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Departamento de Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), CEP 13083–970, Campinas, São Paulo, Brazil;
*
Departamento de Ciência de Alimentos, Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas (UNICAMP), CP 6121, CEP 13081–970, Campinas, São Paulo, Brazil; and
Departamento de Clínica Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), CEP 13081–970, Campinas, São Paulo, Brazil.
2To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • insulin receptor • insulin receptor substrate-1 • magnesium deficiency • magnesium supplementation • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
Kimura et al. 1996
, Lowney et al. 1995
).
Studies in diabetic patients have attempted to correlate hypomagnesemia
and reduced erythrocyte Mg concentration with poor glycemic control and
the development of complications (Bloomgarden 1995
,
Paolisso et al. 1990
, Tosiello 1996
).
Because a low Mg concentration may be a consequence or a cause of
insulin resistance (Paolisso et al.1990
), it may be
premature to assign a primary role to Mg in abnormal carbohydrate
metabolism (Garber et al. 1992
). We demonstrated recently that rats fed a Mg-deficient diet for a long period showed increased insulin sensitivity, accompanied by a reduction in insulin secretion (unpublished data). However, the molecular mechanism responsible for the change was not established.
Insulin action in target tissues is mediated by the heterotetrameric
insulin receptor
(IR)3
.After ligand binding, the receptor tyrosine kinase domain is activated,
resulting in receptor autophosphorylation and tyrosine phosphorylation
of several intermediate proteins, including insulin receptor substrate
1 (IRS-1), a cytoplasmic protein with an apparent molecular weight of
160–185 kDa (Cheatam and Kahn 1995
, Myers and White 1996
). Tyrosine phosphorylated IRS-1 then couples the
insulin receptor to downstream signaling pathways by acting as a
docking protein for the src homology-2 (SH2) domain–containing
proteins. SH2 proteins are the link between upstream tyrosine kinases
and downstream signaling elements. One of the substrates of tyrosine
phosphorylated IRS proteins is the lipid metabolizing enzyme
phosphatidylinositol (PI) 3-kinase (Kelly and Ruderman 1993
). In addition to its role in the regulation of
mitogenesis, cellular transformation, differentiation, chemotaxis and
membrane ruffling (Lange et al. 1998
), activated PI
3-kinase is involved in insulin-stimulated glucose uptake and
glycogen synthesis (Holman and Kasuga 1997
). Thus, the
pathway involving the insulin receptor, IRS-1 proteins and PI 3-kinase
plays some role in glucose clearance.
In this study, we investigated the phosphorylation state of IR and IRS-1, as well as the association of the latter with PI 3-kinase in the liver and muscle of Mg-deficient and Mg-supplemented rats treated acutely with insulin.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Monoclonal anti-phosphotyrosine antibody and anti-PI 3-kinase (p85)
antibody were from Santa Cruz (Santa Cruz, CA). The anti-IRS-1 and
anti-IR antisera were previously described (Sun et al. 1992
). [125I] Protein A was from Amersham
(Buckinghamshire, UK) and Protein A Sepharose 6 MB from Pharmacia
(Uppsala, Sweden). Nitrocellulose (BA85, 0.2 mm) was from Schleicher &
Schuell (Keene, NH) and the chemicals were from Sigma Chemical (St.
Louis, MO).
Buffers.
Buffer A consisted of 100 mmol/L Tris, 10 g/L SDS, 50 mmol/L (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) (pH 7.4), 100 mmol/L sodium pyrophosphate, 100 mmol/L sodium fluoride, 10 mmol/L ethylenediaminetetraacetic acid and 10 mmol/L sodium vanadate. Buffer B was similar to buffer A except that 10 g/L of Triton X-100 replaced 10 g/L of SDS, and 2 mmol/L phenylmethylsulfonylfluoride (PMSF) and 0.1 g aprotinin/L were added. Buffer C contained 100 mmol/L Tris, 10 mmol/L sodium vanadate, 10 mmol/L EDTA and 10 g/L Triton X-100.
