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Abstract

We evaluated the effects of supplementation with oral l-glutamine in Walker-256 tumor–bearing rats. A total of 32 male Wistar rats aged 54 days were randomly divided into four groups: rats without Walker-256 tumor, that is, control rats (C group); control rats supplemented with l-glutamine (CG group); Walker-256 tumor rats without l-glutamine supplementation (WT group); and WT rats supplemented with l-glutamine (WTG group). l-Glutamine was incorporated into standard food at a proportion of 2 g/100 g (2%). After 10 days of the experimental period, the jejunum and duodenum were removed and processed. Protein expression levels of key enzymes of gluconeogenesis, that is, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, were analyzed by western blot and immunohistochemical techniques. In addition, plasma corticosterone, glucose, insulin, and urea levels were evaluated. The WTG group showed significantly increased plasma glucose and insulin levels (p < 0.05); however, plasma corticosterone and urea remained unchanged. Moreover, the WTG group showed increased immunoreactive staining for jejunal phosphoenolpyruvate carboxykinase and increased expression of duodenal glucose-6-phosphatase. Furthermore, the WTG group presented with less intense cancer cachexia and slower tumor growth. These results could be attributed, at least partly, to increased intestinal gluconeogenesis and insulinemia, and better glycemia maintenance during fasting in Walker-256 tumor rats on a diet supplemented with l-glutamine.

Keywords

Antitumor effect phosphoenolpyruvate carboxykinase glucose-6-phosphatase glycemia insulinemia intestinal gluconeogenesis

Introduction

One of the most marked negative effects of cancer is cachexia, a complex syndrome characterized by anorexia, and increased proteolysis with loss of muscle and adipose mass.^1–10 The prevalence of cachexia in gastric, pancreatic, lung, advanced colorectal, or prostate neoplasias is more than 50%, whereas it is estimated to be between 30% and 50% in non-Hodgkin lymphoma, breast cancer, sarcomas, and leukemias.¹¹ Moreover, a cachectic status worsens the prognosis and implies increased mortality, being responsible for the death of 22% of cancer patients.^12–14 The development of cachexia in cancer depends upon signaling promoted by both tumor and host tissues, resulting in the stimulation of catabolic pathways and inhibition of metabolic pathways.^8,12

Cachexia can dramatically affect the small intestine, resulting in failures of glucose and l-glutamine homeostasis, cell proliferation, and intestinal absorption.¹³ The small intestine has a crucial role in glucose homeostasis^14–20 due to the expression of key enzymes of gluconeogenesis, that is, glucose-6-phosphatase (G6Pase)^21–23 and phosphoenolpyruvate carboxykinase (PEPCK).^24–29 Furthermore, the small intestine controls food intake, as well as the release of glucose into the portal circulation and the reduction of food intake induced by hyperproteic diets or gastric bypass surgery.¹⁷

l-Glutamine is an amino acid that plays an essential role as the main intestinal fuel. In addition, l-glutamine participates in other functions of the renal, neuronal, pancreatic, and immune systems.³⁰ This amino acid is a precursor of intestinal gluconeogenesis during prolonged fasting when liver gluconeogenesis becomes reduced.²² Furthermore, l-glutamine is an important precursor of peptides, proteins, and carbohydrates, as well as purines and pyrimidines that are essential for the synthesis of nucleic acids and nucleotides.^30,31 In addition, l-glutamine has a central role in preventing oxidative stress due to its importance in the formation of l-glutathione, a powerful antioxidant involved in neuroprotection. In the intestinal mucosa, l-glutamine acts as the main source of energy for cell migration and proliferation.^32,33

Although cancer cells are avid glutamine consumers, the nutritional support supplied by l-glutamine added to the diet improves clinical responses in cancer patients.³¹ Such clinical improvements are consistent with a role for l-glutamine in stimulating immune function and augmenting protein synthesis in skeletal muscle.³⁰ In agreement with these clinical studies, we previously demonstrated that glutamine supplementation restored the proliferation of intestinal mucosa in Walker-256 tumor–bearing (WT) rats.³³ Here, we have expanded this previous study investigating the effect of glutamine supplementation on intestinal gluconeogenesis, cachexia index, blood corticosteronemia, glycemia, insulinemia, and uremia in WT rats.

