The effect of insulin on macrophage metabolism and function

CELL BIOCHEMISTRY AND FUNCTION VOL. 14 33-42 (1996)

The Effect of Insulin on Macrophage Metabolism and Function

LUIS F. B. P. COSTA ROSA, DANILO A. SAFI, YARA CURY+ AND RUI CURIT

Cellular Physiology Group, Department of Physiology and Biophysics, Biomedical Sciences Institute, University of Siio Paulo, Brazil; 'Physiopathology Laboratory, Butantan Institute, SCo Paulo, Brazil

This study examined the effect of insulin on rat macrophage metabolism and function. The following parameters were studied: cell migration in response to thioglycollate and BCG stimuli, macrophage phagocytic capacity, H2,02 production, glucose and glutamine metabolism as indicated by the measurement of enzyme activities, the utilization of metabolites and production and oxidation of substrates. The results indicate that insulin: (1) did not affect cell migration in response to thioglycollate and BCG; (2) enhanced the phagocytic capacity of macrophages and the production of H 2 0 2 by macrophages; (3) increased the metabolism of glucose and reduced that of glutaminase.

KEY WORDS -insulin; macrophage function; glutaminolysis; glycolysis; Krebs' cycle; pentose-phosphate pathway

INTRODUCTION Monocytes and macro hages, cells involved in the

lin in a manner that mirrors that of major target tissues, such as the hepatocytes. These cells obtained from insulin-resistant patients with obesity or diabetes mellitus bind less insulin than normal cells due to a decreased concentration of insulin recep- t o r ~ . ~ , ~ Monocytes, however, present a different pattern of glucose transporter-dependent hexose uptake when compared to other cell types, such as adipocytes and muscle cells, and express, in the presence of insulin, an increased number of GLUT1 and GLUT3 transporter^."^ Macro- phages are also able to control the expression of insulin receptors in T and B-lymphocytes, adjust- ing the impact afforded by insulin for a given immunobiologic f ~ n c t i o n . ~ However, the effect of insulin on macrophage metabolism and function has not been examined. Indeed, in spite of several studies on macropha e function and metabolism in the diabetic state8- there is no report concerning the effect of insulin itself.

Many studies have shown that macrophages uti- lize glucose and glutamine at high rates.' ' Elicited

inflammatory reaction, 72 bind and internalize insu-

P

peritoneal macrophages satisfy their energy requirements through anaerobic glycolysis and aerobic oxidation of glutamine and fatty acid.'* The high demand for such fuels shown by macrophages and lyrnphocyte~'~ may explain the increased rates of protein degradation in skeletal muscle during injury, trauma and Recent reports have shown that macrophage metabolism is markedly modified by various hormones," as well as by the lipid composition of the diet.16

The effect of insulin on macrophage metabolism and function was examined. The metabolic studies were performed by measuring the activities of: hexokinase, glucose 6-phosphate dehydrogenase, phosphate-dependent glutaminase and citrate synthase and the rates of glucose consumption and decarboxylation of glucose and glutamine. The effect of insulin on H202 production and the phagocytic capacity of macrophages were also investigated. The experiments were done in alloxan-induced diabetic and insulin-treated rats and in BCG-activated and resident macrophages cultured for 48 h in the presence of insulin.

Addressee for correspondence: Dr rui Curi, Department of Phy- siology and Biophysics, Instituto de CiSencias Biomtdicas, Uni- versidade de SZo Paulo, Av. Lineu Prestes, 1524, CEP 05508- 900, Cidade Universitaria, S6o Paulo, SP, Brasil. Tel. 813- 6944 ext. 7245. Fax: 55-11-8130845.

MATERIALS AND METHODS

Animals Male Wistar rats weighing 130-15Og (about 2

CCC 0263-6484/96/010033-10 1996 by John Wiley & Sons, Ltd.

34 L. F. B. P. COSTA ROSA ET AL.

Table 1 Plasma insulin and glucose levels in control rats and control rats treated with insulin, alloxan-diabetic rats and alloxan-diabetic rats treated with insulin or fasted for 24 h.

Groups Insulin (,u Uml-') Glucose (mg IOOml-')

Control Not-T 108 f 10 98 f 6 I-T > 200* 31 f 4 *

Not-T 6.2 zk 0.87 251 i 14t I-T 101 f 12* 91 f 4 *

Alloxan-diabetic

24 h-fasted 8.9 * 07t 94 i 7* ~~~ ~ -~~~ ~~

* p < 0.05 in comparison with rats without treatment in the same group. t p < 0.05 in comparison with the control group without treatment. I - T = insulin treated rats. Not - T = not treated rats.

months of age) were obtained from Instituto Butantan, Sgo Paulo. The rats were maintained at 23°C and a light/dark cycle of 12/12h, lights on at 7:OO a.m.