Animals.
Male Wistar rats (21 d old, 40–60 g), bred at the State University of
Campinas animal breeding center, were allowed 1 wk to adapt to housing
conditions during which they were fed a nonpurified diet (Purina
laboratory diet, Paulinia, São Paulo, Brazil). Thereafter, the
rats were fed either a control diet (CO-6 group) containing 507 mg
Mg/kg or a Mg-deficient diet (DF-6 group) containing 70 mg Mg/kg
(Table 1
). After 6 wk of treatment, some rats from the CO-6 and DF-6 groups
(referred to as CO-11 and DF-11) were maintained on their respective
diets for an additional 5 wk (Fig. 1
). The remaining rats from both groups were changed to a
Mg-supplemented diet containing 2100 mg Mg/kg (Table 1)
for the
same period (referred to as supplemented control, SCO, and supplemented
deficient, SDF groups). The rats had free access to deionized water and
food throughout the study. They were housed individually, and rooms
were maintained at 21 ± 2°C on a 12-h light:dark cycle (lights
on from 0600 to 1800 h). Food deprivation began at 1800 h.
The rats were weighed at the end of the first and second phases. At the
end of each experimental period and after 12 h of food
deprivation, blood samples were collected from the abdominal cava vein,
allowed to clot and the sera stored at -20°C for the subsequent
measurement of insulin by RIA (Scott et al. 1981
) and
for the measurement of magnesium and calcium. The minerals in the sera
and diets were measured by inductively coupled plasma emission
spectroscopy (ICP) using an argon plasma emission spectrophotometer
(BAIRD ICP 2000; Baird , Deford, MA). Mg and Ca serum levels were
measured after digestion of the samples in 10 mol/L HNO3
and appropriate dilution with deionized water (Slavin et al. 1975
). Preparatory to analysis, diets were dried, calcinated at
450°C and diluted in 0.8 mol/L HNO3 (AOAC
1997
). All experiments involving animals were approved by the
State University of Campinas Ethics Committee (São Paulo,
Brazil).
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An intravenous insulin-tolerance test (ivITT) was performed in both
phases. After 12 h of food deprivation, insulin (0.5 mL of a
10-5 mol/L solution) was injected intravenously, and
samples for plasma glucose determination were collected from the cut
tip of the tail at 0 (basal), 4, 8,12 and 16 min after hormone
injection. Plasma glucose levels were determined by the glucose oxidase
method. The glucose disappearance rate
(Kitt) during the ivITT was calculated using
the formula 0.693/t1/2 (Lundbæk 1962
).
The plasma glucose half-time (t1/2) was
calculated from the slope of the least-square analysis of the
plasma glucose concentrations during the linear decay phase. On the
basis of data from Bonora et al. (1989)
, a high degree
of correlation between the insulin-tolerance test (ITT) and clamp
studies confirmed the reliability of this study.
Tissue extraction, immunoblotting and immunoprecipitation.
The rats were anesthetized with sodium amobarbital (15 mg/kg body
weight, intraperitoneally) and used 10–15 min later, as soon as
anesthesia was ensured by loss of the pedal and corneal reflexes. After
the portal vein was exposed, a bolus injection of 0.5 mL of normal
saline (9 g/L NaCl), with or without insulin (10-5 mol/L)
was given. The insulin dose was chosen on the basis of previous work in
which the amount of insulin required to achieve a large signal was
determined (Saad et al. 1995
). At 30 s
postinjection, the bolus injection of insulin leads to a transient rise
in the peripheral concentration of this hormone that is five to ten
times the postprandial levels. The very high levels of insulin attained
suggest that different levels of circulating insulin cannot explain
some of the variations observed. A fragment of the liver was excised
30 s later, minced coarsely and immediately homogenized in freshly
prepared boiling buffer A for immunoblotting, or freshly prepared
ice-cold buffer B for immunoprecipitation. Approximately 90 s
after injection, hind-limb muscle (musculus gastrocnemius) was
quickly excised and homogenized as described for liver. The insoluble
material in both extracts was removed by centrifugation for 45 min at
50,000 x g at 4°C. The protein concentration in
the supernatants was determined by the Bradford method (Bradford 1976
).
