Diabetes causes marked changes in function and metabolism of rat neutrophils

in Journal of Endocrinology

Several studies have shown impairment of neutrophil function, a disorder that contributes to the high incidence of infections in diabetes. Since glucose and glutamine play a key role in neutrophil function, we investigated their metabolism in neutrophils obtained from the peritoneal cavity of streptozotocin-induced diabetic rats. The activities of hexokinase, glucose-6-phosphate dehydrogenase (G6PDH), phosphofructokinase (PFK), citrate synthase, phosphate-dependent glutaminase, NAD+-linked and NADP+-linked isocitrate dehydrogenase were assayed. Glucose, glutamine, lactate, glutamate and aspartate, and the decarboxylation of [U-14C], [1-14C] and [6-14C]glucose; [U-14C]palmitic acid; and [U-14C]glutamine were measured in 1-h incubated neutrophils. Phagocytosis capacity and hydrogen peroxide (H2O2) production were also determined. All measurements were carried out in neutrophils from control, diabetic and insulin-treated (2–4IU/day) diabetic rats. Phagocytosis and phorbol myristate acetate (PMA)-stimulated H2O2 production were decreased in neutrophils from diabetic rats. The activities of G6PDH and glutaminase were decreased, whereas that of PFK was raised by the diabetic state. The activities of the remaining enzymes were not changed. Diabetes decreased the decarboxylation of [1-14C]glucose and [U-14C]glutamine; however, [6-14C]glucose and [U-14C]palmitic acid decarboxylation was increased. These observations indicate that changes in metabolism may play an important role in the impaired neutrophil function observed in diabetes. The treatment with insulin abolished the changes induced by the diabetic state even with no marked change in glycemia. Therefore, insulin may have a direct effect on neutrophil metabolism and function.

Abstract

Several studies have shown impairment of neutrophil function, a disorder that contributes to the high incidence of infections in diabetes. Since glucose and glutamine play a key role in neutrophil function, we investigated their metabolism in neutrophils obtained from the peritoneal cavity of streptozotocin-induced diabetic rats. The activities of hexokinase, glucose-6-phosphate dehydrogenase (G6PDH), phosphofructokinase (PFK), citrate synthase, phosphate-dependent glutaminase, NAD+-linked and NADP+-linked isocitrate dehydrogenase were assayed. Glucose, glutamine, lactate, glutamate and aspartate, and the decarboxylation of [U-14C], [1-14C] and [6-14C]glucose; [U-14C]palmitic acid; and [U-14C]glutamine were measured in 1-h incubated neutrophils. Phagocytosis capacity and hydrogen peroxide (H2O2) production were also determined. All measurements were carried out in neutrophils from control, diabetic and insulin-treated (2–4IU/day) diabetic rats. Phagocytosis and phorbol myristate acetate (PMA)-stimulated H2O2 production were decreased in neutrophils from diabetic rats. The activities of G6PDH and glutaminase were decreased, whereas that of PFK was raised by the diabetic state. The activities of the remaining enzymes were not changed. Diabetes decreased the decarboxylation of [1-14C]glucose and [U-14C]glutamine; however, [6-14C]glucose and [U-14C]palmitic acid decarboxylation was increased. These observations indicate that changes in metabolism may play an important role in the impaired neutrophil function observed in diabetes. The treatment with insulin abolished the changes induced by the diabetic state even with no marked change in glycemia. Therefore, insulin may have a direct effect on neutrophil metabolism and function.

Introduction

Patients with diabetes mellitus have increased susceptibility to and severity of infections. Several studies have shown alterations in neutrophil function, an effect that contributes to the high incidence of infections in diabetic patients. The observed changes in neutrophils include impairment of the following: adhesion to endothelium and migration to the site of inflammation (Pereira et al. 1987), chemotaxis (Mowat & Baum 1971), bactericidal activity (Tan et al. 1974), phagocytosis (Nolan et al. 1978) and production of reactive oxygen species (ROS) (Sagone et al. 1983).

Neutrophil functions require energy (Mowat & Baum 1971, McMurray et al. 1990, Walrand et al. 2004), which is produced mainly by the metabolization of glucose to lactate (Beck & Valentine 1952, Borregaard & Herlin 1982). Only 2–3% of glucose is oxidized through the Krebs cycle in neutrophils (Beck 1958, Wood et al. 1963). These cells also utilize glutamine at high rates, which is mainly converted to glutamate, aspartate, lactate and CO2 (Pithon-Curi et al. 1997).

High levels of glucose and ketone bodies seem to influence neutrophil function through production of polyols (Wilson et al. 1986). Decreased rates of glycolysis and glycogen synthesis were observed in leukocytes of diabetic patients. These changes are abolished by in vivo insulin administration (Esmann 1983). Walrand et al.(2004) postulated that insulin may normalize neutrophil functions, not only by reestablishing the control of the intermediary metabolism, but also through a direct effect of the hormone on the cells. These authors postulated that changes in neutrophil metabolism might play a key role in the impaired function of these cells in diabetes.

In the present study, the metabolism of glucose and glutamine in neutrophils obtained from streptozotocin (STZ)-induced diabetic rats was investigated. Key enzyme activities of glycolysis (hexokinase and phosphofructokinase), the pentose-phosphate pathway (glucose-6-phosphate dehydrogenase), the Krebs cycle (citrate synthase) and glutaminolysis (phosphate-dependent glutaminase) were determined. In addition, the activities of NAD-linked and NADP-linked isocitrate dehydrogenase were also determined. Decarboxylation of [U-14C], [1-14C] and [6-14C]glucose; [U-14C]palmitic acid; and [U-14C]glutamine was measured in incubated neutrophils. Glucose and glutamine utilization, and production of lactate, glutamate and aspartate by incubated neutrophils were determined. Phagocytosis and production of hydrogen peroxide were also evaluated. The measurements were carried out in neutrophils from control, diabetic and insulin-treated diabetic rats.