Chemicals, Enzymes and Hormones All chemicals and enzymes were obtained from

Boehringer Mannheim GmbH, Sussex, England, except NPH insulin, which was purchased from Biobras, Brasil. Culture medium, foetal calf serum, insulin for culture and antibiotics were obtained from Gibco BRL, New York, U.S.A. [U-14C]- glucose and [U-'4C]-glutamine were purchased from Amersham, Buckinghamshire, U.K.

The Induction of Diabetes The animals were fasted for 18 h, then injected

intravenously with a dose of 42mg kg-' body weight alloxan in phosphate buffered saline (PBS).I7 The animals used in the experiments were those with glycemia over of 200mg (100 ml-'). The values of insulinemia and glycemia of the diabetic rats without treatment, and those treated with insulin and fasted for 24 h are shown in Table 1. Diabetic rats fasted for 24 h (presenting with normoglycemia but with low insulinemia), diabetic and normal rats treated with NPH insulin (2 U animal-', for 8 days), as well as normal rats were also included in this study.

Peritoneal Cell Preparation The rats were killed between 8:OO and 1O:OO a.m.

by decapitation without anesthesia. After laparot- omy, macrophages from the peritoneal cavity

were collected. Three types of macrophages were studied: resident thioglycollate-elicited (inflammatory) and BCG-activated (activated). Resident (RES) macrophages were obtained by intraperitoneal lavage with 6 ml of sterile phosphate buffered saline (PBS) at pH 7.2. Inflammatory macrophages were obtained in the same way, 4 days after the injection of 3 ml of sterile thioglycollate broth (4 per cent). The procedure was also used to obtain activated macrophages, after the intraperitoneal injection of 25 mg ONCO-BCG (Bacille Calmette- Gukrin), 7 days prior to the harvest. These two sti- mulating agents were adopted since they provoke slightly different responses.'7 Macrophages obtained from BCG-injected rats show enhanced rates of 0; and H202 production and, thus, enhanced antimicrobial activity and increased capacity to kill tumours, besides spreading faster on glass, ruffling their membranes more promi- nently, increasing their size, and their content of lysosomes, as well as showing a higher rate of phagocytosis. For these reasons these cells are termed activated macrophages. On the other hand, macrophages obtained from animals injected with Bre- wer's thioglycollate broth display many of the same metabolic, phagocytic, plasma membrane, enzyme and lysosomal changes as described for activated macrophages, but they have a much reduced capacity for 0, and H202 production, thus lacking an enhanced antimicrobial and antitu- mour activity. These cells are termed inflammatory or elicited macrophages.

Macrophages were purified by adherence to plas- tic Petri dishes. The cells were suspended in Eagle's minimum essential medium (MEM) supplemented with 10 per cent (v/v) foetal calf serum (FCS), 2 m~ glutamine and 20 pg penicillin- streptomycin ml-' and were added to 100-mm tissue culture Petri dishes at a density of 1.0 x lo7 cells dish-'. After incubation for 4 h at 37°C in 5 per cent C02/95 per cent air, adherent macrophages were washed vigorously three times with PBS. The population of cells that remained adhered to the plate was at least 98 per cent macrophages (as determined by differential staining). l8

The macrophages were removed from the plates by gentle scraping with a rubber policeman into 2-3ml of PBS. The cell viability was determined as > 95per cent by the exclusion of Trypan blue.

Incubation Procedure Macrophages obtained from control, thoglycollate-

INSULIN AND MACROPHAGE METABOLISM 35

and BCG-injected rats were incubated (1.0 x lo6 cells flask-') at 37°C in Krebbs-Ringer medium (NaCl 11 5 mM, K2HP04 5 mM, CaC12, 1 mM, MgS04.6H20 I ~ M , KC1 1 2 4 m ~ , NaHC03 24mM) with 2 per cent (w/v) bovine serum albumin (fatty acid-free) and in the presence of glucose ( 5 m ~ ) or glutamine ( 2 m ~ ) as indicated in the Results section. After 1 h of incubation, the cells were disrupted by 0-2ml of 25 per cent perchloric acid. Protein was removed by centrifugation and the supernatant was neutralized with 40 per cent KOH and a Tris-(hydroxymethyl) aminomethane/ KOH (0.5 to 2 . 0 ~ ) solution for the measurement of metabolites.

