Full text of DOI:10.1385/BTER:77:1:53

©Copyright 2000 by Humana Press Inc.
All rights of any nature, whatsoever, reserved.
0163–4984/00/7701–0053 $13.00
Chromium-Induced Glucose Uptake,
Superoxide Anion Production,
and Phagocytosis in Cultured
Pulmonary Alveolar Macrophages
of Weanling Pigs
1
2
3
DER-NAN LEE, HOUNG-TA YEN, TIAN-FUH SHEN,
,3
AND BAO-JI CHEN*
1Department of Animal Science, National I-Lan Institute
of Technology, I-Lan, Taiwan, Republic of China;
2Pig Research Institute Taiwan, Chunan, Miaoli, Taiwan,
Republic of China; and 3Department of Animal Science,
National Taiwan University, Taipei, Taiwan,
Republic of China
Received January 25, 2000; Accepted February 8, 2000
ABSTRACT
The dose-dependent effects of chromium chloride (CrCl3) and
chromium picolinate (CrPic) were evaluated for their glucose uptake,
superoxide anion (O−2) production, activity of glucose-6-phosphate
dehydrogenase, and phagocytosis of incubated pulmonary alveolar
macrophages in medium containing no or 5 × 10−8M insulin. Glucose
uptake was found to increase in cells treated with 20 µg/L CrCl3.
Incubation with 20 µg/L of CrPic enhanced glucose uptake and O−2
production in an insulin-dependent manner. However, the inclusion
of CrPic to 100 µg/L in the medium absent of insulin also increased
O−2 production. The activity of glucose-6-phosphate dehydrogenase
was not affected by either the addition of Cr or insulin. The phago-
cytosis of Escherichia coli by macrophages was enhanced significantly
(p < 0.05) in medium containing 10–100 µg/L CrCl3 or 20–100 µg/L
CrPic in the presence of insulin. These results suggest that the addi-
tion of 10–20 µg/L CrCl3 enhances directly the cellular activity of
*Author to whom all correspondence and reprint requests should be addressed.
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Lee et al.
macrophages, whereas the effect of CrPic requires the cooperative
action of insulin in enhancing their glucose uptake and phagocytosis.
Index Entries: Chromium; weanling pigs; macrophage; glucose
uptake; superoxide anion; phagocytosis.
INTRODUCTION
The trace element chromium (Cr) was first proposed in 1959 by
Schwarz and Mertz (1) to maintain normal glucose tolerance. Recently,
Cr has been recognized as an essential dietary mineral required for nor-
mal metabolism of carbohydrate, proteins, and lipids in livestock and
laboratory animals (2,3). These beneficial actions of supplemental Cr
have traditionally been attributed to a general improvement in insulin
sensitivity and glucose tolerance in peripheral tissues (2,4). Several inves-
tigations using growing pigs and cows have shown that glucose homeo-
stasis was more stable with both glucose and insulin challenges in the
chromium picolinate (CrPic)-supplemented group (5,6).
Previous work from this (7,8) and other laboratories (9) has sug-
gested that dietary CrPic supplementation inclines to increase the lev-
els of immunoglobulins and anti-Pseudorabies virus titer and to alleviate
stress caused by lipopolysaccharide challenge in weanling pigs. How-
ever, the mechanisms of Cr-enhanced immune responses have not been
established.
The blastogenesis of peripheral blood mononuclear cells (PBMC)
was found to be enhanced in dairy cows supplemented with dietary Cr
(10). The PBMC culture medium containing serum from Cr-fed cows also
induced the enhanced blastogenesis, however; the hormonal profiles in
serum revealed no change in this trial (11). Furthermore, it has been
reported that the addition of Cr from chelated Cr or CrCl3 increased blas-
togenesis and that the level of chelated Cr needed to enhance blastogen-
esis was higher than that of CrCl3 in the presence of insulin (12,13).
Evans and Bowman (14) reported that glucose and amino acid uptake by
muscle cells was more efficient with CrPic than that with chromium
nicotinate or CrCl3 in the presence of insulin. Therefore, we postulated
that the different forms of Cr inducing cellular activity of immune cells
might be by way of different pathways.