Materials and Methods

Animals

Male Wistar rats weighing 200 ± 20 g (about 2 months of age) were obtained from the Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo. The rats were maintained at 23 ± 2 °C under a cycle of 12-h light:12-h darkness, and were allowed free access to food and water. The animal ethical committee of the Institute of Biomedical Sciences approved the experimental procedure of this study.

Induction of diabetes

The experimental type 1 diabetes was induced by intravenous injection of 65 mg/kg STZ dissolved in citrate buffer (pH 4·2). Control rats were injected with buffer only. At 48 h after STZ injection, the diabetic state was confirmed by blood glucose levels above 200 mg/dl estimated with the aid of a glucose meter (Roche). Blood samples were obtained from the cut tip of the animal’s tail. One group of diabetic rats was treated with neutral protamine Hagedorn (NPH) insulin. The daily dose was 2–4 IU by unique s.c. injection at 0900–1000 h for 2 weeks. The NPH insulin treatment was not sufficient to restore the glucose blood levels to control values.

Experimental procedure

After 2 weeks of diabetes induction, fed rats were killed by decapitation without anesthesia at 1100–1200 h. Neutrophils were obtained by intraperitoneal lavage with 30 ml PBS, 4 h after the intraperitoneal injection of 10 ml 1% (w/v) glycogen solution (Sigma type II, from oyster) in PBS. The cell suspension was centrifuged at 4 °C (500 g for 10 min). The number of viable cells (> 95% neutrophils) was determined in a Neubauer chamber under an optical microscope by Trypan blue exclusion (Pires de Melo et al. 1998, Pithon-Curi et al. 2002).

Phagocytosis

Neutrophils (1 × 106) were incubated for 40 min at 37 °C in 1 ml RPMI 1640 medium with opsonized particles of zymosan. The particles (1 × 107) were opsonized by incubation in the presence of control rat serum for 30 min at 37 °C. The cells that were capable of phagocytizing three or more particles were determined in a Neubauer chamber under an optical microscope by the violet crystal coloring method. The percentage of phagocytosis was expressed by the number of cells that had three or more particles of zymosan per total number of cells counted.

Hydrogen peroxide production

Hydrogen peroxide (H2O2) production was measured by the method of Pick and Mizel (1981), which is based on horseradish peroxidase-dependent conversion of phenol red by H2O2 to a colored compound. Briefly, the cells were incubated in the presence of 5 mM glucose and a solution of phenol red (0·5%) and horseradish peroxidase (5 mg/ml) (222 units/mg) at 37 °C for 1 h. The production of H2O2 was measured at rest and after stimulation with phorbol myristate acetate (PMA) (20 nM). The reaction was terminated by addition of 10 μl 1 M NaOH solution, and the amount of product formed was measured by spectrophotometry at 620 nm.

Assay of the enzyme activities

The activities of hexokinase (E.C. 2·7·1·1), glucose-6-phosphate dehydrogenase (G6PDH) (E.C. 1·1·1·49), phosphofructokinase (PFK) (E.C. 2·7·1·11), citrate synthase (E.C. 4·1·3·7), phosphate-dependent glutaminase (E.C. 3·5·1·2), NAD+-linked isocitrate dehydrogenase (E.C. 1·1·1·41) and NADP+-linked isocitrate dehydrogenase (E.C. 1·1·1·42) were determined as previously described (Mansour 1963, Crabtree & Newsholme 1972, Curthoys & Lowry 1973, Bergmeyer & Bernt 1974, Sugden & Newsholme 1975, Alp et al. 1976). Enzyme activities were expressed as nmol of substrate utilized/min per mg protein. A similar procedure was used in our previous studies (Guimarães et al. 1993, Costa-Rosa et al. 1996, Otton et al. 2002).

Cell incubation for measurement of consumption and production of metabolites

Neutrophils (1·0 × 106) were incubated for 1 h at 37 °C in PBS with glucose (5 mM) or glutamine (2 mM). After incubation, the cells were disrupted by the addition of 0·2 ml 25% (w/v) trichloroacetic acid solution. Protein was removed by centrifugation, and the supernatant fluid was neutralized with a Tris (hydroxymethyl-aminomethane)/KOH (0·5–2·0 M) solution for the measurement of the metabolites.

Assays of the metabolites

Samples of the incubation medium were used for measurements of glucose (Berham & Trinder 1972), glutamine (Windmueller & Spaeth 1974), lactate (Engel & Jones 1978), glutamate (Bernt & Bergmeyer 1974) and aspartate (Bergmeyer et al. 1974).

Spectrophotometric conditions of the enzyme assays and metabolite measurements

Activities of hexokinase, G6PDH, citrate synthase and PFK were assayed at 25 °C and activity of glutaminase at 37 °C. Citrate synthase activity was assayed by following the rate of change in absorbance at 412 nm and the remaining enzymes at 340 nm. The final volume of the assay mixtures in all cases was 1·0 ml. The production of NADH or NADPH was monitored in a Pharmacia Biotech spectrophotometer (model: Ultrospec 3000).

Incubation procedure for determination of metabolite decarboxylation

Neutrophils (1 × 107) were incubated for 60 min at 37 °C in 1 ml PBS with the following labeled metabolites: [U-14C], [1-14C] or [6-14C]glucose (5·0 mM and 0·2 μCi/ml); [U-14C]palmitic acid (0·1 mM and 0·2 μCi/ml); or [U-14C]glutamine (2·0 mM and 0·2 μCi/ml). Palmitic acid was previously dissolved in ethanol (20 mM) for addition to incubation medium. The incubation was stopped by adding 0·2 ml chloridric acid. 14CO2 produced from these metabolites was collected as previously described (Leighton et al. 1985) in a special apparatus containing phenylethylamine and methanol solution (1:1 v/v), and the radioactivity was measured in a Beckman-LS 5000TD scintillator (Beckman Instruments, Fullerton, CA, USA). A similar procedure was used in our previous studies (Otton et al. 2002, Hirabara et al. 2003). Another procedure was used to estimate the palmitic acid decarboxylation. The decrease of [U-14C]palmitic acid from the medium and cell preparation after 1-h incubation was determined.

