Glutamine or whey-protein supplementation on alloxan-induced diabetic rats. Effects on CD4+ and CD8+ lymphocytes

Efeitos da oferta de glutamina ou de proteína do soro de leite sobre os linfócitos CD4+ e CD8+ em ratos diabéticos aloxano induzidos

ORIGINAL ARTICLE

Glutamine or whey-protein supplementation on alloxan-induced diabetic rats. Effects on CD⁴⁺ and CD⁸⁺ lymphocytes¹

Efeitos da oferta de glutamina ou de proteína do soro de leite sobre os linfócitos CD⁴⁺ e CD⁸⁺ em ratos diabéticos aloxano induzidos

Renato Motta Neto^I; Sergio Botelho Guimarães^II; Sônia Leite da Silva^III; José Napoleão da Cruz^IV; Thiago Dias^V; Paulo Roberto Leitão de Vasconcelos^VI

^IMaster in Patology, Doctoral Fellow Post-Graduate Student, UFC, Faculty of Pharmacy, University of Fortaleza (UNIFOR). Brazil

^IIAssociate Professor, Department of Surgery. Head, LABCEX. Federal University of Ceará (UFC). Brazil

^IIIAssociate Professor, Department of Nephrology, UNIFOR. Brazil

^IVPharmacist, Central Laboratory of Ceará (LACEN-CE). Brazil

^VGraduate student, UFC. Brazil

^VIAssociate Professor, Department of Surgery. Coordinator, Post-Graduation Program in Surgery, UFC. Brazil

^{Correspondence}

ABSTRACT

PURPOSE: To evaluate the effects of glutamine (L-Gln) or whey-protein supplementation on CD⁴⁺ and CD⁸⁺ lymphocytes in alloxan-induced diabetic rats.

METHODS: Thirty-two healthy male Wistar rats were used in the experiment. Eight rats served as baseline controls (G-1). The remaining 24 animals received alloxan 150mg/Kg intraperitonially dissolved in buffer solution and were equally distributed in 3 subgroups, upon induction of diabetes mellitus, and treated as follows: (G2): saline, 2.0ml; (G3): glutamine solution (0.7g/kg), 2.0 ml; and (G4): whey-protein (WPS) solution (0.7g/kg), 2.0 ml. All solutions were administered by daily 7:00 AM gavages during 30 days. Next, arterial blood samples (3.0 ml) were collected from anesthetized rats for CD⁴⁺ and CD⁸⁺ lymphocyte count through flow cytometry technology.

RESULTS: CD⁴⁺ and CD⁸⁺ counts decreased significantly in all groups compared with baseline values (G1). G2 rats CD⁴⁺/CD⁸⁺ ratio decreased significantly compared with G1. CD⁴⁺/CD⁸⁺ ratio increased significantly (>260%) in L-Gln treated group (G3) compared with saline-treated rats (G2). There were no statistical differences in lymphocyte counts (CD⁴⁺ and CD⁸⁺) between L-Gln (G3) and saline-treated (G2) groups. There was a significant reduction in CD⁸⁺ cell count compared with CD⁴⁺ cell count in L-Gln treated rats (G3).

CONCLUSION: The offer of L-Gln to experimental diabetic rats enhances the immunologic response to infection.

Key words: Glutamine. T-Lymphocytes. CD4-Positive T-Lymphocytes. CD8-Positive T-Lymphocytes. CD4-CD8 Ratio. Rats.

RESUMO

OBJETIVO: Avaliar os efeitos da suplementação de glutamina ou proteína do soro de leite ( PSL) sobre os linfócitos CD⁴⁺ e CD⁸⁺ em ratos diabéticos aloxano induzidos.

