protein expression leads to an increase in its activity and therefore to the electrochemical potential gradient of Na⁺ through the plasmalemma. This could in turn lead to an increase in Na⁺/H⁺ exchanger activity in an attempt to resolve the acid-base decompensation by bringing the pH back to the physiological values. In this regard, it should be noted that there is no additivity between the effect of chronic acidosis and that of creatine, so that chronic treatment with creatine in the simultaneous presence of conditions of chronic acidosis would not exert any enhancement on the expression of Na⁺/K⁺-ATPase, since acidosis by itself should have induced a compensatory increase in the expression of the carrier.

** p = 0.05 vs. control.
Western blot analysis for Na⁺/K⁺-ATPase was performed on jejunum total proteins and was carried out on 4 experiments. Densitometric analysis of the bands reveals significant statistical difference in the intensity of the signals.

Figure 6: Effects of 11 days administration of 20 mM creatine on Na⁺/K⁺- ATPase expression in jejunal mucosal homogenates in control condition and in chronic acidosis.

Figure 7 shows the results of western blot assays on GLUT2 expression. In all experimental conditions the immunoblots show the presence of a band at the expected weight of about 53 kDa. The administration of creatine does not determine differences in the expression of GLUT2 with respect to the control condition. The molecule, therefore, does not appear to show any ergogenic effect on the levels of this carrier, at least at physiological pH. As we previously showed, chronic acidosis causes a reduction in the expression of GLUT2 [6], confirming the data in literature [57] according to which the expression of this transporter is influenced by perturbations of the physiological conditions related to stress of different nature. This reduction, anyway, is abolished by creatine. In fact, following the simultaneous administration of NH₄Cl and creatine, the expression levels of GLUT2 are comparable to those of the control; in these conditions, therefore, creatine would exert on the expression of GLUT2 a protective action able to counteract the negative effects of chronic acidosis.

** p = 0.05 vs. control.
Western blot analysis for GLUT2 was performed on jejunum total proteins and was carried out on 4 experiments. Densitometric analysis of the bands reveals significant statistical difference in the intensity of the signals.

Figure 7: Effects of 11 days administration of 20 mM creatine on GLUT2 expression in jejunal mucosal homogenates in control condition and in chronic acidosis.

Figure 8 shows the results of Western blot assays on SGLT1 expression. In all the experimental conditions considered, the immunobots show the presence of a band at the expected molecular weight of 72 kDa. Following chronic treatment with creatine at physiological pH there are no significantly changes in the signal, which is instead significantly reduced (approximately -20%) following the administration of NH₄Cl and does not undergo further modifications following simultaneous treatment with NH₄Cl and creatine. The chronic acidosis, therefore, determines a certain reduction of the expression of the protein, both in the absence and in the presence of creatine that, contrary to what observed for the Na⁺/K⁺-ATPase, would not induce any strengthening effect on the expression of SGLT1 neither at physiological pH nor in conditions of chronic acidosis.

** p = 0.05 vs control.
Western blot analysis for SGLT1 was performed on jejunum total proteins and was carried out on 4 experiments. Densitometric analysis of the bands reveals significant statistical difference in the intensity of the signals.

Figure 8: Effects of 11 days administration of 20 mM creatine on SGLT1 expression in jejunal mucosal homogenates in control condition and in chronic acidosis.

To sum up, chronic treatment with creatine has shown beneficial effects for jejunal epithelium. Creatine seems to have antioxidant properties that would be realized through direct interaction of the molecule with ROS. In fact, the antioxidant kit of the cell is not influenced by its administration, except for CAT, whose activity, both in presence and in the absence of acidosis, is significantly reduced. The administration of creatine seems to make the tissue more resistant: its presence in fact determines a functional strengthening of the tissue, increasing the expression of Na⁺/K⁺-ATPase. Chronic treatment with creatine also counteracts the inhibitory effect of acidosis on GLUT2, whose level of expression does not differ from the control if creatine is present in acidosis condition. There is no involvement of HSP70 in the effects shown for creatine, as their expression does not change in its presence.

These observations provide a compelling argument for creatine supplementation as an adjuvant therapy to ameliorate intestinal epithelial cells response to oxidative stress, which is implicated in the pathogenesis of various gastro-intestinal diseases, including post-ischemic reperfusion injury of the small intestine, gastric ulcers, ulcerative colitis, Crohn’s disease, cancer and inflammation.

We are most grateful to Prof. M. N. Orsenigo and Dr. M. Tosco for helpful suggestions.

All authors contributed in equal part performing experiments and writing the manuscript.

This research was supported by Ministero dell’Istruzione, dell’Università e della Ricerca, Italy.