1 Departments of Physiology and 2 Internal Medicine, University of Manitoba, Winnipeg R3E 0W3, Canada
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ABSTRACT |
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Glucose homeostasis was
examined in male transgenic (Tg) mice that overexpressed the human
insulin-like growth factor (IGF)-binding protein (IGFBP)-3 cDNA, driven
by either the cytomegalovirus (CMV) or the phosphoglycerate
kinase (PGK) promoter. The Tg mice of both lineages
demonstrated increased serum levels of human (h) IGFBP-3 and
total IGF-I compared with wild-type (Wt) mice. Fasting blood glucose
levels were significantly elevated in 8-wk-old CMV-binding protein
(CMVBP)-3- and PGK binding protein (PGKBP)-3-Tg mice compared with Wt
mice: 6.35 ± 0.22 and 5.22 ± 0.39 vs. 3.99 ± 0.26 mmol/l, respectively. Plasma insulin was significantly elevated only in CMVBP-3-Tg mice. The responses to a glucose challenge were
significantly increased in both Tg strains: area under the glucose
curve = 1,824 ± 65 and 1,910 ± 115 vs. 1,590 ± 67 mmol · l1 · min for CMVBP-3, PGKBP-3,
and Wt mice, respectively. The hypoglycemic effects of insulin and
IGF-I were significantly attenuated in Tg mice compared with Wt mice.
There were no differences in adipose tissue resistin, retinoid X
receptor-
, or peroxisome proliferator-activated receptor-
mRNA
levels between Tg and Wt mice. Uptake of 2-deoxyglucose was reduced in
muscle and adipose tissue from Tg mice compared with Wt mice. These
data demonstrate that overexpression of hIGFBP-3 results in fasting
hyperglycemia, impaired glucose tolerance, and insulin resistance.
insulin resistance; diabetes; resistin; peroxisome
proliferator-activated receptor-
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INTRODUCTION |
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ALTHOUGH THE INSULIN-LIKE GROWTH FACTORS (IGFs) can exert hypoglycemic effects when administered to rodents or human subjects (2, 22), the role of endogenous IGF-I and -II in glucose homeostasis remains unclear. Although IGF-I and -II are present in much higher concentrations in the circulation than insulin, the presence of high-affinity binding proteins limits the free concentration of the IGFs in plasma to levels comparable with insulin (3).
In experiments with rodents and human subjects, the IGFs are ~5-15% as potent as insulin in terms of their acute hypoglycemic effects (2, 22). Thus the 2- to 8-ng/ml plasma concentrations of free IGF-I and -II (8) are unlikely to have a major hypoglycemic effect compared with the 1- to 4-ng/ml of insulin normally present in human plasma. However, hyperglycemia has been convincingly demonstrated after the injection of IGF-binding protein (IGFBP)-1 in rats (13) and in transgenic (Tg) mice that overexpress IGFBP-1 (20). In these situations, the hyperglycemia could be attributable to a reduction in free IGF-I levels, although this was not documented in either study (13, 20). Furthermore, in mice carrying a liver-specific null mutation in the IGF-I gene, there is an increase in serum insulin levels despite normal blood glucose levels (24), possibly indicating that the reduction in the tonic hypoglycemic effect of free IGF-I resulted in a compensatory increase in insulin levels.
Conversely, hypoglycemia is observed when there are increased levels of free IGF present in the circulation. The pathological condition of nonislet tumor-associated hypoglycemia is due to synthesis and secretion of a 15-kDa IGF-II variant (7). In this condition, elevated levels of "big IGF-II" are associated with suppressed growth hormone (GH) levels and, as a consequence, reduced levels of IGF-I, IGFBP-3, and the acid-labile subunit (32). This results in a decreased proportion of the IGFs present in the ternary complex and presumably increased levels of free IGFs, particularly free IGF-II, in the blood (32). Taken together, these data suggest that the free IGF-I and -II may have a tonic hypoglycemic effect and that modulation of the IGFBPs by regulation of the proportion of free and bound IGF may be important in glucose homeostasis.