Culture Procedure To test whether the main effects of insulin

observed in vivo could be reproduced in vivo, experiments with macrophages cultured for 48 h were done. Macrophages obtained from control and BCG-injected rats were left to adhere to the plates for 4 h. After this period, the adhered cells were cultured in MEM supplemented with 10 per cent FCS and 20 pg ml-' penicillin-streptomycin, for 48 h at 37"C, in an artificially humidified atmo- sphere of 5 per cent C 0 2 in air under sterile conditions. Insulin (200mU mi-') was added to the culture medium in the experiments for the assess- ment of its effects. The cultures were maintained in a LAB-LINE Microprocessor C 0 2 incubator (LAB-LINE, U.S.A.). After 48 h of culture, more than 98 per cent of macrophages were viable, as measured by Trypan blue exclusion.

Enzyme Activity Assays Enzyme activities were measured as previously

The cells obtained from the rats or from the culture dishes were homogenized in a glass homogenizer containing 0.4 ml of extraction medium for each enzyme. Hexokinase (EC 2.7.1. l), which provides information on glucose phosphorylation and so the capacity for glucose utilization by the was measured in an extraction medium containing 50 mM Tris-HC1, 1 mM EDTA, 30 mM mercaptoethanol and 20 mM MgC12 at pH 7-4. Phosphate-dependent glutaminase (EC 3.5.1.2) which provides information on the flux through the glutaminolysis path- way2' was measured in the following medium: 150 mM potassium phosphate, 1 mM EDTA and 50mM Tris-HC1 at pH 8.6. Citrate synthase (EC

4.1.3.7) which provides a qualitative index of the flux through the Krebs' cycle23 and glucose 6-phosphate dehydrogenase (EC 1.1.1.49) which provides a qualitative index of the maximum flux through the pentose-phosphate pathway24 were measured in an assay medium consisting of 5 0 m ~ Tris- HC1 and 1 mM EDTA; the final pH was 7.4 for citrate synthase and pH 8.0 for glucose 6-phosphate dehydrogenase. For all the enzyme assays, 0.05 per cent (v/v) Triton X-100 was added to the assay system to complete the extraction of the enzyme. For the assay of hexokinase (the activity of the enzyme was measured through the production of NADPH via glucose 6-phosphate dehydrogenase), the following medium was used: 75 mmol Tris-HC1, 7.5mmol MgC12, 0.8 mmol EDTA, 1.5 mmol KCI, 4.0 mmol P-mercaptoethanol, 0.4mmol creatine phosphate, 1.8 U ml-' creatine kinase, 1.4 U ml-' glucose 6-phosphate dehydrogenase, 0.4mmol NADP at pH 7.5. The assay of citrate synthase was determined by following the production of a yellow compound which results from the reaction between DTNB and the CoA released. The medium consisted of 100 mmol Tris- HCl, 0.2 mmol 5,5'-ditho-bis-2-nitrobenzoic acid, 15 mmol acetyl CoA and 0.5 mmol oxaloacetate at pH 8-1. The assay medium for glutaminase (the activity of the enzyme was evaluated through the production of NADH in the conversion of glutamate to a-ketoglutarate catalysed by glutamate dehydrogenase) consisted of 50 mmol phosphate buffer (an equimolar mixture of KH2P04 and K2HP04), 0.2mmol EDTA, 50mmol Tris-HC1 and 20mmol glutamine, at pH 8.6. The final volume of the assay mixtures in all cases was 1.0ml. Citrate synthase was assayed by following the rate of change in absor- bance at 412nm, and the other enzymes were assayed by following the rate of change in absor- bance at 340nm. All enzyme activities were measured at 25°C except for glutaminase which was determined at 37°C.

Phagocytosis by Macrophages Macrophages were incubated with 10 ml of PBS

containing opsonized zymosan for 30 min at 37"C, so that phagocytosis could be determined by counting (in a counting chamber) the cells that had pha- gocytosed more than four particles of zymosan.

The Production of Hydrogen Peroxide. The production of hydrogen peroxide in incubated


Table 2. Cell migration into the peritoneal cavity in response to thioglycollate (TG) or BCG injection (B-A) in rats from the five groups.