For immunoblotting, samples of 150 µg of total protein
were suspended in 50 µL of Laemmili sample buffer,
boiled for 5 min and subjected to SDS-PAGE (6% bis-acrylamide) in
a Bio-Rad (Hercules, CA) miniature slab gel apparatus
(Mini-Protean). For immunoprecipitation, samples containing 3
mg of total protein were incubated with 15 µL of
anti-IRS-1 or anti-IR antiserum at 4°C overnight. The immune
complexes were precipitated for 1 h with protein A-Sepharose 6
MB and were repeatedly washed in buffer C (five times). The pellets
were resuspended in 50 µL of Laemmli sample buffer and
boiled for 5 min before loading onto the gel. Electrotransfer of
proteins from the gel to nitrocellulose was performed as described by
Towbin et al. (1979)
. The membranes were blocked, probed
and developed as described previously (Saad et al. 1995
).
Statistics.
The results are presented as means ± SD for the number of rats (n) indicated. Student’s t test for unpaired data was used for direct comparisons between the CO-6 and DF-6 groups. Levene’s test followed by one-way or two-way ANOVA and the Tukey-Kramer test for multiple comparisons among groups were used in the second phase. P-values < 0.05 were considered to indicate significant differences.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After 6 wk, the serum Mg concentration in DF-6 rats (0.4 ± 0.02
mmol/L) was significantly lower (P < 0.001) than that
in CO-6 rats (1.05 ± 0.2 mmol/L), but there was no difference in
the serum Ca concentrations of the two groups. By wk 11 of treatment,
the serum Mg concentrations of DF-11 rats were significantly
(P < 0.01) lower than those of CO-11, SDF and SCO rats
(Table 2
). Two-way ANOVA revealed a significant effect of the previous diet
(df 1; F 25; P < 0.001) and supplementation (df 1; F
47; P < 0.001), as well as a two-way interaction
between the previous diet and supplementation (df 1; F 85; P
< 0.001) (Table 2)
. The serum Ca concentrations during the second
phase did not differ among the groups. A significant effect of the
previous diet (df 1; F 7; P = 0.03) and supplementation
(df 1; F 29.7; P = 0.005) was observed, but with no
significant two-way interaction (Table 2)
. The classic signs of Mg
deficiency were observed from wk 3 to 5 of treatment, after which
hyperemia of the ears and ulcerative lesions around the head and neck
disappeared. During the first phase, the body weight of DF-6 rats was
5% lower than that of CO-6 rats (DF-6, 294.4 ± 17.8 g,
n = 13, and CO-6, 310 ± 17.6 g, n
= 12, P < 0.05). However, at the end of the
second phase, the body weights did not differ among the groups (CO-11
rats, 413 ± 36.7 g, n = 6; DF-11 rats 385
± 20.4 g, n = 7; SDF rats, 399.5 ± 35.3 g, n = 6; SCO rats, 413.6 ± 17.5 g, n = 6).
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There were no significant differences in the
Kitt and plasma glucose and serum
insulin concentrations of food-deprived rats from the first phase
(data not shown). By wk 11 of treatment, rats in the second phase had
basal plasma glucose and serum insulin concentrations that did not
differ, and two-way ANOVA showed no significant main effect or
interaction (data not shown). In contrast, the
Kitt of DF-11 rats was significantly
higher (P < 0.05) than that of the other groups (Table 2)
. Two-way ANOVA showed a significant main effect of the previous
diet (df 1; F 4.4; P = 0.047) and supplementation (df
1; F 8.2; P = 0.008), as well as a two-way
interaction between the previous diet and supplementation (df 1; F
8.24; P = 0.008).