Methods

Animal model of cachexia and supplementation with l-glutamine

All procedures were performed in accordance with the ethical principles adopted by the Brazilian Society for Laboratory Animal Science and were approved by the Ethics Committee for Animal Experimentation of the State University of Maringá (protocol number: 099/2012).

A total of 32 male Wistar rats (Rattus norvegicus) aged 54 days were randomly divided into four groups: rats without a WT, that is, control rats (C group); control rats supplemented with l-glutamine (CG group); rats with a WT (WT group); and WT rats supplemented with l-glutamine (WTG group).

The rats were kept in individual cages for the experimental period (10 days), with a 12-h dark/12-h light cycle and a controlled temperature (24°C). Food and water were available ad libitum. The non-supplemented animals (C and WT groups) received standard chow (Nuvilab, Colombo, Brazil), whereas the supplemented rats (CG and WTG groups) received l-glutamine (Fagron of Brazil Pharma Ltda, São Paulo, Brazil) incorporated in the standard chow at a proportion of 2 g/100 g.³²

Walker-256 tumors were induced according to the methodology described by Guarnier et al.¹⁰ by subcutaneously inoculating 8.0 × 10⁷ viable tumor cells in 0.5 mL phosphate-buffered saline (PBS), 16.5 mM PBS, 137 mM NaCl, and 2.7 mM KCl, pH 7.4/animal in the right rear flank. The groups without tumors (C and CG groups) received the same volume of PBS inoculated in the same location.

After the experimental period, all rats were fasted for 48 h, weighed, and anesthetized with an intraperitoneal (40 mg/kg) injection of sodium thiopental (Abbott Laboratories, Chicago, IL, USA). Body weights and food intake (24 h) of rats were measured before fasting.

Calculation of cachexia index

After an animal’s death, each tumor was carefully dissected and weighed, and the percentage of body weight loss was calculated according to the equation below. The animals were considered cachectic when the body mass loss was greater than 10% according to Guarnier et al.¹⁰

Body mass loss (%) = [ibm - fbm + (tm) + mgcg] \times 100 / (ibm + mgcg) .

where ibm refers to initial body mass of the WT rat, fbm refers to final body mass of the WT rat, tm refers to tumor mass, and mgcg refers to mass gain of the control group.

Plasma concentration of corticosterone, glucose, insulin, and urea

Blood was collected by cardiac puncture, and plasma was obtained by using potassium fluoride as an anticoagulant (Laborclin, Pinhais, Brazil) for the determination of glucose, corticosterone, insulin, and urea concentrations. The following kits were used: corticosterone EIA kit (ADI-900-097; Enzo Life Sciences GmbH, Lorrach, Germany) and DetectX urea nitrogen detection kit (K024-H1; Arbor Assays, Ann Arbor, MI, USA). Insulin was measured by radioimmunoassay (Wizard2 Automatic Gamma Counter, TM-2470; PerkinElmer^®, Shelton, CT, USA). Human insulin, monoclonal anti-insulin antibody from rats (I2018; Sigma-Aldrich, St Louis, MO, USA) and ¹²⁵I-labeled recombinant human insulin (NEX420010UC; PerkinElmer) were used. The limit of detection was 0.006 ng/mL.³⁴ Blood glucose was measured using a glucose oxidase technique.³⁵