Groups TG ( x lo4 cells)

RES B-A

~ ~ ~ ~~~~~~

Control 1254 i 98 2398 f 101' 2559 i 95* Diabetic 1001 f 297 2031 f 51*,t 2221 f 23*,t Diabetic-fasted 1008 i 14t 2008 i 32*.t 2201 f 16*,t Insulin-treated 1231 f 15 2215 f 29* 2678 i 17* Diabetic-insulin treated 1100 i 18 2209 i 28* 2445 f 22*

~ ~ ~~~~~~~~

The values are presented as the means f SEM of 15 rats. * p < 0.5 in comparison with resident macrophages. t P < 0.5 in comparison with the control group RES, resident macrophages.

activated macrophages for 1 h was monitored by the reduction of horseradish peroxidase (HRP0)- dependent oxidation of phenol red.25

The Measurement of Metabolites

Neutralized extracts of the incubation medium were analysed for lactate26 and glucose.27 The assay of lactate was done in the presence of lactate dehydrogenase and NAD, whereas glucose was measured using a hexokinase/glucose 6-phosphate dehydrogenase mixture with the production of NADPH. The plasma insulin concentration was measured by means of a modified radioimmunoas- say,28 in which polyethyleneglycol was used instead of the second antibody. The 14C02 produced from [U-'4C]-glucose and [U-'4C]-flutamine was collected as described previously and expressed as nmol of substrate converted into C02.

Expression of the Results The enzyme activities, glucose utilization, lactate

or glucose production, glutamine oxidation and hydrogen eroxide production are presented as nmol min-7 mg-1 protein.

St at is t ical Analysis The results are expressed as the means j, SEM.

Comparisons between the groups were assessed by ANOVA and the Student's t-test at a significance level of p < 0.05.

in order to evaluate whether these treatments could interfere with the interpretation of our findings. The results obtained (pU ml-', presented as mean f SEM of five rats) were 121 f 21 for control, 118 f 18 for BCG-injected rats and 87 3~ 16 for thioglycollate-treated rats and indicate that both stimuli per se do not seem to affect significantly the plasma insulin levels under these conditions.

To evaluate the effect of insulin upon the parameters studied we used alloxan-induced diabetic rats and normal and diabetic rats treated with insulin. Alloxan-diabetic rats were used since in this model there is a decreased level of plasma insulin with no change in the activation state of the immune cells as has been reported for spontaneously diabetic BB rats".

The total number of cells in the peritoneal cavity was significantly lower in both the diabetic and the diabetic normoglycemic groups (Table 2). As caused by the thioglycollate injection, there was a

Table 3. The macrophage phagocytic capacity ("A) of resident (RES), thioglycollate-injected (TG) or BCG-injected (B-A) rats of the five groups. ~~~~~~~~~~~ ~

TG Groups RES (phagocytosis %) B-A

Control 39 + 4 54 i 2* 58 i 3* Diabetic 3 4 i l t 42 i I*,$ 42 f 3*,f D-F 32 i 3 t 41 i l*,f 44 i 4*t I-T 44 f 3 67 i 4*,t 71 i 3*,t D-I-T 38 i 2 5 2 f 1 * 57 i 4*

RESULTS AND DISCUSSION

The effect of BCG or thioglycollate injection on the plasma insulin levels of control rats was determined

The values are expressed as the means i SEM of 11 rats. * p < 0.05 in comparison with resident macrophages. t p < 0.05 in comparison with the control group. D-F, diabetic fasted rats; I-T, insulin-treated rats; D-I-T, diabetic rats treated with insulin.


two-fold increase in the number of cells in all the groups and this response was not modified by insulin. The administration of BCG provoked a similar increase of approximately 2.2-fold in all the groups. Therefore it may be assumed that insulin does not alter the capacity of cell migration either in response to thioglycollate or to BCG.

The phagocytic capacity of the macrophages was reduced significantly in the three cell types of the diabetic rats as compared to the controls (Table 3): 10 per cent resident, 22 per cent for inflammatory and 28 per cent for activated macrophages. An increment of approximately 20 per cent in the percentage of phagocytosis as provoked by hyper- insulinemia was observed in the three cell types. The conclusive evidence of the importance of insulin for phagocytosis by macrophages in vivo was obtained by insulin injection in diabetic rats. This treatment fully reverted the alterations observed in the diabetic state. Corroborating these findings, the addition of insulin to the culture medium also promoted a marked increase in the percentage of macrophage phagocytosis, by 27 per cent for resident and by 39 per cent for BCG-activated macrophages (Table lo).

The possible metabolic mechanisms involved in these alterations of macrophage function were examined. Hexokinase activity was augmented by at least two-fold in the inflammatory and activated macrophages in comparison to the resident cells, suggesting that glucose might play an important role in macrophage function. In fact, studies devel- oped by Meszaros et al.29 have previously shown that injection of endotoxin provokes a marked increase of the uptake of glucose by the Kupfer cells in the liver. The activity of hexokinase was reduced by 50 per cent as a consequence of diabetes in the three cell types (Table 4). These changes were totally reverted by the administration of insulin. Furthermore, insulin given in excess raised the hexokinase activity by 60 per cent in BCG-activated macrophages both in vivo and in vitro (Tables 4 and 10, respectively). These observations show that insulin does in fact regulate hexokinase activity in macrophages.