MÉTODOS: Trinta e dois ratos Wistar machos, saudáveis, foram utilizados no estudo. Oito ratos foram usados como controles basais (G1). Os 24 animais remanescentes foram equitativamente distribuídos em 3 subgrupos, após indução do diabetes mellitus por injeção intraperitonial de aloxano (150mg/Kg) e tratados como se segue: (G2): salina; (G3): 2,0 ml de solução de glutamina (0,75g/Kg);(G4): PSL, (0,7g/Kg), 2,0ml. Todas as soluções foram administradas por gavagem, diariamente, cada 7:00 h, durante 30 dias. Após esse período, foram coletadas amostradas de sangue arterial para contagem de linfócitos CD⁴⁺ e CD⁸⁺ por citometria de fluxo.

RESULTADOS: A população de linfócitos CD⁴⁺ e CD⁸⁺ diminuiu significantemente em todos os grupos em comparação aos valores encontrados no grupo G1. A razão CD⁴⁺/CD⁸⁺ foi significantemente maior (>260%) nos ratos tratados com L-Gln que nos ratos tratados com salina (G2). Não se observaram diferenças significantes na população de linfócitos CD⁴⁺ e CD⁸⁺ entre os grupos G3 e G2. Houve redução significante do número de células CD⁸⁺ quando comparado ao número de células CD⁴⁺ nos ratos tratados com L-Gln (G3).

CONCLUSÃO: A oferta de L-Gln em ratos diabéticos aloxano-induzidos melhora a resposta imunológica à infecção.

Descritores: Glutamina. Linfócitos T. Linfócitos T CD4-Positivos. Linfócitos T CD8-Positivos. Relação CD4-CD8. Ratos.

Introduction

The major cause of mortality and morbidity in diabetic subjects is due to immune dysfunction ¹. The four important factors that make diabetic subjects more prone to complications are: susceptibility to infections, hyperglycemia, vascular disease and nerve damage ². In diabetic patients, infection occurs with greater frequency and severity than in non-diabetics due to the impairment of both humoral and cellular immune responses ³. The severity of dysfunction increased susceptibility to infections in diabetic patients led the World Health Organization to classify diabetes mellitus as a secondary immunodeficiency disease ⁴. In recent years, the molecular biology of lymphocytes (CD⁴⁺and CD⁸⁺), macrophages, and neutrophils and the process of chemical communication between them has attracted considerable interest, and much progress has been made in our understanding of some aspects of fundamental importance not only in preventing or limiting infection, but also in the overall process of repair and clinical conditions of trauma, sepsis, burns, and recovery from surgery ⁵. Whey-protein is widely used by athletes due to its high caloric value and its influence on mass gain. Another function of this protein is the immune function through the tissue reparation and stimulation in the production of immunoglobulin ⁶. A recent development in the metabolic support of critically ill patients has been the evolution of a concept termed "immunonutrition". Nutrients have been identified that stimulate cells in such a manner as to enhance immunologic responses and potentially improve outcome. The amino acid glutamine is usually included in the list of "immunonutrients"that possess these biological effects ⁷. Glutamine is used at a high rate by lymphocytes, even in the resting state ⁸; the rate of glutamine use is increased if lymphocytes are activated, for example after stimulation with mitogens ⁹. The high rate of glutamine use and its increase upon activation are suggestive that glutamine plays na important role in these cells. Plasma glutamine concentrations are lowered in a variety os "stress" conditions, such as after burns ¹⁰, during sepsis ¹¹, after surgery ¹² and endurance exercise ¹³, and in athletic overtraining ¹⁴. These situations are associated with an increased susceptibility to infections, and it has been suggested that this partly may be due to the decreased supply of glutamine to immunocompetent cells such as lymphocytes ¹⁵. The aim of this study is to evaluate the effects of glutamine (L-Gln) or whey-protein supplementation on CD⁴⁺ and CD⁸⁺ lymphocytes in alloxan-induced diabetic rats.