Protein determination

The total protein content of neutrophils was measured by the method of Bradford (1976), using BSA as standard.

Statistical analysis

All results are expressed as means ± s.e.m. Student’s t-test or ANOVA followed by the Tukey–Kramer test was used to assess the significance of differences between groups. Data were considered as statistically significant at P ≤ 0·05.

Results

Relative to controls, animals rendered diabetic by STZ injection exhibited a significant reduction in body weight gain during the experimental period (Table 1). Blood glucose levels were significantly elevated in comparison to controls (by 3·5-fold). The treatment of diabetic rats with NPH insulin (2–4 IU/day) did not restore the blood glucose levels to the values of the control group (Table 1).

Neutrophil counts were performed 4 h after the intra-peritoneal injection of 10 ml 1% (w/v) glycogen solution. In all groups, a similar number of cells had migrated into the peritoneal cavity. The mean values (× 107; ± s.e.m.) were as follows: 23·3 ± 2·7 (control, n=11), 21·1 ± 2·6 (diabetic, n=6) and 23·4 ± 2·5 (insulin treated, n=7).

Phagocytosis and PMA-stimulated hydrogen peroxide production by neutrophils from the diabetic group were decreased (by 17% and 31% respectively) as compared with controls. The daily treatment with insulin restored phagocytosis and H2O2 production to the values found in neutrophils from control rats (Fig. 1A and B).

G6PDH activity was decreased (by 59%) in neutrophils from diabetic rats as compared with controls. The diabetic state also decreased phosphate-dependent glutaminase activity (by 22%) and increased PFK activity (by 32%) (Fig. 2). The treatment with insulin abolished the effect of diabetes on G6PDH, glutaminase and PFK activities. Hexokinase, citrate synthase and NAD+-linked and NADP+-linked isocitrate dehydrogenase activities were not changed by diabetes (Fig. 2).

Glucose and glutamine utilization by incubated neutrophils was not markedly affected by the diabetic state. Lactate production was decreased (by 24%) in neutrophils from diabetic rats as compared with controls, but the production of glutamate and aspartate was not modified. The treatment of diabetic rats with insulin restored the production of lactate by neutrophils to the values of the control group (Table 2).

Diabetes decreased the decarboxylation of [1-14C]glucose (by 31%) and [U-14C]glutamine (by 22%) by 1-h incubated neutrophils. On the other hand, [6-14C]glucose (by 55%) and [U-14C]palmitic acid (by 83%) decarboxylation was higher in neutrophils obtained from diabetic rats. The treatment with insulin abolished the changes induced by diabetes (Fig. 3). Similar results for palmitic acid decarboxylation (data not shown) were obtained by measuring the decrease of [U-14C]palmitic acid in the medium and cell preparation; there was an increase of 61% (P < 0·01) in neutrophils obtained from diabetic rats, and the treatment with insulin abolished the changes induced by the diabetic state. The decarboxylation of [U-14C]glucose was not affected by the diabetic state; glucose decarboxylation by neutrophils of the control and diabetic groups was 14·9 ± 0·6 and 14·0 ± 1·1 nmol/h per 107 cells (mean ± s.e.m. of two determinations from six animals in each group) respectively.

Discussion

Neutrophils constitute the first line of defense against bacterial and fungal infections. Phagocytosis stimulates the respiratory burst of neutrophils as a result of activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Bellavite 1988). The superoxide anion (O2) is the first metabolite generated through reduction of molecular oxygen by NADPH oxidase (Tauber et al. 1985, Bellavite 1988). The O2 formed dismutates spontaneously or via superoxide dismutase to H2O2, which is converted to hypochlorous acid by myeloperoxidase released from neutrophil granules (Tauber et al. 1985).

Conflicting results have been reported about the production of H2O2 by neutrophils in diabetes. In un-stimulated neutrophils from diabetic patients, no significant effect on H2O2 production was found (Noritake et al. 1992, Inoue et al. 1996), while Zozulinska et al.(1996) found it to be increased. After incubation with PMA, neutrophils from diabetic patients have shown low (Noritake et al. 1992, Inoue et al. 1996) or unchanged (Zozulinska et al. 1996) production of H2O2 as compared with cells from healthy subjects. Neutrophils from diabetic patients with nephropathy have shown increased production of H2O2 as compared with those without kidney complications (Watanabe 1992). In our study, neutrophils from diabetic rats showed low production of H2O2 after PMA stimulation was abolished by treatment with insulin. There was no other study on H2O2 production by rat neutrophils in diabetes. An increase in H2O2 production by nonactivated neutrophils in the insulin-treated group was observed. Some studies have shown that insulin activates a plasma membrane enzyme system that presents properties of a Nox (NADPH oxidase enzymes). The enzyme system catalyzes the reduction of oxygen to superoxide (2 O2 +NADP++H+), which is spontaneously or by action of superoxide dismutase converted to H2O2 (Mukherjee et al. 1978, Rhee et al. 2003, Goldstein et al. 2005).

Impairment of phagocytosis and decreased release of lysosomal enzymes in neutrophils from diabetic patients have been observed by many researchers (Bybee & Rogers 1964, Bagdade et al. 1972, Sagone et al. 1983, Wilson & Reeves 1986). Studies with diabetic rats and mice also showed a decreased neutrophil phagocytosis capacity (Canturk et al. 1998, Panneerselvam & Govindasamy 2003). The lowering of blood glucose levels by insulin treatment has been reported to have significant correlation with the improvement of phagocytosis capacity by neutrophils (Jakelic et al. 1995, Delamaire et al. 1997). In our study, the decreased capacity of neutrophils from diabetic rats to phagocytize zimosan particles was reversed by insulin treatment even under high blood glucose levels.