The effect of this hormone on glucose consumption by macrophages was then investigated. As shown in Table 5 , macrophages obtained from diabetic rats (diabetic and diabetic normoglycemic) showed a decreased rate of glucose utilization regardless of the conditions of stimulation. These changes were abolished when the diabetic animals received insulin. It is interesting to note that this

Table 4. Maximal activity of hexokinase of resident macrophages (RES), and cells from thioglycollate-injected (TG) or BCG-injected (B-A) rats of the five groups.

Hexokinase activity (nmol min-' mg-' of protein)

Groups RES TG B-A

Control 267 i 10 632 f 13* 554 f 9* Diabetic 150 i 167 300 f 23*,f 244 f 17*,t D-F 148 i 117 293 f 18*,t 253 i 16*,t I-T 274 i 31 639 f 34* 865 i 39*,t D-I-T 263 i 18 614 f 19* 526 f 14*

The results are expressed as the means f SEM of 11 rats. * p < 0.5 in comparison with resident macrophages. t p < 0.05 in comparison with the control group. D-F, diabetic fasted rats; I-T, insulin-treated normal rats; D-I-T, diabetic rats treated with insulin. 1 mg of protein corresponds to approximately 10' cells.

hormone added to the culture medium did not modify the rates of glucose utilization by resident macrophages but increased it by 30 per cent in BCG-activated cells (Table 10). These finding show that the response to insulin appears to vary with the cell state in addition to the cell type.

The rate of lactate formation was not the same in all the cells from the alloxan-injected rats. In resident macrophages, diabetes provoked a reduction

Table 5. Glucose consumption and lactate production in resident (RES) macrophages and cells obtained from thioglycollate-injected (TG) or BCG-injected (B-A) rats of the five groups.

Groups RES TG B-A

Glucose utilization (nmol min-' mg-' of protein) Control 32-5 * 1.5 38.6 f 0.8* 49.6 f 1. I *

D-F 21-5 i 0.71 31-1 i 0-6*,7 42% f 1.1*,7 I-T 33-5 f 0.9 44.6 f 1.2*,t 57.5 f 1.4*,t D-I-T 32-1 f 0.8 40.8 f 1.4*

Diabetic 22- I i 0.9 30.3 f 0,9*,t 40.8 f 1. I *,t

50.6 i 1.6* Y

Lactate formation (nmoI min-' mg-' of protein) Control 63-0 f 1.3 71.5 & 1 4 * 51.3 f 1.7*

D-F 35-1 f 151 63.1 f 2.0*,t 54.3 i 1.6* I-T 63-5 i 1.6 79.8 i 2.2*,? 668 & 2- 1 t D-I-T 61-5 i 1.8 72.8 * 1.9* 53.3 f 1.7*

Diabetic 34-5 i O % t 61.6 i 1.9*,t 53.6 1.8"

The values are expressed as the means f SEM of 11 rats. * p < 0.5 in comparison with resident macrophages. t P < 0.5 in comparison with the control group. D-F, cells obtained from diabetic fasted rats, I-T, cells from insulin-treated normal rats; D-I-T, cells obtained from diabetic rats treated with insulin.

38 L. F. B. P. COSTA ROSA E T A L .

in the lactate formation form glucose by 45 per cent, whereas in inflammatory macrophages there was a decrease of only 12 per cent. (Table 5). Macrophages obtained from diabetic rats injected with BCG showed the same rates of lactate formation as those found for the cells of control rats. Insulin injections in normal rats raised the rate of lactate production by 42 per cent in the inflammatory macrophages and by 30 per cent in the activated cells as compared to resident macrophages. The in vivo observations were confirmed in cultured BCG-activated cells; insulin raised lactate production by 47 per cent (Table 10).