Methods

Thirty two healthy male albino Wistar rats obtained from Faculty of Medicine (Federal University of Ceará) Small Animals Laboratory, weighing 150-200g (average 180g) were used in this study. All animals were in compliance with the University of Ceará ethical guidelines for International Organization of Medical Sciences (CIOMS) ethical code for animal experimentation (WHO Chronicle 1985;39(2): 51-6). Approval for experimental use of laboratory animals was obtained from the Commission of Ethics in Animal Research, Federal University of Ceará. The animals were housed in polypropylene cages at ambient temperature of 24ºC on a 12 h light-dark cycle. Rats were allowed free access to food (Purina chow) and fasted overnight before the experimental procedure. All procedures were performed under inhalatory diethyl ether anesthesia.

Induction of diabetes

Experimental diabetes was induced by intraperitoneal injection (150mg/kg) of Alloxan monohydrate (Acros Organics Research Laboratories, Inc., New Jersey, USA) dissolved in 0.1M sodium citrate buffer (pH 3,0) . After a period of 2 weeks, rats with marked hyperglycemia (fasting blood glucose > 250mg/dL) were selected and used for study.

Experimental design

Thirty-two healthy male Wistar rats were used in the experiment. Eight rats served as baseline controls (G-1). The remaining 24 animals received alloxan 150mg/Kg ip. dissolved in buffer solution. Upon induction of diabetes mellitus those rats were equally distributed in 3 subgroups and treated as follows: (G2): Saline, 2.0ml; (G3): glutamine solution (0.7g/kg), 2.0 ml; and (G4): whey-protein (WPS) solution (0.7g/kg), 2.0 ml. All solutions were administered by daily 7:00 AM gavages during 30 days. Next, arterial blood samples (3.0 ml) were collected from anesthetized rats for CD⁴⁺ and CD⁸⁺ lymphocyte count through flow cytometry technology.

Analysis of blood leukocyte subsets

Samples of 3.0ml of total blood in EDTA were collected through arterial punch until immunophenotyping was made. The rehearsals of Flow Citometrics for immunophenotyping of the peripheral lymphocytes were conducted according to a protocol developed specifically for this study as follows: in 5 ml tubes containing 15µl of anti-CD⁴⁺ monoclonal antibodies and anti-CD⁸⁺ marked with fluorescein isothiocyanate 30 µl of blood collected in ethylene diamine tetracetic acid (EDTA) were added. After a 20-minute room temperature incubation, all cell preparations were submitted to lysing of erythrocytes by adding 2ml of a lysing Solution (BD FACS Lysing Solution Catalog number 349202, BD Biosciences, Califórnia, U.S.A.); next, the leucocytes were washed in phosphate buffer solution (PBS) and fixed with 200 µl of a solution containing 1% paraformaldehyde in buffered saline 10g/l, pH 7.2. Analysis was carried out on a FACS Calibur Flow Citometer (Becton Dickinson Instruments, Cambridge, Mass) for a minimum of 10000 gated events.

Statistical analysis

Data were analyzed to assess the significance of differences between groups. ANOVA with post hoc Tukey's Multiple Comparison Test and Kruskal-Wallis/Dunn non-parametric tests were used for statistical analysis, as adequate. Statistical significance was accepted as p<0.05.

Results

CD4+ and CD8+ counts decreased significantly in all groups compared with baseline values (G1). G2 rats CD4+/CD8+ ratio decreased significantly compared with G1. CD4+/CD8+ ratio increased significantly (>260%) in L-Gln treated group (G3) compared with saline-treated rats (G2). There were no statistical differences between L-Gln (G3) and saline-treated (G2) groups (Figures 1 - 3). CD⁸⁺ cell count was significantly reduced compared with CD⁴⁺ cell count in control rats (G1). CD⁴⁺ and CD⁸⁺ counts in diabetic rats treated with saline or whey-protein were not different. However CD⁸⁺ cell count was significantly reduced (p<0.001) compared with CD⁴⁺ cell count in L-Gln-treated rats (Figure 4).