Macrophages are the important immune cells acting on phagocyto-
sis and respiratory burst (15). The increased level of glucose in culture
medium is related to the increased capacity of phagocytosis and the level
of interleukin-1β (IL-1β) production (16,17). The aim of the present study
was to examine the relationship among various Cr compounds and
insulin by investigating the influence of the dose-dependent effects of
CrPic and CrCl3 in the presence or absence of insulin on glucose uptake,
superoxide anion (O−2) production, activity of glucose-6-phosphate dehy-
drogenase, and phagocytosis in cultured pulmonary alveolar macro-
phages of weanling pigs.
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Chromium on Macrophages of Weanling Pigs
MATERIALS AND METHODS
Cell Culture
55
Macrophages used in the study were isolated from pulmonary alve-
oli of 6 to 8 wk-old crossbred weanling pigs as described previously by
Hsiao (18). The macrophages collected from the tissues were washed and
resuspended in RPMI 1640 medium (Life Technologies, Inc., USA) con-
taining 10% (v/v) dimethylsulfoxide (DMSO) (Sigma Chemical Co., USA)
and 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies,
Inc.) before freezing at −140°C. When performing the assay, macrophages
were thawed and resuspended in Dulbecco’s modified Eagle medium
(DMEM; Life Technologies, Inc.) with 10 mM glucose, 2% heat-inactivated
FBS, and 1% antibiotic–antimycotic solution (Life Technologies, Inc.).
Cells were grown in an incubator under 5% CO2 atmosphere at 37°C for
12 h, and then washed and suspended in DMEM with 2 mM glucose.
The macrophages were then incubated with chromium chloride (CrCl3;
Sigma Chemical Co.) or chromium picolinate (CrPic; Prince Co., USA) in
0–100 µg/L Cr medium containing no or 5 × 10−8M insulin for 4 h at 37°C.
Glucose Uptake Studies
The glucose uptake of macrophages was measured according to the
method of Morris et al. (19). After incubating 1 × 106 cells/well (n = 8)
in a 24-well microplate with Cr and insulin for 4 h, the medium was aspi-
rated and cells were washed three times with DMEM. The uptake of glu-
cose was initiated by the addition of 200 µL DMEM containing 0.2 µCi
of 2-deoxy-D-[1-3H] glucose (specific activity 16.0 µCi/mmol, Amersham
Life Sciences, UK) with “cold” anhydrous glucose as the carrier (2 mmol/L).
This produced a final volume per well of 2 mL and a final glucose con-
centration of 200 µmol/L. Glucose uptake was allowed to continue for
15 min at 37°C. After that, the cells were washed rapidly three times
with phosphate-buffered saline (PBS) and finally lysed and digested with
500 µL of 1 M sodium hydroxide. Aliquots of the solubilized cells were
counted by a liquid scintillation counter (Packard Instruments Co., USA).
The remainder was taken for protein determination by the method of
Bradford (20).
Superoxide Anion Production Assay
Superoxide anion production in macrophages was measured by the
method described by Hsiao (18). After incubating 5 × 105 cells/well (n = 8)
in a 96-well microplate with Cr and insulin for 4 h, 50 µL cytochrome-c
(4 mg/mL; Sigma Chemical Co.) and 50 µL phorbol 12-myristate 13-
acetate (PMA) (5 µg/mL; Sigma Chemical Co.) were added in the DMEM
and incubated for 30 min at 37°C. The reactions were terminated by plac-
ing them on ice. The mixtures were centrifugated at 3000g for 3 min at 4°C,
and the suspensory fractions were taken for measurement of reduced
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Lee et al.
cytochrome-c production by using a microplate spectrophotometer at
550 nm (Bio-Tek 310, USA). Absorbance values were converted to nano-
moles of cytochrome c reduced/h using the extinction coefficient
21.1 × 104 M cm.
Glucose-6-Phosphate Dehydrogenase Assay
Macrophages were plated at a density of 2 × 107 cell per 75T flask
in 20 mL of culture medium (n = 3). After incubation for 4 h at 37°C in
5% CO2, adherent macrophages were washed vigorously with PBS and
1 mL of the extraction medium containing 50 mmol Tris-HCl/L, 1 mmol
EDTA/L, and 0.05% (v/v) Triton X-100, pH 8.0, was added. Glucose-6-
phosphate dehydrogenase (G-6-PDH; E.C. 1.1.1.49) was assayed by using
a G-6-PDH assay kit (Sigma Chemical Co.) according to the modified
method of Costa Rosa et al. (21). The amount of enzyme was assayed by
following the rate change in absorbance at 340 nm within 5 min at 30°C
(Hitachi-2000, USA). A reduction of 1 nmol of NADP+ in 1 s was deter-
mined as 1 unit of G-6-PDH.