Concerning the rates of conversion of glucose to lactate (Table 5) , the following considerations might be put forward: in the resident macrophages from the control rats, 97 per cent of the glucose was converted into lactate. Low circulating levels of insulin reduced this proportion to 79 per cent and the administration of insulin reverted it back to 96 per cent. Cells of thioglycollate-injected rats were not affected by insulin and around 90 per cent of the glucose was converted into lactate. In incubated activated cells, 50 per cent of the glucose was transformed into lactate in the control and insulin-injected groups and this proportion was slightly enhanced in the diabetic rats. In resident and BCG-activated macrophages cultured for 48 h, the proportion of glucose converted to lactate was 97 per cent and 53 per cent, respectively; these results were not affected by insulin (Table 10). These findings demonstrate that the proportion of lactate formation form glucose decreases in inflammatory and even more dramatically in activated macrophages in comparison to the resident cells. Clearly, in activated macrophages, 50 per cent of the pyruvate generated from glucose is oxidized through the Krebs' cycle or directed either towards the pentose-phosphate pathway, or alternatively, to the production of lipids such as triacylglycerols, phospholi ids and cholesterol as reported for lymphocytes.' The possoibility of the generation of lipids from this precursor has to be mentioned, since a previous study14 shows a low decarboxylation of pyruvate in incubated inflammatory macrophages. In this case, acetyl-CoA formed from pyruvate, through pyruvate deh drogenase would be diverted to lipid synthesis. I' Further experiments have to be performed to evaluate this point.

To examine further the metabolic regulation of macrophages by insulin, the activities of citrate synthase and of phosphate-dependent glutaminase were determined. As shown in table 6, citrate

Table 6. Maximal activities of citrate synthase, and phosphate- dependent glutaminase, of resident (RES) macrophages and cells obtained from thioglycollate-injected (TG) or BCG- injected (B-A) rats of the five groups.

Groups RES TG B-A _ _ _ _ ~ ~ ~ ~ ~

Citrate synthase activity (nmol min-' mg-' of protein) Control 36.1 f 1.2 54-2 f 1.6* 50.3 f 2.3* Diabetic 30.3 f 1.5 52.4 f 2.1* 55.7 f 2.8* D-F 32.2 f 1.1 50-9 f 1.2* 52.3 f 1-9* I-T 81.0 f 4.27 97.4 f 3.9*,7 84.7 f 5-I t

52.4 f 2-1* D-I-T 33.9 f 1.1 55.9 i 1.9

Glutaminase activity (nmol min-' mg-' of protein) Control 248 f 7.3 112 f 3.9* 157 f 2.3* Diabetic 260 f 3.3 149 f 4.5*,t 156 f 4.9* D-F 262 f 9.8 153 f 6.7*,7 159 i 5.3* I-T 140 f 5.3t 68 f 5.1*,7 129 f 2.77 D-I-T 268 f 5.9 123 f 6.8* 160 * 6.l*

The values are expressed as the means f SEM of 11 rats * p < 0.5 in comparison with resident macrophages. t P < 0.5 in comparison with the control group. D-F, macrophages obtained from diabetic fasted rats; I-T, cells from insulin-treated rats; D-I-T, cells from diabetic rats treated with insulin.

synthase activity was not altered in the cells obtained from diabetic rats but, conversely, was markedly increased in the hyperinsulinemic state (insulin-treated normal rats): 2.2-fold in resident, by 73 per cent in inflammatory and 63 per cent in activated macrophages, as compared to cells from the control rats. The experiments done on cultured cells confirm these observations; insulin enhanced citrate synthase activity two-fold in resident cells and by 78 per cent in BCG-activated macrophages (Table 10). Insulin administered to diabetic rats did not provoke a similar response of citrate synthase activity as that observed for control animals. The significance of these findings remains unclear.

It is interesting to note that both thioglycollate and BCG caused a 44 per cent elevation of citrate synthase activity in macrophages. These findings point to the importance of the Krebs' cycle for the macrophage response to both stimuli and the effect of insulin, when given in excess, is to increase the capacity for Krebs' cycle activity.

Diabetes affected phosphate-dependent glutaminase activity only in the macrophages from (thioglycollate-injected rats, where an increment of 33 per cent was found (Table 6). However, administration of insulin to control rats decreased the activity of this enzyme by 40 per cent in resident and inflammatory cells and by 18 per cent in activated macrophages. An inhibitory effect of insulin


Table 7. Rates of conversion of [U-'4C]-glucose and [U-'4C]-glutamine to I4CO2 in resident (RES) macrophages and cells obtained from thioglycollate-injected (TG) or BCG-injected (B-A) rats of the five groups.