Discussion

High CD⁴⁺ and CD⁸⁺ lymphocyte counts are seen in healthy individuals (G1). It has been demonstrated that diabetes mellitus leads to a reduction in the number of CD⁴⁺ and CD⁸⁺ lymphocytes ^16-17 Insulin administration to diabetic individuals may change this picture by stimulating the glucose transporters and enabling the passage of the carbohydrate to the interior of the cell, and by inhibiting proteolysis and bloking the liberation and oxidation of aminoacids ¹⁶. Low levels of CD⁴⁺and CD⁸⁺ lymphocytes found in all diabetic rats was expected, as no insulin treatments were carried out in this study. It has been widely demonstrated that the decrease in lymphocyte cells play an important role in the immunologic response to infections. Diabetic patients show functional loss in the defense mechanism attributed to these cells such as quimiotaxy, adhesion and fagocytosis ¹⁷. In vitro studies using rat macrophage cells have demonstrated that fagocytosis is directly related to the concentration of glutamine available¹⁰. Juretic et al. ¹⁸ have demonstrated that glutamine acts as an activator of Killer cell promoting the lysing of the aggressing agent. Malfunctions found in macrophages are associated with the depletion of NADPH and with the increase in the concentrations and the advance of proteic glicolisation which lead to the induction and liberation of cytocines like the interleukines-1 (IL-1) and tumor necrosis factor ¹⁰. The first (IL-1) promotes the liberation of the free radicals resulting in cell injury ¹⁹. Diabetic patients have reduced capacity to produce interferon gama (IFN-á), resulting in decreased T lymphocytes production with direct effects on in the CD⁴⁺ and CD⁸⁺ cells ²⁰. In vitro studies have showed an increase in CD⁴⁺ lymphocytes and a decrease in CD⁸⁺ lymphocytes counts when glutamine is added to the culture media; this effect is not noticed when another amino acid is added ⁷. The offer of glutamine to G3 rats promoted a significant reduction in CD⁸⁺ lymphocytes count. According to Ardavi in vitro lymphocytic proliferation of rat cells is dependent on glutamine ²¹. Glutamine stimulates the synthesis of nucleic acids and provides energy to the proliferation of mononuclear cells like the CD⁴⁺ lymphocytes. Besides, it is an important source of nitrogen for the synthesis of some intermediate constituents ²² CD⁴⁺/CD⁸⁺ ratio has been used for assessment of individuals immunodeficiency state¹⁰. The significant increase in G3 CD⁴⁺/CD⁸⁺ ratio (Fig. 3) compared with G2 rats and the significant reduction in CD⁸⁺ lymphocytes count in L-Gln treated rats (G3) found in this study suggests that the offer of L-Gln to diabetic individuals may alter the immunologic response to infection.

Conclusion

The offer of L-Gln to experimental diabetic rats enhances the immunologic response to infection.