Previous studies on the effect of diabetes on glucose metabolism in neutrophils are controversial. Esmann (1972) and Munroe & Shipp (1965) did not observe differences in glucose utilization by neutrophils from healthy and diabetic patients. Nevertheless, decreased utilization of glucose by neutrophils from diabetic patients was found by others (Martin et al. 1954, Esmann 1983). Munroe & Shipp (1965) did not observe alteration, whereas Martin et al.(1954) and Esmann (1983) found decreased production of lactate by neutrophils from diabetic patients. In the present study, there was no alteration in glucose utilization by neutrophils from diabetic rats, whereas lactate production was decreased. These findings support the proposition that the diabetic state reduces glycolysis activity in neutrophils.

Neutrophils from diabetic rats had no significant change in glucose oxidation, citrate synthase, and NAD+-linked and NADP+-linked isocitrate dehydrogenase activities, suggesting that the flux of substrates through the Krebs cycle was not altered. Decreased citrate synthase activity was observed in lymphocytes from diabetic rats (Otton et al. 2002), whereas no significant change was found in macrophages (Costa-Rosa et al. 1996). There was no study on glucose oxidation, or NAD+-linked and NADP+-linked isocitrate dehydrogenase activities by rat neutrophils in diabetes.

The pentose-phosphate pathway oxidizes glucose-6-phosphate to intermediates of the glycolytic pathway, generating NADPH and ribose-5-phosphate for fatty acid and nucleotide synthesis respectively (Casazza & Veech 1986). NADPH is important for NADPH oxidase activity and for glutathione reductase to recycle oxidized glutathione in neutrophils (Pithon-Curi et al. 1998, 1999). Similarly, glutamine is also probably very important in neutrophils to provide glutamate for glutathione synthesis (Newsholme et al. 2003).

To examine the flux of glucose through the pentose-phosphate pathway of neutrophils from diabetic rats, G6PDH activity and decarboxylation of [1-14C] and [6-14C]glucose were determined. The difference between 14CO2 production from [1-14C] and [6-14C]glucose estimates the flux of glucose through the pentose-phosphate pathway (Larrabee 1989). The difference for neutrophils (107; ± s.e.m.) from the diabetic group was 2·35 ± 0·2 nmol/h compared with 3·79 ± 0·15 nmol/h for the control group (decrease of 38%; P < 0·001). Insulin treatment abolished the difference between the decarboxylation of [1-14C] and [6-14C]glucose. The activity of G6PDH was decreased in neutrophils from diabetic rats, as was also found in macrophages and lymphocytes (Costa-Rosa et al. 1996, Otton et al. 2002). These findings support the proposition that the activity of the pentose-phosphate pathway is decreased in leukocytes from diabetic rats. Leukocytes with deficiency of G6PDH activity present impaired phagocytosis, bactericidal capacity and superoxide production (Gray et al. 1973, Roos et al. 1999). Moreover, decreased flux of glucose through the pentose-phosphate pathway is expected to reduce the production of NADPH and ribose-5-phosphate. Therefore, reduced pentose-phosphate pathway activity may be related to impaired neutrophil function in the diabetic state. Although the production of NADP-linked isocitrate dehydrogenase activity was not affected by the diabetic state, this may not be sufficient to compensate for the reduced flux of substrates in the pentose-phosphate pathway.

Although decreased lactate production was observed in neutrophils from diabetic rats, the PFK maximal activity was increased. Similar results were observed in mesenteric lymph nodes and thymus lymphocytes from diabetic rats (Moreno-Aurioles et al. 1996, Otton et al. 2002). The activity of PFK is stimulated by fructose 2,6-biphosphate (Wegener & Krause 2002) and inhibited by ATP at low fructose 6-phosphate content, but not at high fructose 6-phosphate concentration (Mansour 1963). In this study, we did not observe changes in the glucose consumption and oxidation in neutrophils from STZ-induced diabetic rats, but lactate production and pentose-phosphate pathway activity were markedly reduced. Therefore, it is expected that the content of intermediates of glycolysis, such as fructose 6-phosphate and fructose 2,6-biphosphate, are elevated in this condition. This may partially explain the increase in the PFK activity. In accordance with this proposition, Moreno-Aurioles et al.(1996) found increased fructose 2,6-biphosphate content and PFK activity in neutrophils from STZ-induced diabetic rat.

The production of glutamate and aspartate was not altered in neutrophils from diabetic rats incubated in the presence of 2 mM glutamine. Nevertheless, glutamine oxidation and glutaminase activity were significantly decreased in neutrophils from diabetic rats. Glutamine plays an important role in protein (as amino-acid source), lipid (through NAD(P)H production) and nucleotide synthesis (through purine and pyrimidine production), and in NADPH oxidase activity (Newsholme et al. 2003, Curi et al. 2005). Glutamine raises the in vitro bacterial killing activity and the rate of ROS production by neutrophils (Ogle et al. 1994, Pithon-Curi et al. 1998, 2002). Pithon-Curi et al.(2003) showed that glutamine has a protective effect on neutrophil apoptosis. Therefore, decreased glutamine utilization may contribute to the impaired function by increasing the occurrence of apoptosis in neutrophils from diabetic rats. This issue remains to be investigated.

Diabetes leads to an increase in plasma levels of free fatty acids and triacylglycerols (Boden 1999, Kelley & Mandarino 2000). We have previously demonstrated increased fatty acid oxidation in lymphocytes from diabetic rats (Otton et al. 2002). Herein, we observed that palmitic acid decarboxylation is also elevated in neutrophils from diabetic rats.

In summary, neutrophils from diabetic rats present impaired metabolism of glucose and glutamine. On the other hand, palmitic acid oxidation is increased, and this may compensate for the reduction in glucose and glutamine utilization for ATP production. These metabolic changes may be related to impaired functions of these cells, such as phagocytosis and hydrogen peroxide production. The changes in metabolism and function of neutrophils from diabetic rats are fully abolished by insulin treatment, even with no marked changes in glycemia. These findings show that insulin may have a direct effect on neutrophil metabolism and function.