Groups RES TG B-A

[U-'4C] Glucose converted into 14C02 (nmol min-' mg-' of protein)

Control 6.9 i 0.3 10.1 f 0.2* 1 1.7 zk 0.4* Diabetic 6.5 f 0-2 7.9 f 0.4*t 8.2 f 0.2*,t D-F 6.2 f 0.4 8.2 f 0.57 8.4 f 0.4* I-T 7.1 i 0.5 10.3 f 0.1* 12-5 f 0.7* D-I-T 7.0 i 0-8 9.9 f 0.8 11.4 f 06*

[U-14C] Glutamine converted into I4CO, (nmolmin-' mg-' of protein)

Control 5.7 f 0.4 6-3 f 0.3 8.9 f 0.2* Diabetic 7.2 f 0.27 10.7 i 0.3*t 13.2 f 0.3*,t D-F 7.0 i 04t 11.0 f O.S*,t 13.6 f O.S*,t I-T 5.0 i 0.5 6.7 f 0.4 8.6 f 06* D-I-T 6.0 zk 0.7 6.5 f 0.5 9-2 f 0.6*

The values are expressed as the means f SEM of 10 incuba- tions of five cell preparations. * p < 0.5 in comparison with resident macrophages. t P < 0.5 in comparison with the control group. D-F, macrophages obtained from diabetic fasted rats; I-T, cells from insulin-treated; D-I-T, cells from diabetic rats treated with insulin.

on glutaminase activity was also obtained in the in vitro experiments; 43 per cent for resident and 26 per cent for BCG-activated macrophages (Table 10). Hence, the combined results in Table 6 and 10 indicate that high levels of plasma insulin were able to reduce glutaminase activity in macrophages, whereas low insulinemia caused only a minor effect.

The results of glucose and glutamine oxidation in macrophages obtained from the rats rendered diabetic or treated with insulin are presented in Table 7. Diabetes caused the rate of glucose decarboxylation in macrophages from thioglycollate or BCG-injected rats to decrease by approximately 20 per cent. In contrast, this condition enhanced the rate of glutamine oxidation by 26 per cent, 69 percent and 48 per cent in resident, inflammatory and activated macrophages, respectively. In cultured macrophages, insulin did not affect glucose and glutamine oxidation (Table lo). These findings of changes in hexokinase and glutaminase activities suggest that insulin might stimulate the metabolism of glucose and suppress that of glutamine in rat macrophages.

The production of hydrogen peroxide was markedly decreased in BCG-activated macrophages obtained from diabetic rats (Table 8), both in the presence and absence of phorbol-myristate acetate (PMA). This effect was not observed in resident cells. Insulin administration fully reversed these changes, confirming the importance of this hormone for H202 production in BCG-activated macrophages and therefore for their antimicrobial and antitumoural a~t iv i ty .~"~ ' The reduction in hydrogen peroxide production by activated macrophages obtained from alloxan-diabetic rats is not comparable to those observed in other ~ t u d i e s , ~ ~ , ~ ' which involved an autoimmune diabetes with a different aetiology. In the latter case, macrophages participate in an activated state during which they are able to produce large amounts of H,02 and 02-, this being the mechanism for the establish-

Table 8. Hydrogen peroxide production of macrophages obtained from BCG-treated (B-A) rats of the five groups.

Hz02 Production (nmol min-' mg-' of protein)

RES B-A

Groups Without PMA PMA Without PMA PMA

Control 0,042 * 0-004 0.06 1 f 0.0 15 2.26 f 0.19 6.91 f 0.57 Diabetics 0.046 f 0005 0.052 f 0-006 0.054 f 0.004* 0.061 i 0.008*,? D-F 0.040 f 0.003 0.051 f 0006 0.051 f 0.003* 0.13 f 0.01 l*,t

2.13 f0-15 6 6 6 f 0,457 I-T 0.050 f 0.004 0.071 f 0021 D-I-T 0.049 f 0.003 0.064 f 0-01 8 2.01 f 0 . 1 2 6.73 f 0.60t

The values are expressed as the means f SEM of 10 incubation of five cell preparations. * p < 0.05 in comparison with the control group. t p < 0.05 in comparison with the group without PMA. D-F, macrophages obtained from diabetic fasted rats; I-T, cells obtained from insulin-treated; D-I-T cells from diabetic rats treated with insulin. RES, resident; PMA, phoxbol-myristate acetate.


Table 9. Maximal activity of glucose 6-phosphate dehydrogenase (G6PDh) of resident (RES) macrophages and cells from thioglycollate-injected (TG) or BCG-injected (B-A) rats of the five groups.

G6PDh activity (nmol min-' mg-' of protein)

Groups RES TG B-A ~ ~

Control 8.6 f 1.4 15.2 i 1.5* 17.9 + 0.7* Diabetic 8-1 i 0 . 9 21.1 f 1,7*,t 12.1 *0.6*,t D-F 7.9 * 0.6 20.0 f I.l*,t 11.3 f 0.7*,t I-T 8.3 z t 1.1 9.2 f 0.47 25.8 f 1.4*,t D-I-T 8.2 f 0.9 13-9 f 1.2* 18.4 f 0.9*

The values are expressed as the means f SEM of 11 rats. * p < 0-5 in comparison with resident macrophages.