Western blot for G6Pase and PEPCK

After a celiotomy, the duodenum and the jejunum were removed and gently washed with Krebs–Ringer buffer solution pH 7.4. Then, the homogenates were prepared, and total proteins of the duodenum and jejunum mucosal were isolated after centrifugation at 10,000g for 10 min in homogenization buffer (50 mM Tris–HCl, 600 mM NaCl, 1 mM EDTA (ethylenediaminetetraacetic acid), and 1% protease inhibitor solution, pH 7.4), and total protein concentration was determined by the method of Bradford.³⁶ Proteins (30 µg per well) were separated using 12% sodium dodecyl sulfate –polyacrylamide gel electrophoresis (SDS-PAGE) according to standard procedures of Bio-Rad Mini Protean System^® and transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) during 30 min at 25 V. Subsequently, immunostaining was performed after checking the transfer efficiency by Ponceau-S staining (Sigma-Aldrich), blocking the membranes in solution containing 5% non-fat dried milk and 0.1% Tween 20 in Tris-buffered saline (TBS) buffer (2.24 g/L Tris base, 8 g/L NaCl, pH 7.6) for 1 h at room temperature; then, the membranes were incubated with primary rabbit polyclonal anti-G6Pase antibody at 1:2000 dilution (sc-25840; Santa Cruz Biotechnology, Santa Cruz, CA, USA), primary rabbit polyclonal anti-PEPCK antibody at 1:2000 dilution (sc-32879; Santa Cruz Biotechnology), or mouse monoclonal anti-β-actin antibody at 1:5000 dilution (ab6276; Abcam, Cambridge, MA, USA) for 12 h, followed by incubation in goat anti-rabbit IgG secondary antibody conjugated with peroxidase at 1:2000 dilution (31460; Pierce, Rockford, IL, USA) for 2 h. Subsequently, the immunoreaction was visualized with colorimetric reagent 4CN Chromogenic Plus (NEL300001EA; PerkinElmer, Waltham, MA, USA). The level of immunoreactivity was measured as peak intensity using the scanned image of the immunostaining and image analysis software ImageJA^® version 1.43 (National Institutes of Health (NIH), Bethesda, MD, USA). Western blot normalization was done using β-actin as control protein. Thus, the relative amount of G6Pase or PEPCK was evaluated by calculating the ratio of the intensity of the signal for these proteins to that of β-actin. The results were expressed as arbitrary units.

Immunohistochemical technique for G6Pase and PEPCK

After a celiotomy, the jejunum and the duodenum were removed, gently washed with saline, and processed for immunohistochemical analysis of G6Pase and PEPCK. Segments were then opened longitudinally on the mesenteric border and immersed in Zamboni’s fixative solution (4% paraformaldehyde and 0.4% picric acid in phosphate buffer) at 4°C for 18 h. The samples were washed successively in 0.1 M PBS, pH 7.4, for 12 h, followed by cryoprotection in 18% sucrose solution in 0.1 M PBS, pH 7.4, for 24 h. After cryoprotection, samples were embedded in OCT 4583 (Cryomatrix Shandon, Pittsburgh, PA, USA), frozen rapidly in liquid nitrogen, and stored at −80°C. Samples were then cut in semi-serial 10-µm-thick sections in a cryostat microtome, arranged on previously prepared slides with 2% organosilane adhesive in acetone, and stored at −18°C.

For immunohistochemical analysis, sections at room temperature were washed three times in a solution containing 0.5% Triton X-100 (Sigma-Aldrich) in 0.1 M PBS, pH 7.4, and blocked for 6 h in solution containing 2% bovine serum albumin (BSA; Sigma-Aldrich), 0.5% Triton X-100, and 10% goat serum in 0.1 M PBS, pH 7.4. The immunostaining was performed in different blades. Sections were then incubated in a humidified chamber with a solution containing the following primary antibodies: rabbit polyclonal anti-G6Pase at a dilution of 1:1000 (sc-25840; Santa Cruz Biotechnology) or rabbit polyclonal anti-PEPCK at a dilution 1:1000 (sc-32879; Santa Cruz Biotechnology), diluted in 2% BSA solution containing Triton X-100 and 0.5% goat serum in 0.1 M PBS, pH 7.4, for 48 h at 4°C. After incubation, sections were washed three times with a solution containing Triton X-100 in 0.1 M PBS, pH 7.4. The sections were incubated in a dark humid chamber, in solution containing polyclonal goat anti-rabbit IgG secondary antibody 1:1000 (A-11011, Alexa Fluor^® 568; Molecular Probes, Invitrogen Co., Carlsbad, CA, USA), 2% BSA solution, Triton X-100, and 0.5% goat serum in 0.1 M PBS, pH 7.4, for 48 h at 4°C. Sections were then washed with a solution containing Triton X-100 and 0.5% goat serum in 0.1 M PBS, pH 7.4, and mounted on slides with 10% PBS in glycerol.