Phagocytosis
Phagocytosis was studied by coincubation of macrophages with live
Escherichia coli (ATCC 23815) (22). E. coli was grown in 5 mL trypic soy
broth (Difco Labs, Detroit, MI, USA) at 37°C for 18 h. After centrifuga-
tion at 1000g for 25 min, the bacterial pellet was washed twice and resus-
pended in PBS. The bacterial suspension was sonicated for 10 s to break
up clumps of bacteria; the optical density was measured as a basis to ad-
just the concentration to 2 × 108 bacteria/mL. Immediately prior to
assays, bacteria were treated (opsonized) with 10% pig serum for 10 min
at 37°C to facilitate phagocytosis. The opsonized bacterial suspension
was sonicated again.
Assay was performed (n = 6 wells/treatment) by using a 96-well
microplate. After incubating 1 × 105 cells/well with Cr and insulin for
4 h, all wells received E. coli of 1 × 107 bacteria/well suspended in
DMEM. Plates were incubated at 37°C in a shaking water bath for 60 min
followed by the addition of 100 µL of DMEM : H2O (1 : 1) solution and
10 µCi [3H] uridine (specific activity 37.0 Ci/mmol, Amersham) in 25 µL
to all wells. The [3H] uridine was incorporated into nonphagocytized bac-
teria and used to calculate phagocytosis. After 30 min incubation, 20 µL
of 1 M sodium hydroxide was added to cultures for 3 min, and cells then
were harvested (Parkard Inst. Co.) onto glass microfiber filters (934-AH,
Whatman, USA). The amount of radioactivity was measured by a liquid
scintillation counter. The results were calculated as the phagocytic index
(PI), by the following equation:
(cpm bacteria + macrophages)
bacteria


PI = 1 −
×




(cpm bacteria alone + cpm macrophage alone)
macrophage ratio
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Chromium on Macrophages of Weanling Pigs
57
Statistical Analysis
The variance for each set of data was analyzed by using the GLM
procedure of SAS (23). All data were expressed as mean S.E. The sig-
nificance among treatments was analyzed by Duncan’s multiple range
test (p < 0.05).
RESULTS AND DISCUSSION
The dose-dependent effect of insulin on increasing glucose uptake
by macrophages is presented in Table 1. The glucose uptake in cells incu-
bated with 5 × 10−8M insulin was 0.62 nmol/min/mg protein, which was
higher than that of the non-insulin-treated control with 0.45 nmol/min/mg
protein. Therefore, 5 × 10−8M insulin was chosen for further assays.
Chromium-stimulated changes in glucose uptake of cells differenti-
ated in CrCl3 and CrPic are presented in Fig. 1. The highest glucose up-
take occurred after cells were incubated with 20 µg/L Cr of medium
containing CrCl3 or CrPic. However, the glucose uptake declined with
the addition of 100 µg/L Cr by CrCl3 or CrPic. The increased glucose
uptake by the addition of CrCl3 was not dependent on the presence of
insulin, but the increased glucose uptake by the addition of CrPic
behaved in an insulin-dependent manner.
In Table 2, we tested the effect of the same level of picolinic acid and
CrPic (60 µg/L picolinic acid) on glucose uptake. The data showed that
the addition of picolinic acid had no effect on glucose uptake. This
demonstrated that the enhancing effect of CrPic on glucose uptake was
accomplished by CrPic. In an experiment on rat skeletal muscle cells, a
similar result was also found (24).