Table 1

Body weight gain and blood glucose levels of the control, diabetic and insulin-treated diabetic groups

nBody weight gain (g)Blood glucose levels (mg/dL)
Rats were rendered diabetic by streptozotocin injection (65 mg/kg, i.v.) and the measurements were carried out 2 weeks afterward. NPH insulin (2–4 IU/day) was subcutaneously administered during the experiment period. Values are presented as means ± s.e.m.. n indicates the number of rats used in each group. aP < 0·001 as compared to diabetic and insulin-treated rats; bP < 0·05 as compared to insulin-treated rats.
Groups
Control1552 ± 5a81 ± 4a
Diabetic1025 ± 4b365 ± 25
Insulin-treated diabetic1144 ± 2301 ± 13
Table 2

Consumption and production of metabolites by 1-h incubated neutrophils from the control, diabetic and insulin-treated diabetic groups

nGlucose consumptionLactate productionGlutamine consumptionGlutamateProduction of aspartateLactate
Consumption of glucose and glutamine and production of lactate, glutamate and aspartate were determined in 1 h incubated neutrophils. The cells were obtained from the peritoneal cavity of the rats, and the results are expressed as nmol/h per mg protein. The values are presented as means ± s.e.m.n represents the number of rats per group. aP > 0·05 as compared to control and insulin-treated diabetic rats.
Groups
Control6855 ± 170945 ± 651110 ± 95390 ± 5515 ± 50215 ± 5
Diabetic5755 ± 150700 ± 65a1015 ± 70380 ± 10555 ± 65230 ± 40
Insulin-treated diabetic6815 ± 150935 ± 351115 ± 85390 ± 5510 ± 60230 ± 35
Figure 1
Figure 1

Phagocytosis capacity (A) and hydrogen peroxide production (B) by neutrophils. The values are presented as means ± s.e.m. of 10 determinations from at least five animals in each group. aP < 0·05 as compared to control, bP < 0·01 as compared to insulin-treated diabetic rats and cP < 0·05 as compared to diabetic rats.

Citation: Journal of Endocrinology 188, 2; 10.1677/joe.1.06438

Figure 2
Figure 2

Enzyme activities of neutrophils. The results are expressed as nmol/min per mg protein, and the values are presented as means ± s.e.m. of 10 determinations from five animals in each group. aP < 0·01 as compared to control, bP < 0·05 as compared to diabetic rats, cP < 0·05 as compared to control and dP < 0·01 as compared to diabetic rats.

Citation: Journal of Endocrinology 188, 2; 10.1677/joe.1.06438

Figure 3
Figure 3

Decarboxylation of labeled metabolites by 1 h incubated neutrophils. The values are presented as means ± s.e.m. of eight determinations from four animals in each group. aP < 0·01 as compared to control, bP < 0·05 as compared to diabetic rats and cP < s0·05 as compared to control and insulin treated diabetic rats.

Citation: Journal of Endocrinology 188, 2; 10.1677/joe.1.06438

The authors are indebted to the constant assistance of E P Portiolli and G de Souza. This research is supported by FAPESP, CNPq and CAPES. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

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  • InoueS Lan Y Muran J & Tsuji M 1996 Reduced hydrogen peroxide production in neutrophils from patients with diabetes. Diabetes Research and Clinical Practices33119–127.

    • Search Google Scholar
    • Export Citation
  • JakelicJ Kokic S Hozo I Maras J & Fabijanic D 1995 Nonspecific immunity in diabetes: hyperglycemia decreases phagocytic activity of leukocytes in diabetic patients. Medical Archives499–12.

    • Search Google Scholar
    • Export Citation
  • KelleyDE & Mandarino LJ 2000 Fuel selection in human skeletal muscle in insulin resistance: a reexamination. Diabetes49677–683.

  • LarrabeeMG1989 The pentose cycle (hexose monophosphate shunt). Rigorous evaluation of limits to the flux from glucose using 14CO2 data with applications to peripheral ganglia of chicken embryos. Journal of Biological Chemistry26415875–15879.

    • Search Google Scholar
    • Export Citation
  • LeightonB Challiss RA Lozeman FJ & Newsholme EA 1985 The effect of prostaglandins E1 E2 and F2 alpha and indomethacin on the sensitivity of glycolysis and glycogen synthesis to insulin in stripped soleus muscles of the rat. Biochemistry Journal227337–340.

    • Search Google Scholar
    • Export Citation
  • MansourTE1963 Studies on heart phosphofructokinase: purification inhibition and activation. Journal of Biological Chemistry2382285–2292.

    • Search Google Scholar
    • Export Citation
  • MartinSP Chaudhuri SN Green R & McKinney GR 1954 The effect of adrenal steroids on aerobic lactic acid formation in human leukocytes. Journal of Clinical Investigation33358–360.

    • Search Google Scholar
    • Export Citation
  • McMurrayRW RW Bradsher RW Steele & NS Pilkington 1990 Effect of prolonged modified fasting in obese persons on in vitro markers of immunity: lymphocyte function and serum effects on normal neutrophils. American Journal of Medical Science299379–385.

    • Search Google Scholar
    • Export Citation
  • Moreno-AuriolesVR Montano R Conde M Bustos R & Sobrino F 1996 Streptozotocin-induced diabetes increases fructose 26-biphosphate levels and glucose metabolism in thymus lymphocytes. Life Sciences58477–484.

    • Search Google Scholar
    • Export Citation
  • MowatAG & Baum J 1971 Chemotaxis of polymorphonuclear leukocytes from patients with diabetes mellitus. New England Journal Medicine284621–627.

    • Search Google Scholar
    • Export Citation
  • MukherjeeSP Lane RH & Lynn WS 1978 Endogenous hydrogen peroxide and peroxidative metabolism in adipocytes in response to insulin and sulfhydryl reagents. Biochemical Pharmacology272589–2594.

    • Search Google Scholar
    • Export Citation
  • MunroeJF & Shipp JC 1965 Glucose metabolism in leucocytes from patients with diabetes mellitus with and without hypercholesteremia. Diabetes14584–590.