Analysis of immunostaining for G6Pase and PEPCK

This analysis was performed by using 25 images per segment (duodenum and jejunum) per animal with a ×10 magnification. The images were captured by high-resolution camera Moticam^® 2500 5.0 Mega Pixel (Motic China Group Co., Shanghai, China) coupled to an optical microscope Olympus BX40 fluorescence (Olympus Co., Tokyo, Japan), transferred to a computer via the software, Motic Images Plus^® 2.0 ML (Motic China Group Co.), and recorded. ImageJA 1.43 (NIH) was used to quantify the immunoreactive staining of G6Pase and PEPCK in recorded images by measuring the intensity of brightness in RGB (red–green–blue).

Statistical analysis

Results were statistically analyzed using Statistica 8.1 and GraphPad Prism 6.1 software, and expressed as mean ± standard error of the mean (SEM). For quantitative data, we performed one-way analysis of variance (ANOVA), followed by Fisher’s post-test. For the analysis of cachexia and tumor mass index, data were analyzed using Student’s t test. The significance level was 5%.

Results

Body weight gain (Table 1) was similar (p = 0.978) in the C and CG groups, whereas the body mass was significantly reduced (p < 0.0001) in the WT group compared with C and CG groups. l-Glutamine supplementation significantly decreased (61%; p = 0.026) body weight loss in the WTG group compared with the WT group. However, food intake (Table 1) did not differ among groups (WTG vs WT vs C vs CG).

Table 1.

Initial body weight, final body weight, body weight gain, and food intake (between 54 and 64 days of age).

Parameters	C	CG	WT	WTG
Initial body weight (g)	230.1 ± 1.97	230.6 ± 2.61	238.9 ± 6.15	234.0 ± 2.49
Final body weight (g)	239.3 ± 3.18	239.9 ± 3.67	221.5 ± 5.01*	225.8 ± 3.90^#
Body weight gain (g)	9.13 ± 3.11	9.25 ± 2.31	−17.38 ± 4.45*	−6.75 ± 2.45^#
Food intake (g/animal × day)	18.2 ± 1.74	18.5 ± 1.66	19.0 ± 1.61	19.3 ± 0.57

SEM: standard error of the mean; ANOVA: analysis of variance.

Results are expressed as mean ± SEM (n = 8 animals per group). Significant differences between means were analyzed by one-way ANOVA test followed by Fisher’s post-test for multiple comparisons, *p < 0.05 versus C; #p < 0.05 versus WT. Rats without Walker-256 tumor (WT), that is, control rats (C group); control rats supplemented with 2% l-glutamine (CG group); rats with WT (WT group); and WT rats supplemented with 2% l-glutamine (WTG group).

The WTG group exhibited a lower (76%; p = 0.023; Figure 1(a)) tumor growth mass and lower (p = 0.014; Figure 1(b)) cachexia index (WTG group vs WT group).

Figure 1.

(a) Tumor mass and (b) cachexia index in Walker-256 tumor–bearing (WT) rats and WT rats supplemented with 2% l-glutamine (WTG) in their diet for 10 days. Results are expressed as mean ± standard error of the mean (n = 8 animals per group). The significant differences between means were analyzed by Student’s t test.

Plasma concentration of corticosterone, glucose, insulin, and urea

The plasma concentrations of corticosterone and urea did not exhibit a significant difference (p > 0.05) between C, CG, WT, and WTG groups (Table 2).

Table 2.

Plasma concentrations of corticosterone, glucose, insulin, and urea (64 days of age).