Received: January 15, 2007

Review: February 13, 2007

Accepted: March 19, 2007

Conflitc of interest: none

Financial source: CAPES

• 1. Fairchild RS, Kyner JL, Abdou NJ, Specific immunoregulation abnormality in insulin dependent diabetes mellitus. J Lab Clin Med. 1982;99:175-85.
• 2. Pozzilli P, Signore A, Leslie RDG. Infections, immunity and diabetes. In: Aberti KGMM, Zimmet P, De Fronzo RA, Keen H, (Eds). International textbook of diabetes mellitus. Wiley; 1997. p.1231-41.
• 3. Smitherman KO, Peacock JE. Infectious emergencies in patients with diabetes mellitus. Med Clin North Am. 1995; 79: 53-77.
• 4. WHO. Immunodeficiency: report of scientific group. Technical report series 630, World Health Organization. Genova; 1978.
• 5. Tsiauou A, Hatziagelaki E,.Chaidaroglou A, Koniavitou KK, Degiannis D, Raptis SA. Correlation between intracellular interferon-gama (IFN-g) production by CD4 and CD8 lymphocytes and IFN-g gene polymorphism in patients with type 2 diabetes mellitus and latent autoimune diabetes of adults (LADA). Cytokine. 2005;31:135-41.
• 6. Michalski MC, Januel C. Does homogenization affect the human health properties of cows milk?. Food Sci Technol. 2006; 17:423-37.
• 7. Wilmore DW, Shabert JK. Role of glutamine in Immunologic responses. Nutrition. 1998;14:618-26.
• 8. Rourker AM, Rider LC. Glucose, glutamine and ketone body utilization by resting and concanavalin A activated rat splenic lymphocytes. Biochim Biophys Acta. 1989; 1010-342.
• 9. Calder PC. Fuel utilisation by cells of the immune system. Proc Nutr Soc. 1995;54-65.
• 10. Parry-Billings M, Evans J, Calder PC, Newsholme EA. Does glutamine contribute to immunosuppression after burn?. Lancet. 1990; 336:523-5.
• 11. Askanazi J, Carpantier YA, Michelsen CB. Muscle and Plasma amino acids following injury: Influence of intercurrant infection. Ann Surg. 1980;:192:78-85.
• 12. Parry-Billings M, Baigrie RJ, Lamont P.M, Morris PJ, Newsholme EA. Effects of major and minor surgery on plasma glutamine and cytokine levels. Arch Surg. 1992; 127:1237-.40.
• 13. Powell H, Castall L.M, Parry-Billings S.M, Desborough J.P, Hall G.M, Newsholme E.A. Growth hormone suppression and glutamine flue associated with cardiac surgery. Clin Physiol. 1994; 14:569-80.
• 14. Parry-Billing S.M, Budgett R, Koutedakisy. Plasma amino acid concentrations in the overtraining syndrome: possible effects on the immune system. Med Sci Sport Exer. 1992;24:1353-8.
• 15. Newsholme E.A, Newsholme P, Curi R, Crabtree B, Ardawi M.S.M. Glutamine metabolism in different tissues: its physiological and pathological importance. In: Kinney JM, Borum PR. (Eds). Perspectives in clinical nutrition. Baltimore: Urban and Schwarzenberg; 1989.
• 16. Gupta S, Lymphocytes response in diabetes mellitus. In: Gupta S. (Ed), Immunology of clinical and experimental diabetes. New York: Plenum Publishing Corp; 1984. p.326-29.
• 17. Pozzilli P, Nagh M, Visalli N, Pagoni S, .Beales P, Andreani D, Impaired CD4/CD8 lymphocytes ratio in patients with long standing diabetes mellitus, IRCS Med Sci. 1986; 14:648-49.
• 18. Mandrop PT, Bendtzenk N, Dinareld CA, Nerup J, Man tumor necrosis factor potentiates human interleukin mediated rat pancreatic b-cell cytotoxicity, JImmunol.1987; 139:4080-82.
• 19. Juretic A, Spagnoli GC, Horig H. Glutamine requerements in the generation of lymphokine activated killer cells. Clin Nutr. 1994;13:42-9.
• 20. W.Marhoffer M, Stein E. Maeser, E. Federlin, Impairment of polymorphonuclear leukocyte function and metabolic control of diabetes, Diabetes Care. 1992;15:256-60.
• 21. Ardawi M.S. Effect of glutamine-enriched total parenteral nutrition on septic rats. Clin Sci. 1991; 81(2):215-22.
• 22. Calder P.C. Glutamine and system. Clin Nutr. 1994; 13(1):2-8.

1

Research performed at Experimental Surgical Research Laboratory (LABCEX), Department of Surgery, Post-Graduation Program

, Federal University of Ceará (UFC). Brazil.

Publication Dates

• Publication in this collection
  24 May 2007
• Date of issue
  June 2007

History

• Accepted
  19 Mar 2007
• Reviewed
  13 Feb 2007
• Received
  15 Jan 2007