Effect of a Mg-deficient diet on IR and IRS-1 protein and phosphorylation levels and IRS-1/PI 3-kinase association in rat liver and muscle.
The protein levels of IR and IRS-1 and the rate of IR and IRS-1 phosphorylation in response to insulin, as well as the rate of IRS-1/p85/PI 3-kinase association, did not differ between the CO-6 and DF-6 groups in either tissue studied (data not shown).
Effect of Mg deficiency and supplementation on IR and IRS-1 protein and phosphorylation levels and IRS-1/PI 3-kinase association in rat muscle.
Mg deficiency and subsequent supplementation had no effect on the early steps of insulin signaling in muscle from CO-11, DF-11 and SDF rats. There were no differences in the amount of IR protein (100 ± 23%, n = 8 for CO-11; 85 ± 51%, n = 7 for DF-11, and 74 ± 36%, n = 7 for SDF), or IRS-1 protein (100 ± 16.5%, n = 9 for CO-11, 121 ± 15%, n = 5 for DF-11, and 125 ± 24%, n = 7 for SDF) among rats in the second phase. Similarly, there were no significant differences in the insulin-stimulated phosphorylation of IR (100 ± 24%, n = 5 for CO-11, 84 ± 24%, n = 4 for DF-11, and 94 ± 15%, n = 4 for SDF), and IRS-1 (100 ± 48%, n = 8 for CO-11, 131 ± 31%, n = 5 for DF-11, and 118 ± 25%, n = 14 for SDF), or in the IRS-1/p85/PI 3-kinase association (100 ± 31%, n = 8 for CO-11, 93 ± 18.5%, n = 4 for DF-11, and 124 ± 21%, n = 12 for SDF).
Effect of Mg deficiency and supplementation on IR and IRS-1 protein and phosphorylation levels and IRS-1/PI 3-kinase association in rat liver.
Figure 2A
shows the IR protein levels in the liver of second-phase rats as
detected by immunoblotting. Higher IR levels were observed in DF-11
than in CO-11 rats (P < 0.001). In SDF rats, the IR
level was not different from that of CO-11 rats, but was significantly
lower than that in DF-11 rats (P < 0.005).
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), the level of this protein was 62.5 ± 24% greater
in the liver of DF-11 rats (P < 0.002) compared with
CO-11 rats. In the SDF group, the IRS-1 levels were not significantly
different from those in either CO-11 or DF-11 rats.
Liver samples previously immunoprecipitated with anti-insulin
receptor antibody and immunoblotted with antiphosphotyrosine antibody
(Fig. 3A
) showed a 88.5 ± 26% greater insulin-stimulated IR
phosphorylation in DF-11 rats compared with CO-11 rats (P
< 0.02), whereas in SDF rats, the values were not significantly
different from those of the other two groups.
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).
To examine the association of the 85-kDa subunit of PI 3-kinase with
IRS-1, blots of samples that had been previously immunoprecipitated
with anti-IRS-1 antibodies were incubated with anti-PI 3-kinase
antibody (Fig. 4
). As expected, a greater (41 ± 22%) insulin-stimulated
IRS-1-p85/PI 3-kinase association was detected in the liver of DF-11
rats compared with CO-11 rats. SDF rats tended (P = 0.195) to have a greater (18.5 ± 10%) IRS-1-p85/PI
3-kinase association than CO-11 rats.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Nadler et al. 1993
). Our results demonstrate that insulin sensitivity,
measured by the glucose disappearance rate
(Kitt) during the 15 min ITT, was not
different in DF-6 rats and CO-6 rats. In addition, DF-6 rats had a
normal glucose uptake at maximal insulin concentration, as previously
described (Suárez et al. 1995
). On the other hand,
after an additional period of deficiency, DF-11 rats showed an
improvement in insulin sensitivity, as revealed by the elevated
Kitt compared with the CO-11, SDF and
SCO groups, suggesting an increased glucose uptake during Mg
deficiency. The mechanism underlying the improvement in insulin
sensitivity induced by Mg deficiency is not fully understood. The
increased glucose disposal observed in Mg-deficient rats has been
associated with a noninsulin-mediated glucose uptake and an
improvement in hepatic insulin sensitivity (Lowney et al. 1995
).