Plasma Parameters	C	CG	WT	WTG
Corticosterone (ng/mL)	253.5 ± 2.81	256.9 ± 1.56	258.7 ± 1.06	255.0 ± 3.09
Glucose (mg/dL)	105.6 ± 6.42	96.3 ± 2.98	106.7 ± 2.99	120.5 ± 4.12^*,#
Insulin (ng/mL)	0.059 ± 0.008	0.049 ± 0.009	0.078 ± 0.016	0.116 ± 0.015^*,#
Urea (mg/dL)	37.3 ± 1.84	37.1 ± 1.07	38.9 ± 2.42	37.2 ± 2.34

SEM: standard error of the mean; ANOVA: analysis of variance.

Results are expressed as mean ± SEM (n = 8 animals per group). Significant differences between means were analyzed by a one-way ANOVA test followed by Fisher’s post-test for multiple comparisons, *p < 0.05 versus C; #p < 0.05 versus WT. Rats without a Walker-256 tumor (WT), that is, control rats (C group); control rats supplemented with 2% l-glutamine (CG group); rats with WT (WT group); and WT rats supplemented with 2% l-glutamine (WTG group).

Insulin levels in the WTG group compared with the WT group were significantly increased (49%; p = 0.044; Table 2). However, there were no significant differences (p > 0.05; Table 2) between C, CG, and WT groups. In addition, the WTG group showed significantly increased (13%, p = 0.019; Table 2) glycemia when compared with the WT group. On the contrary, C, CG, and WT groups showed similar (p > 0.05; Table 2) glycemia.

Western blot

The migration of β-actin, G6Pase, and PEPCK proteins in duodenum and jejunum samples is shown in Figures 2 and 3, with their molecular weights of 42, 36, and 62 kDa, respectively.

Figure 2.

Expression of (a) glucose-6-phosphatase (G6Pase) and (b) phosphoenolpyruvate carboxykinase (PEPCK) by western blot (n = 4) and quantification of immunoreactive (c) G6Pase and (d) PEPCK in the duodenum (n = 8) of rats without a Walker-256 tumor (WT), that is, control rats (C group); control rats supplemented with 2% l-glutamine (CG group); rats with WT (WT group); and WT rats supplemented with 2% l-glutamine (WTG group). Results are expressed as mean ± standard error of the mean. Significant differences between means were analyzed by a one-way ANOVA test followed by Fisher’s post-test for multiple comparisons, *p < 0.05 versus C; #p < 0.05 versus WT. Photomicrographs illustrate the immunoreactivity of duodenal G6Pase in tissues from (e) C, (f) CG, (g) WT, and (h) WTG groups. Photomicrographs reveal the immunoreactivity of duodenal PEPCK in tissues from (i) C, (j) CG, (k) WT, and (l) WTG groups. Calibration bar: 50 µm.

Figure 3.

Expression of (a) glucose-6-phosphatase (G6Pase) and (b) phosphoenolpyruvate carboxykinase (PEPCK) by western blot (n = 4) and quantification of immunoreactive (c) G6Pase and (d) PEPCK in the jejunum (n = 8) of rats without Walker-256 tumors (WT), that is, control rats (C group); control rats supplemented with 2% l-glutamine (CG group); rats with WT (WT group); and WT rats supplemented with 2% l-glutamine (WTG group). Results are expressed as mean ± standard error of the mean. Significant differences between means were analyzed by a one-way ANOVA test followed by Fisher’s post-test for multiple comparisons, *p < 0.05 versus C; #p < 0.05 versus WT. Photomicrographs illustrate the immunoreactivity of jejunal G6Pase in tissues from (e) C, (f) CG, (g) WT, and (h) WTG groups. Photomicrographs represent the immunoreactivity of jejunal PEPCK in tissues from (i) C, (j) CG, (k) WT, and (l) WTG groups. Calibration bar: 50 µm.