In the present study, the Cr level in medium was 0.46 µg/L. The pos-
itive effect of inorganic Cr on glucose uptake to a lesser extent was seen
in the absence of insulin, suggesting perhaps an insulin-mimic function
of CrCl3. Glucose uptake was lower with the addition of 100 µg/L Cr than
20 µg/L from CrCl3 or CrPic, suggesting a possible saturable mechanism
at work or a toxic effect of Cr. In this study, addition of Cr 20 µg/L with
CrCl3 increased glucose uptake by 20% and 10% in insulin-treated and
control groups, respectively. This result was similar to those in the myo-
tube cells of mouse, in which the addition of 1 µg/L Cr with CrCl3 may
have enhanced the maximum response of glucose uptake (19). Chromium
picolinate increased glucose uptake only in the presence of insulin (Fig. 1).
This showed that the effect of CrPic on glucose uptake was insulin-
dependent in macrophages. This result was similar to a study involving
liposomal membranes, in which CrPic was shown to enhance insulin inter-
nalization (14). Increasing insulin internalization may enhance glucose up-
take of muscle cells (24), suggesting that CrPic may act as an essential trace
element in insulin-dependent glucose uptake. It has been found that the
macrophage surface contains glucose transport proteins (25). Insulin may
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Lee et al.
Table 1
Effect of Insulin on 2-Deoxy-glucose Uptake in Pig
Pulmonary Alveolar Macrophages
+Values are mean S.E. (n = 8).
*Significantly different from basal level (p < 0.05).
increase the number of glucose transport proteins; thus, this results in in-
creased glucose uptake in the presence of insulin (26,27).
Changes in O−2 production following the addition of Cr are shown in
Fig. 2. An increase of 1.24-fold and 1.40-fold O−2 production occurred in
those cells incubated with 100 µg/L CrCl3 medium with and without
insulin, respectively. The addition of 20 µg/L CrPic increased O−2 pro-
duction in the presence of insulin. However, when supplementation
100µg/L CrPic to the medium had enhanced O−2 production regardless
the presence of insulin, the O−2 production increased 1.27-fold and 1.23-fold
in 100 µg/L Cr with CrPic in the incubating medium with and without
insulin, respectively. Insulin treatment has no effect on O−2 production.
The addition of CrCl3 or CrPic increased O−2 production. This result
is similar to previous studies that have suggested the dose-dependent
effects of CrPic and chromium nicotinate on enhancing O−2 production in
the cultured cell line of macrophages (28). The addition of CrCl3 and CrPic
resulted in a significant increase in the production of O−2, and the concen-
tration used in this study was 1000 times lower than the toxic level of Cr
(3+) in human macrophage tests (28–30).
The activity of G-6-PDH was not affected by Cr treatment and the
addition of insulin to the medium tended to raise the activity of G-6-
PDH (p < 0.12) (Fig. 3). The average activities of G-6-PDH were 47.43 and
36.26 nmol/min/mg protein in the presence and absence of insulin,
respectively. Insulin also has increased the activity of G-6-PDH of bovine
microvascular endothelial cells, as a result of stimulating the activity of gly-
colysis and pentose cycle enzymes (31). The supplementation of 600 ng/g
CrPic in the diet resulted in enhanced activity of hexose kinase to normal
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Fig. 1. Effect of chromium chloride (A) and chromium picolinate (B) on
uptake of 2-deoxy-glucose in pig macrophages. Data presented as percentage of
basal uptake (basal = 100%) are cells cultured in insulin 0 (᭿) and 5 × 10−8M (᭹).
Values are mean
S.E. (n = 8). Significant differences from basal level:
**p < 0.01 and *p < 0.05.
Table 2
Effect of Chromium Picolinate and Picolinic Acid
on 2-Deoxy-glucose Uptake in Pig Pulmonary
Alveolar Macrophages
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Lee et al.
Fig. 2. Effect of chromium chloride (A) and chromium picolinate (B) on
superoxide anion production in pig macrophages. Data presented are cells cul-
tured in insulin 0 (᭿) and 5 × 10−8M (᭹). Values are mean S.E. (n = 8). Signif-
icant difference from basal level: *p < 0.05.
in insulin-dependent diabetes mellitus rats (32). The patterns of G-6-PDH
and O−2 production are well correlated with respect to Cr addition (Figs.
2 and 3). The increased activity of G-6-PDH has been implicated in en-
hancing NADPH production, which may be associated with more O−2
production (33).