    • Search Google Scholar
    • Export Citation
  • NewsholmeP Lima MM Procopio J Pithon-Curi TC Doi SQ Bazotte RB & Curi R 2003 Glutamine and glutamate as vital metabolites. Brazilian Journal of Medical Biology and Research36153–163.

    • Search Google Scholar
    • Export Citation
  • NolanCN Beaty HN & Bagdade JD 1978 Further characterization of the impaired bactericidal function of granulocytes in patients with poorly controlled diabetes. Diabetes27889–894.

    • Search Google Scholar
    • Export Citation
  • NoritakeM Katsura Y Shinomiya N Kanatani M Uwabe Y Nagata N & Tsuru S 1992 Intracellular hydrogen peroxide production by peripheral phagocytes from diabetic patients. Dissociation between polymorphonuclear leucocytes and monocytes. Clinical and Experimental. Immunology88269–274.

    • Search Google Scholar
    • Export Citation
  • OgleCK Ogle JD Mao JX Simon J Noel JG Li BG & Alexander JW 1994 Effect of glutamine on phagocytosis and bacterial killing by normal and pediatric burn patient neutrophils. Journal of Parenteral and Enteral Nutrition18128–133.

    • Search Google Scholar
    • Export Citation
  • OttonR Mendonça JR & Curi R 2002 Diabetes causes marked changes in lymphocyte metabolism. Journal of Endocrinology17455–61.

  • PanneerselvamS & Govindasamy S 2003 Sodium molybdate improves the phagocytic function in alloxan-induced diabetic rats. Chemical and Biological Interactions145159–163.

    • Search Google Scholar
    • Export Citation
  • PereiraMAA Sannomiya P & Garcia-Leme J 1987 Inhibition of leukocyte chemotaxis by factor in alloxan-induced diabetic rat plasma. Diabetes361307–1314.

    • Search Google Scholar
    • Export Citation
  • PickE & Mizel D 1981 Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader. Journal of Immunological Methods46211–226.

    • Search Google Scholar
    • Export Citation
  • Pires de MeloM Curi TC Miyasaka CK Palanch AC & Curi R 1998 Effect of indole acetic acid on oxygen metabolism in cultured rat neutrophil. General Pharmacology31573–578.

    • Search Google Scholar
    • Export Citation
  • Pithon-CuriTC Melo MP Azeveda RB Zorn TMT & Curi R 1997 Glutamine utilization by rat neutrophils: presence of phosphate-dependent glutaminase. American Physiology Society273C1124–C1129.

    • Search Google Scholar
    • Export Citation
  • Pithon-CuriTC Melo MP Palanch AC Miyasaka CK & Curi R 1998 Percentage of phagocytosis production of O2 H2O2 and NO and antioxidant enzyme activities of rat neutrophils in culture. Cell Biochemistry and Function1643–49.

    • Search Google Scholar
    • Export Citation
  • Pithon-CuriTC Levada AC Lopes LR Doi SQ & Curi R 2002 Evidence that glutamine is involved in neutrophil function. Cell Biochemistry and Function2081–86.

    • Search Google Scholar
    • Export Citation
  • Pithon-CuriTC Schumacher RI Freitas JJS Lagranha CJ Newsholme P Palanch AC Doi SQ & Curi R 2003 Glutamine delays spontaneous apoptosis in neutrophils. American Journal of Physiology284C1355–1361.

    • Search Google Scholar
    • Export Citation
  • RheeSG Chang TS Bae YS Lee SR & Kang SW 2003 Cellular regulation by hydrogen peroxide. Journal of the American Society of Nephrology14S211–S215.

    • Search Google Scholar
    • Export Citation
  • RoosD van Zwieten R Wijnen JT Gomez-Gallego F de Boer M Stevens D Pronk-Admiraal CJ de Rijk T van Noorden CJ Weening RS et al.1999 Molecular basis and enzymatic properties of glucose 6-phosphate dehydrogenase volendam leading to chronic nonspherocytic anemia granulocyte dysfunction and increased susceptibility to infections. Blood942955–2962.

    • Search Google Scholar
    • Export Citation
  • SagoneAL JR Greenwald J Kraut EH Bianchine J & Singh D 1983 Glucose: a role as a free radical scavenger in biological systems. Journal of Laboratory and Clinical Medicine10197–104.

    • Search Google Scholar
    • Export Citation
  • SugdenPH & Newsholme EA 1975 Activities of citrate synthase NAD+-linked and NADP+-linked isocitrate dehydrogenase glutamate dehydrogenase aspartate aminotransferase and alanine aminotransferase in nervous tissues from vertebrates and invertebrates. Biochemistry Journal150105–111.

    • Search Google Scholar
    • Export Citation
  • TanJS Anderson JL Watanakunakorn C & Phair JP 1974 Neutrophil dysfunction in diabetes mellitus. Clinical Experiments8526–33.

  • TauberAI Wright J Higson FK Edelman SA & Waxman DJ 1985 Purification and characterization of the human neutrophil NADH-cytochrome b5 reductase. Blood66673–678.

    • Search Google Scholar
    • Export Citation
  • WalrandS Guillet C Boirie Y & Vasson M-P 2004 In vivo evidences that insulin regulates human polymorphonuclear neutrophil functions. Journal of Leukocyte Biology761104–1110.

    • Search Google Scholar
    • Export Citation
  • WatanabeA1992 The production of hydrogen peroxide by neutrophilic polymorphonuclear leukocytes in patients with non-insulin dependent diabetes mellitus Nippon Jinzo Gakkai Shi341219–1225.

    • Search Google Scholar
    • Export Citation
  • WegenerG & Krause U 2002 Different modes of activating phosphofructokinase a key regulatory enzyme of glycolysis in working vertebrate muscle. Biochemistry Society Transactions30264–270.

    • Search Google Scholar
    • Export Citation
  • WilsonRM & Reeves WG 1986 Neutrophil phagocytosis and killing in insulin-dependent diabetes. Clinical and Experimental Immunology63478–484.