The level of G6Pase expression (C group vs CG group) was not significantly different in the duodenum (p = 0.875; Figure 2(a)) and the jejunum (p = 0.289; Figure 3(a)). WT rats showed a marked increase (122%; p = 0.025; Figure 2(a)) in the expression of duodenal G6Pase (WT group vs C group). In contrast, WT rats showed a substantial reduction (78%; p < 0.0001; Figure 3(a)) in the expression of jejunal G6Pase (WT group vs C group).

l-Glutamine supplementation in control rats (CG group) promoted a significant decrease (22%; p = 0.006; Figure 2(b)) and increase (48%; p = 0.008; Figure 3(b)) in the duodenal and jejunal expression of PEPCK, respectively (C group vs CG group). Furthermore, the WT group showed a significant increase in PEPCK expression (C group vs WT group) in the duodenum (24%; p = 0.003; Figure 2(b)) and jejunum (65%; p = 0.0005; Figure 3(b)). However, l-glutamine supplementation (WTG group vs WT group) did not influence PEPCK expression in the duodenum (p = 0.299; Figure 2(b)) and jejunum (p = 0.350; Figure 3(b)).

Immunoreactive intensity of G6Pase and PEPCK in histological sections

A similar intensity of immunoreactive G6Pase (IR-G6Pase) was observed in tissues of C and CG groups, either in the duodenum (p = 0.456; Figure 2(c)) or in the jejunum (p = 0.082; Figure 3(c)). A similar level of IR-G6Pase was also observed (WTG group vs WT group) in the duodenum (p = 0.372; Figure 2(c)) and jejunum (p = 0.721; Figure 3(c)).

The WT group exhibited a significant increase (50%; p < 0.0001; Figure 2(c)) in duodenal IR-G6Pase (WT group vs C group). In contrast, we observed a significant reduction (52%; p < 0.0001; Figure 3(c)) in jejunal IR-G6Pase (WT group vs C group).

Glutamine supplementation promoted a significant reduction (13%; p = 0.013; Figure 2(d)) of duodenal IR-PEPCK and its marked increase in the jejunum (92%; p < 0.0001; Figure 3(d)) when comparing C and CG groups. Furthermore, WT and WTG groups exhibited a similar level of (p = 0.580; Figure 2(d)) IR-PEPCK in the duodenum. However, the WTG group showed a significantly higher (17%; p = 0.0002; Figure 3(d)) jejunal IR-PEPCK level compared to the WT group. Representative photomicrographs illustrating the immunoreactive intensity of G6Pase and IR-PEPCK staining in the duodenum and jejunum are illustrated in Figures 2(e)–(l) and 3(e)–(l), respectively.

Discussion

The development of a cancer leads to a loss of energy balance in response to the intensification of catabolism, which, in turn, results in cancer-associated cachexia.^1–13 The lower cachexia index and reduced body weight loss in WTG rats compared to the WT group confirmed our previous studies demonstrating the beneficial effects of l-glutamine supplementation in the diet, particularly in the small intestine.^33,37

These anti-cachectic effects could be attributed to the higher levels of l-glutamine available in the blood. This, in turn, would have led to an improvement in the antioxidant status of healthy cells in the small intestine by reestablishing their levels of l-glutamine,^13,38 crucial in maintaining their digestive and absorptive capacities.^30,31 Moreover, an improvement of the immune system occurs in response to l-glutamine via a reduction of tumor necrosis factor (TNF)-α, IL-1, and IL-6 levels.³⁸ Furthermore, l-glutamine prevents proteasomal degradation and apoptosis³⁴ and reduces hypoxia,³⁸ nitric oxide synthesis, and oxidative stress.^37,39 Such effects may be considered positive and help explain the inhibition of tumor growth by l-glutamine.