Figure 4 shows the effects of both Cr salts and insulin supplementa-
tion on phagocytosis in macrophages. The cumulative effect of the addition
of CrCl3 with insulin was highly significant on phagocytosis of macro-
phages (p < 0.01) (Fig. 4). In the presence of insulin, the addition of Cr
above 10 µg/L with CrCl3 increased the phagocytosis of macrophages.
However, no difference in the addition of CrCl3 without insulin was
observed. The addition of 100 µg/L CrPic resulted in higher phagocy-
tosis than that of the 1 and 10 µg/L CrPic groups in the presence of
insulin (p < 0.05). The phagocytic indexes were 43.29% and 26.99% with
the addition of 100 and 1 µg/L CrPic, respectively. However, there were
no differences among the addition of CrPic 0 to 10 µg/L in the medium.
Supplementation of chelated 0.5 ppm Cr in the diet did not affect the
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Chromium on Macrophages of Weanling Pigs
61
Fig. 3. Effect of chromium chloride (A) and chromium picolinate (B) on
glucose-6-phosphate dehydrogenase activity in pig macrophages. Data pre-
sented are cells cultured in insulin 0 (᭿) and 5 × 10−8M (᭹). Values are mean
S.E. (n = 3).
neutrophil phagocytosis of cows (13). However, Cr has been reported to
increase the blastogenesis of PBMC (11,12).
Chromium has been known as both a glucose tolerance factor and a
low-molecular-weight Cr binding substance, and to exist in trace levels
in free form in the body (34). Chromium ions can affect cellular activity
by catalyzing the removal of pyrophosphate from nucleoside triphos-
phate molecules (35). That the addition of CrCl3 resulted in increased
glucose uptake and O−2 production regardless of insulin in this experi-
ment was similar to a previous experiment conducted with muscle cells
by Morris et al. (19). The present study confirmed the direct effect of Cr
on O−2 production and phagocytosis in macrophages of weanling pigs.
The compound in the Cr–pyridine carboxylate complexes with best
affinity is CrPic. In addition, CrPic has a higher glucose uptake capacity
in muscle cells than those of Cr niacinate or CrCl3 (14,24). Further, that
CrPic induced glucose uptake in an insulin-dependent manner similar to
previous experiment shows that CrPic increased cellular function only in
the presence of insulin or in insulin-sensitive tissues (4).
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Lee et al.
Fig. 4. Effect of chromium chloride (A) and chromium picolinate (B) on
phagocytic index in pig macrophages. Data presented are cells cultured in
insulin 0 (᭿) and 5 × 10−8M (᭹). Values are mean S.E. (n = 6). Significant dif-
ference from basal level: *p < 0.05.
The Cr concentrations of plasma were 3.45 and 5.48 µg/L in the
groups supplemented with 0 and 400 ppb Cr from CrPic in weanling
pigs, respectively (7). In this study, the measurement of glucose uptake
(Fig. 1), superoxide anion production (Fig. 2), glucose-6-phosphate dehy-
drogenase activity (Fig. 3), and phagocytic index (Fig. 4) showed that there
were no differences between CrCl3 and CrPic in vitro. Under in vitro con-
ditions, the concentration for both CrCl3 and CrPic to induce the physi-
ological response of macrophages was 10–20 µg/L which is much higher
than the plasma levels of Cr-fed pigs. The viability of human J774A.1
macrophage cells was not affected in a medium containing 30–50 µg/mL
CrPic for 24 h (28). This concentration was 1000-fold higher than the
effective concentration used in this study. From this point of view, CrPic
might to postulated to be a safe agent for macrophages.
Use of this system in vitro to evaluate the effects of Cr on the func-
tion of pig macrophages would examine the direct effects of Cr on the
immune system. In this regard, the results suggest that the supplemen-
tation of 10–20 µg/L Cr with CrCl3 medium enhances the activity of
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Chromium on Macrophages of Weanling Pigs
63
macrophages directly, whereas the CrPic requires the cooperative action
of insulin to enhance glucose uptake and phagocytosis.
ACKNOWLEDGMENTS
The authors wish to acknowledge the technical assistance of Prof.
Fu Yu Wu of National I-Lan Institute of Technology and Prof. Victor Fei
Pang of National Taiwan University. We also thank the National Science
Council (NSC87-2313-B-197-003), Republic of China for financial support.
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