The Mg-deficient diet fed to DF-6 rats did not affect the insulin
sensitivity or the amount and action of the proteins involved in the
early steps of insulin action in the two tissues studied. However, a
longer Mg deficiency significantly increased the insulin sensitivity
and the insulin-induced IR and IRS-1 protein and tyrosine
phosphorylation levels in the liver of DF-11 rats. The increased
phosphorylation of IRS-1 was accompanied by an increase in IRS-1/PI
3-kinase association in this tissue. These increases in IR and IRS-1
phosphorylation were observed in the liver, but not in muscle,
suggesting a tissue-specific effect of Mg deficiency. Our results
may be of biological importance because an increase in insulin receptor
and IRS-1 phosphorylation, as well as IRS-1/PI 3-kinase association,
has been associated with enhanced insulin sensitivity in animal models
of insulin resistance (Carvalho et al. 1997
) and in
animal models of protein malnutrition (Latorraca et al. 1998
, Reis et al. 1997
).
Food-deprived and streptozotocin-induced diabetic animals show an
increase in total receptor amount, in IRS-1 phosphorylation and in the
association/activation between IRS-1 and PI 3-kinase, despite the
decreased responsiveness to insulin (Saad 1994
).
However, when the data were normalized for insulin binding in liver and
muscle, reduced receptor phosphorylation (per receptor) was observed in
both animal models (Saad et al. 1992
). These findings
suggest that in insulinopenic diabetes, hypoinsulinemia-induced
insulin receptor up-regulation is associated with desensitization
of the receptor kinase as a result of chronic hyperglycemia
(Sbraccia et al. 1994
). In contrast, when hyperglycemia
is associated with hyperinsulinemia, the receptor kinase defect is
exacerbated, and the insulin resistance is more severe (Mayor et al. 1992
). This alteration in insulin receptor tyrosine kinase
activity has been attributed to protein kinase C activation in
response to elevated intracellular glucose (Draznin et al. 1988
, Koya and King 1998
). Thus, it is possible
that reduced glycemia and insulinemia (Gueux and Rayssiguier 1983
) and reduced total area under the glucose and insulin
curves (Reis et al., unpublished data) in Mg-deficient rats could
act synergistically to hypersensitize the tyrosine kinase of the
insulin receptor.
In addition to increased IR tyrosine phosphorylation, an increased
number of insulin receptors were observed in DF-11 rats compared with
the other groups. This may be explained by an increase in insulin
receptor gene transcription in the presence of long-term, reduced
insulinemia (Tozzo and Desbuquois 1992
), or by
alterations in receptor internalization and intracellular degradation
(Carpentier 1992
).
Although the importance of certain intracellular ions in the control of
several cell functions is well established, the role of Mg is not
completely known. Many components of the protein synthetic machinery
are sensitive to changes in Mg concentrations (Barnes et al. 1995
, Zieve et al. 1977
). However, the presence
of Mg per se is not an absolute requirement for protein synthesis or
function in all cell types (Barnes et al. 1995
).
As with IR, increased IRS-1 protein and tyrosine phosphorylation levels
were observed in the liver of DF-11 rats after insulin stimulation. On
the basis of the concept of IRS-1 as the core molecule of a
multisubunit signaling complex, high levels of IRS-1 expression could
potentially dilute out the effector proteins (Yamauchi and Pessin 1994
) and differentially regulate postreceptor processes
by preserving the phosphorylation of only some substrates and pathways
(Thirone et al. 1998
). Studies in Chinese hamster ovary
cells overexpressing both IRS-1 and insulin receptors have shown that
there may be a delicate balance between the levels of IRS-1 and insulin
receptor in the insulin action cascade (Sun et al. 1992
). There is evidence that increased expression of the
insulin receptor and/or IRS-1 can either enhance or inhibit insulin
signaling, depending on the relative levels of IR and IRS-1 and other
intracellular components (Yamauchi and Pessin 1994
).