The role of intestinal gluconeogenesis in the maintenance of glycemia during fasting is well established.^14–26,40 The ability of the intestine to express G6Pase and PEPCK and respond to signaling molecules that regulate the gene expression of these enzymes has been described previously.^15,18

Increased PEPCK (expression and IR-PEPCK) in the jejunum and duodenum of WT rats can be explained by the development of compensatory responses against the negative energy balance that occurs in cachectic animals (WT group vs C group). In such animals, substances released by the tumor promote the synthesis of glucose that supplies the high energy demand of the tumor.^41,42 For example, according to Croset et al.,¹⁴ the intestinal synthesis of glucose from l-glutamine occurs because the small intestine expresses glutaminase.

l-Glutamine supplementation promoted the increased expression of jejunal IR-PEPCK (WTG rats vs WT rats). In agreement with our results, Mithieux et al.²² also observed the increased activity and expression of jejunal PEPCK compared to that in other segments. These results suggest an important role for l-glutamine in promoting gluconeogenesis, thereby supplying glucose as energy supply to healthy cells without affecting tumor growth.

In contrast to the jejunum, l-glutamine supplementation did not change the expression of PEPCK in the duodenum. In fact, the duodenum and jejunum exhibit different capacities to express and regulate the enzymatic activity of gluconeogenesis.²²

The higher glycemia observed in the WTG group in comparison with WT animals could be attributed to a metabolic adaptation of tumor cells to the higher availability of l-glutamine, leading to a lower glucose uptake as reported by Daye and Wellen.⁴³ The increased glycemia may also be related to increased gluconeogenesis activity in the small intestine due to the increased availability of glutamine as a glucose precursor.^22,26,44,45 Our results are consistent with the higher expression of gluconeogenic enzymes found in the small intestine (G6Pase and PEPCK) of WT rats supplemented with l-glutamine.

WT rats supplemented with l-glutamine in their diet also showed higher insulinemia, probably as a consequence of increased glycemia.^46–48 This could be attributed to the stimulating effect of l-glutamine on insulin secretion, which is reduced in WT animals without supplementation with this amino acid.^49,50 Furthermore, considering that insulin can inhibit Walker-256 tumor growth,⁵¹ the beneficial effects of l-glutamine supplementation on slowing tumor growth in WTG rats could be attributed, at least in part, to the higher insulin levels measured in the WTG group. The mechanism by which insulin slows weight loss and inhibits cachexia associated with Walker-256 tumor growth could involve its classical effects in stimulating lipid and protein anabolism.⁵² It follows that the antitumor activity of insulin may allow this hormone to be used in combination with chemotherapy to treat cancer.^53–55

Conclusion

l-Glutamine supplementation of diet for 10 days in a cancer cachexia animal model promoted increased PEPCK expression in the jejunum that was associated with a significant elevation of glycemia and insulinemia, suggesting a beneficial effect on energy balance. An indirect effect on insulin secretion by l-glutamine is likely to prevent catabolism, thus reducing the harmful effects of cachexia.

Footnotes

Compliance with ethical standards

The authors certify that they comply with the ethical guidelines for authorship and publishing of Tumor Biology.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical approval

All procedures performed in studies involving animals were in accordance with the ethical standards of the Brazilian Society for Laboratory Animal Science (SBCAL) and were approved by the Ethics Committee for Animal Experimentation of the State University of Maringá (protocol number: 099/2012).

Funding

This research was supported by the Brazilian research agencies: Araucária Foundation (grant number: 266/2014) and Research Program for the unified health system—PPSUS (Process 987/2013).

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l -Glutamine supplementation promotes an improved energetic balance in Walker-256 tumor–bearing rats

Abstract

Keywords

Introduction

Methods

Animal model of cachexia and supplementation with l-glutamine

Calculation of cachexia index

Plasma concentration of corticosterone, glucose, insulin, and urea

Western blot for G6Pase and PEPCK

Immunohistochemical technique for G6Pase and PEPCK

Analysis of immunostaining for G6Pase and PEPCK

Statistical analysis

Results

Plasma concentration of corticosterone, glucose, insulin, and urea

Western blot

Immunoreactive intensity of G6Pase and PEPCK in histological sections

Discussion

Conclusion

Footnotes

Compliance with ethical standards

Declaration of conflicting interests

Ethical approval

Funding

References