D-F, macrophages obtained from diabetic-fasted rats; I-T, cells from insulin-treated rats; D-I-T, cells from diabetic rats.

P < 0.5 in comparison with the control group.

ment of the disease.'322 In contrast our study the activation of the macrophages was induced after the induction of the diabetic state which was induced without the involvement of immune cells.

The reaction catalysed by glucose 6-phosphate dehydrogenase produces NADPH for NADPH- oxidase activity and thus for the generation of H202.9310 In fact, activated macrophages which presented a 2 1 I 1 -fold increased capacity for H202 production as compared to resident cells also

showed a higher activity of glucose 6-phosphate dehydrogenase. This activity might ensure that H202 production is stimulated during a process of inflammation or infection. Indeed, the microbi- cidal and antitumoural activity of activated macrophages depend on their capacity to release hydrogen per~xide .~~, ' ' Diabetes reduced glucose 6-phosphate dehydrogenase (G6PD) activity in BCG-activated macrophages by 32 per cent (Table 9). The administration of insulin to control rats enhanced G6PDh activity by 44 per cent in activated macrophages (Table 9). A similar effect of insulin was observed in cultured cells (Table 10). Interestingly, the activity of G6PDh in macrophages from thioglycollate-injected rats was increased in the diabetic group and reduced as a result of insulin administration. These observations indicate that the control of glucose 6-phosphate dehydrogenase by insulin macrophages varies with the activation state of the cells.

The results presented in Table 10 and those previously described for diabetic and insulin-treated rats indicate that the qualitative response of macrophages to insulin found in vivo could be reproduced under culture conditions. Overall insulin enhanced the macrophage phagocytic capacity both in vifro and in vivo and H202 production in vivo. These functional effects were associated with stimulation of the metabolism of glucose and inhibition of that of glutamine.

Table 10. Maximal enzyme activities, macrophage pha ocytic capacity (PC), glucose consumption (GC), lactate (LP) and H202 (HP) production and the formation of I4CO2 from [U-' C]-glucose (GD) and [U-'4C1-glutamine (GLD) in resident and BCG-activated macrophages cultured for 48 h in the presence of insulin.

$

Resident B-A

Control Insulin Control Insulin

PC 37 f 2 47 i 1.7* 54 i 2.1 75 i 3* HEX 258.5 f 8.1 265.2 4~ 12.7 5092 f 21.4 822.3 f 37.9* GC 33.5 f 0.26 32.1 f 0.21 46.8 f 0.28 61.1 f 0.45 LP 64.5 f 0.81 63-3 f 0.76 50.1 f 0.65 64.7 f 054

85.6 f 7.1* cs 38.1 * 2.1 76.9 f 5.3* 108.7 It 6.2* GLUT 239.8 i 13.4 137.8 f 6.6*

G D 7.4 i 0.5 7.7 It 0.3 12.3 + 0.3 13.1 fO.6 GLD 6.3 f 0.5 5.7 * 0.8 8.5 i 0.24 8.8 z t 0.19 HP 6 31 5 0.32 6.53 f 0.45 G6PD 9.1 * 1.1 9.5 * 1.3 14.3 * 0.9 26.1 i 1.8*

The enzyme activities (measured at 25"C, except for glutaminase determined at 37"C), glucose consumption and lactate production, the results of 14C02 produced from [U-'4C]-glucose- and glutamine and the hydrogen peroxide production measured at 37°C and assays in the presence of phorbol myrlstate acetate, are presented as nmol min-' mg-' of protein. The values are presented as the means * SEM of nine culture dishes from three cell preparations. * p < 0.05 in comparison between the control macrophages and those cultured in the presence of insulin (200mUml-'). HEX, hexokinase; CS, citrate synthase; G6PD, glucose 6-phosphate dehydrogenase; GLUT, glutaminase.

48.4 It 1.9 146.9 f 5.5


ACKNOWLEDGEMENTS The authors are grateful to the technical assis- tance of J.R. Mendonga and G. de Souza and to M. Seelaender for the revision of the manu- script. This research has been supported by FAPESP, CNPq and Sandoz Foundation for Gerontological Research.

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Received (revised) 26 June 1995 Accepted 3 July 1995

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The effect of insulin on macrophage metabolism and function