As expected, the increase in IRS-1 phosphorylation observed in the
liver of DF-11 rats was accompanied by an increase in the association
of IRS-1 with the p85 subunit of PI 3-kinase. Although PI 3-kinase
activity was not determined, studies on the regulation of enzymatic
activity have reported a close correlation with IRS-1 phosphorylation.
PI 3-kinase, the best-studied signaling molecule activated by
IRS-1, plays an important role in many insulin-regulated metabolic
processes, including glucose transport by translocating the
intracellular glucose transporter (GLUT 4) to the cell surface
(Cheatam et al. 1994
, Tsakiridis et al. 1995
). Evidence from different sources has demonstrated a
correlation between PI 3-kinase activity and glycogen metabolism
(Welsh et al. 1994
). Thus, the IRS-1/PI 3-kinase pathway
may be linked to the activation of glycogen synthesis in liver, and an
increase in this association in DF-11 rats may have a role in the
enhanced insulin sensitivity of these animals. Knocking out the IR in
mouse liver leads to a more pronounced effect on insulin action (i.e.,
severe insulin resistance) than does knocking out this receptor in
muscle (Bruning et al. 1998
, Michael et al. 1999
). Thus, at least in rodents, the liver plays a more
important role in glucose clearance than muscle, what may explain the
present data.
Mg supplementation in the SDF group maintained the insulin sensitivity in a manner similar to that of CO-11 rats, and reduced it in comparison to the DF-11 group. Because Mg supplementation in the SCO group did not interfere with the insulin sensitivity as determined by the ivITT, the early steps of insulin action were not studied in this group.
To investigate the mechanism behind the phosphorylation-enhancing action of Mg deficiency, we examined the effect of Mg supplementation in regulating the phosphorylation of these proteins. SDF rats showed values between those of the CO-11 and DF-11 groups for the proteins involved in the early steps of insulin action, with no significant alteration in the sensitivity to insulin in either of the tissues studied.
Thus, the moderate Mg deficiency imposed for 6 wk did not change the sensitivity to insulin relative to control rats as determined by the Kitt and Western blot analysis of IR, IRS-1 and the IRS-1/PI 3-kinase association in muscle and liver. In contrast, an enhanced Kitt and hepatic insulin sensitivity were observed in rats fed a Mg-deficient diet for 11 wk. This group showed increased insulin-induced protein and tyrosine phosphorylation levels of the IR and IRS-1, as well as increased IRS-1/PI 3-kinase association compared with the CO-11 group. The maintenance of insulin sensitivity in SDF rats suggested that Mg supplementation in Mg-deficient rats avoided the increase in insulin sensitivity seen in the DF-11 rats. Thus, modulation of the early steps of insulin action in the liver of rats fed a Mg-deficient diet for 11 wk may play a role in the improved sensitivity to insulin seen in this animal model.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: CO, control group; DF, Mg-deficient group; ICP, inductively coupled plasma emission spectroscopy; IR, insulin receptor; IRS-1, insulin receptor substrate-1; ITT, insulin-tolerance test; ivITT, intravenous insulin-tolerance test; Kitt, rate constant for serum glucose disappearance during insulin-tolerance test; PI 3-kinase, phosphatidylinositol 3-kinase; PMSF, phenylmethylsulfonylfluoride; SCO, Mg-supplemented control group; SDF, Mg-supplemented deficient group; SH2, src homology-2.
Manuscript received June 23, 1999. Initial review completed July 12, 1999. Revision accepted October 11, 1999.
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