    • Search Google Scholar
    • Export Citation
  • WilsonRM Clarke P Barkes H Heller SR & Tattersall RB 1986 Starting insulin treatment as an outpatient. Report of 100 consecutive patients followed up for at least one year. Journal of the American Medical Association256877–880.

    • Search Google Scholar
    • Export Citation
  • WindmuellerHG & Spaeth AE 1974 Uptake and metabolism of plasma glutamine by the small intestine. Journal of Biological Chemistry2495070–5079.

    • Search Google Scholar
    • Export Citation
  • WoodHG Katz J & Landau BR 1963 Estimation of pathways of carbohydrate metabolism. Biochemistry Journal338809–847.

  • ZozulinskaDA Wierusz-Wysocka B Wysocki H Majchrzak AE & Wykretowicz A 1996 The influence of insulin-dependent diabetes mellitus (IDDM) duration on superoxide anion and hydrogen peroxide production by polymorphonuclear neutrophils. Diabetes Research and Clinical Practices33139–144.

    • Search Google Scholar
    • Export Citation

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    Phagocytosis capacity (A) and hydrogen peroxide production (B) by neutrophils. The values are presented as means ± s.e.m. of 10 determinations from at least five animals in each group. aP < 0·05 as compared to control, bP < 0·01 as compared to insulin-treated diabetic rats and cP < 0·05 as compared to diabetic rats.

  • View in gallery

    Enzyme activities of neutrophils. The results are expressed as nmol/min per mg protein, and the values are presented as means ± s.e.m. of 10 determinations from five animals in each group. aP < 0·01 as compared to control, bP < 0·05 as compared to diabetic rats, cP < 0·05 as compared to control and dP < 0·01 as compared to diabetic rats.

  • View in gallery

    Decarboxylation of labeled metabolites by 1 h incubated neutrophils. The values are presented as means ± s.e.m. of eight determinations from four animals in each group. aP < 0·01 as compared to control, bP < 0·05 as compared to diabetic rats and cP < s0·05 as compared to control and insulin treated diabetic rats.

References

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    • Export Citation
  • JakelicJ Kokic S Hozo I Maras J & Fabijanic D 1995 Nonspecific immunity in diabetes: hyperglycemia decreases phagocytic activity of leukocytes in diabetic patients. Medical Archives499–12.

    • Search Google Scholar
    • Export Citation
  • KelleyDE & Mandarino LJ 2000 Fuel selection in human skeletal muscle in insulin resistance: a reexamination. Diabetes49677–683.

  • LarrabeeMG1989 The pentose cycle (hexose monophosphate shunt). Rigorous evaluation of limits to the flux from glucose using 14CO2 data with applications to peripheral ganglia of chicken embryos. Journal of Biological Chemistry26415875–15879.

    • Search Google Scholar
    • Export Citation
  • LeightonB Challiss RA Lozeman FJ & Newsholme EA 1985 The effect of prostaglandins E1 E2 and F2 alpha and indomethacin on the sensitivity of glycolysis and glycogen synthesis to insulin in stripped soleus muscles of the rat. Biochemistry Journal227337–340.

    • Search Google Scholar
    • Export Citation
  • MansourTE1963 Studies on heart phosphofructokinase: purification inhibition and activation. Journal of Biological Chemistry2382285–2292.

    • Search Google Scholar
    • Export Citation
  • MartinSP Chaudhuri SN Green R & McKinney GR 1954 The effect of adrenal steroids on aerobic lactic acid formation in human leukocytes. Journal of Clinical Investigation33358–360.

    • Search Google Scholar
    • Export Citation
  • McMurrayRW RW Bradsher RW Steele & NS Pilkington 1990 Effect of prolonged modified fasting in obese persons on in vitro markers of immunity: lymphocyte function and serum effects on normal neutrophils. American Journal of Medical Science299379–385.

    • Search Google Scholar
    • Export Citation
  • Moreno-AuriolesVR Montano R Conde M Bustos R & Sobrino F 1996 Streptozotocin-induced diabetes increases fructose 26-biphosphate levels and glucose metabolism in thymus lymphocytes. Life Sciences58477–484.

    • Search Google Scholar
    • Export Citation
  • MowatAG & Baum J 1971 Chemotaxis of polymorphonuclear leukocytes from patients with diabetes mellitus. New England Journal Medicine284621–627.

    • Search Google Scholar
    • Export Citation
  • MukherjeeSP Lane RH & Lynn WS 1978 Endogenous hydrogen peroxide and peroxidative metabolism in adipocytes in response to insulin and sulfhydryl reagents. Biochemical Pharmacology272589–2594.

    • Search Google Scholar
    • Export Citation
  • MunroeJF & Shipp JC 1965 Glucose metabolism in leucocytes from patients with diabetes mellitus with and without hypercholesteremia. Diabetes14584–590.

    • Search Google Scholar
    • Export Citation
  • NewsholmeP Lima MM Procopio J Pithon-Curi TC Doi SQ Bazotte RB & Curi R 2003 Glutamine and glutamate as vital metabolites. Brazilian Journal of Medical Biology and Research36153–163.

    • Search Google Scholar
    • Export Citation
  • NolanCN Beaty HN & Bagdade JD 1978 Further characterization of the impaired bactericidal function of granulocytes in patients with poorly controlled diabetes. Diabetes27889–894.

    • Search Google Scholar
    • Export Citation
  • NoritakeM Katsura Y Shinomiya N Kanatani M Uwabe Y Nagata N & Tsuru S 1992 Intracellular hydrogen peroxide production by peripheral phagocytes from diabetic patients. Dissociation between polymorphonuclear leucocytes and monocytes. Clinical and Experimental. Immunology88269–274.

    • Search Google Scholar
    • Export Citation
  • OgleCK Ogle JD Mao JX Simon J Noel JG Li BG & Alexander JW 1994 Effect of glutamine on phagocytosis and bacterial killing by normal and pediatric burn patient neutrophils. Journal of Parenteral and Enteral Nutrition18128–133.

    • Search Google Scholar
    • Export Citation
  • OttonR Mendonça JR & Curi R 2002 Diabetes causes marked changes in lymphocyte metabolism. Journal of Endocrinology17455–61.

  • PanneerselvamS & Govindasamy S 2003 Sodium molybdate improves the phagocytic function in alloxan-induced diabetic rats. Chemical and Biological Interactions145159–163.

    • Search Google Scholar
    • Export Citation
  • PereiraMAA Sannomiya P & Garcia-Leme J 1987 Inhibition of leukocyte chemotaxis by factor in alloxan-induced diabetic rat plasma. Diabetes361307–1314.

    • Search Google Scholar
    • Export Citation
  • PickE & Mizel D 1981 Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader. Journal of Immunological Methods46211–226.

    • Search Google Scholar
    • Export Citation
  • Pires de MeloM Curi TC Miyasaka CK Palanch AC & Curi R 1998 Effect of indole acetic acid on oxygen metabolism in cultured rat neutrophil. General Pharmacology31573–578.

    • Search Google Scholar
    • Export Citation
  • Pithon-CuriTC Melo MP Azeveda RB Zorn TMT & Curi R 1997 Glutamine utilization by rat neutrophils: presence of phosphate-dependent glutaminase. American Physiology Society273C1124–C1129.

    • Search Google Scholar
    • Export Citation
  • Pithon-CuriTC Melo MP Palanch AC Miyasaka CK & Curi R 1998 Percentage of phagocytosis production of O2 H2O2 and NO and antioxidant enzyme activities of rat neutrophils in culture. Cell Biochemistry and Function1643–49.

    • Search Google Scholar
    • Export Citation
  • Pithon-CuriTC Levada AC Lopes LR Doi SQ & Curi R 2002 Evidence that glutamine is involved in neutrophil function. Cell Biochemistry and Function2081–86.

    • Search Google Scholar
    • Export Citation
  • Pithon-CuriTC Schumacher RI Freitas JJS Lagranha CJ Newsholme P Palanch AC Doi SQ & Curi R 2003 Glutamine delays spontaneous apoptosis in neutrophils. American Journal of Physiology284C1355–1361.

    • Search Google Scholar
    • Export Citation
  • RheeSG Chang TS Bae YS Lee SR & Kang SW 2003 Cellular regulation by hydrogen peroxide. Journal of the American Society of Nephrology14S211–S215.

    • Search Google Scholar
    • Export Citation
  • RoosD van Zwieten R Wijnen JT Gomez-Gallego F de Boer M Stevens D Pronk-Admiraal CJ de Rijk T van Noorden CJ Weening RS et al.1999 Molecular basis and enzymatic properties of glucose 6-phosphate dehydrogenase volendam leading to chronic nonspherocytic anemia granulocyte dysfunction and increased susceptibility to infections. Blood942955–2962.

    • Search Google Scholar
    • Export Citation
  • SagoneAL JR Greenwald J Kraut EH Bianchine J & Singh D 1983 Glucose: a role as a free radical scavenger in biological systems. Journal of Laboratory and Clinical Medicine10197–104.

    • Search Google Scholar
    • Export Citation
  • SugdenPH & Newsholme EA 1975 Activities of citrate synthase NAD+-linked and NADP+-linked isocitrate dehydrogenase glutamate dehydrogenase aspartate aminotransferase and alanine aminotransferase in nervous tissues from vertebrates and invertebrates. Biochemistry Journal150105–111.

    • Search Google Scholar
    • Export Citation
  • TanJS Anderson JL Watanakunakorn C & Phair JP 1974 Neutrophil dysfunction in diabetes mellitus. Clinical Experiments8526–33.

  • TauberAI Wright J Higson FK Edelman SA & Waxman DJ 1985 Purification and characterization of the human neutrophil NADH-cytochrome b5 reductase. Blood66673–678.

    • Search Google Scholar
    • Export Citation
  • WalrandS Guillet C Boirie Y & Vasson M-P 2004 In vivo evidences that insulin regulates human polymorphonuclear neutrophil functions. Journal of Leukocyte Biology761104–1110.

    • Search Google Scholar
    • Export Citation
  • WatanabeA1992 The production of hydrogen peroxide by neutrophilic polymorphonuclear leukocytes in patients with non-insulin dependent diabetes mellitus Nippon Jinzo Gakkai Shi341219–1225.

    • Search Google Scholar
    • Export Citation
  • WegenerG & Krause U 2002 Different modes of activating phosphofructokinase a key regulatory enzyme of glycolysis in working vertebrate muscle. Biochemistry Society Transactions30264–270.

    • Search Google Scholar
    • Export Citation
  • WilsonRM & Reeves WG 1986 Neutrophil phagocytosis and killing in insulin-dependent diabetes. Clinical and Experimental Immunology63478–484.

    • Search Google Scholar
    • Export Citation
  • WilsonRM Clarke P Barkes H Heller SR & Tattersall RB 1986 Starting insulin treatment as an outpatient. Report of 100 consecutive patients followed up for at least one year. Journal of the American Medical Association256877–880.

    • Search Google Scholar
    • Export Citation
  • WindmuellerHG & Spaeth AE 1974 Uptake and metabolism of plasma glutamine by the small intestine. Journal of Biological Chemistry2495070–5079.

    • Search Google Scholar
    • Export Citation
  • WoodHG Katz J & Landau BR 1963 Estimation of pathways of carbohydrate metabolism. Biochemistry Journal338809–847.

  • ZozulinskaDA Wierusz-Wysocka B Wysocki H Majchrzak AE & Wykretowicz A 1996 The influence of insulin-dependent diabetes mellitus (IDDM) duration on superoxide anion and hydrogen peroxide production by polymorphonuclear neutrophils. Diabetes Research and Clinical Practices33139–144.

    • Search Google Scholar
    • Export Citation

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