In addition to modulating the availability of IGF-I and -II, IGFBP-3
has been shown to have IGF-independent effects on cellular proliferation and apoptosis in a variety of cell lines
(18, 19, 28). These effects may be mediated either via
cell surface binding proteins or nuclear binding sites (19,
22). Thus the potential exists for IGFBP-3 to have both
IGF-dependent and IGF-independent effects on glucose homeostasis. Of
note in this regard is the recent observation that IGFBP-3 interacts
with the retinoid X receptor- (RXR
; Ref. 14). The
latter is an important binding partner for the peroxisome
proliferator-activated receptor-
(PPAR
), a nuclear protein that
is involved in transcriptional regulation of a variety of enzymes
involved in glucose and lipid metabolism (11).
In this report, we investigated the effects of overexpression of
IGFBP-3 in Tg mice on glucose homeostasis. Expression of PPAR,
RXR
, and resistin, a potential mediator of the insulin-sensitizing PPAR
agonists (25), was also assessed in these Tg mice.
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MATERIALS AND METHODS |
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Tg mice. The generation of the Tg mice and the characterization of their phenotype have been described in detail elsewhere (15). A human (h) IGFBP-3 cDNA containing the entire coding region was subcloned downstream of either the mouse phosphoglycerate (PGK) promoter (11) or the cytomegalovirus (CMV) promoter (9). Tg mice were generated by microinjection of the transgene into pronuclei of fertilized CD-1 zygotes. The founders were bred with CD-1 mice. CD-1 mice from the same colony, bred in a similar fashion, provided wild-type (Wt), non-Tg control mice of the same genetic background. The Tg mice of both lineages demonstrated ubiquitous expression of hIGFBP-3 mRNA and serum levels of hIGFBP-3 (~5 µg/ml) compared with Wt mice (15).
Homozygous Tg mice and Wt male mice of 8 wk of age were used for all experiments. Studies on fasting mice were performed starting at 10 AM after an overnight fast. For all experiments, mice were anesthetized with 2.4% Avertin, and blood samples were collected from the retroorbital sinus using heparinized capillaries unless otherwise stated. Samples for insulin, GH, and leptin assays were collected between 9 and 11 AM. Because GH levels were suppressed by avertin-induced anesthesia, blood for GH determinations was collected by cardiac puncture after cervical dislocation. All experiments were performed in accordance with protocols approved by the Animal Care Committee of the Faculty of Medicine, University of Manitoba.Blood glucose determinations and glucose tolerance test. Glucose was measured in whole blood with the use of a glucose analyzer (YSI 2300; Yellow Springs, OH). For the intraperitoneal glucose tolerance test, mice were fasted overnight. Blood samples were collected before and 15, 30, 60, 120, and 180 min after administration of an intraperitoneal injection of glucose (1 mg/g body wt in sterile 0.45% saline). The area under the glucose curve (AUCglucose) was calculated by the trapezoid method. In a separate experiment, the insulin concentration in fasting mice was measured before and 15, 30, and 60 min after an intraperitoneal glucose injection (1 mg/g body wt).
Insulin and IGF-I tolerance tests. Recombinant human insulin and IGF-I were purchased from Boehringer (Mannheim, Germany) and GroPep (Adelaide, Australia), respectively. The insulin and IGF-I tolerance tests were performed on overnight-fasted 8-wk-old male mice. Blood glucose was measured before and 20, 40, 60, 80, 100, and 120 min after the subcutaneous administration of insulin (10 µg/kg body wt) or IGF-I (50 µg/kg body wt).
RNA extraction and RNase protection assays.
Total RNA was isolated from skeletal muscle and hepatic and adipose
tissue with TRIzol reagent (GIBCO-BRL Life Technology, Burlington, ON,
Canada). The concentration of RNA was determined spectrophotometrically, and the integrity of the RNA in all samples was
documented by visualization of the 18S and 28S ribosomal bands after
electrophoresis through a 0.8% formaldehyde-agarose gel. RNase
protection assays (RPA) were used to measure resistin, RXR, and
PPAR
mRNA abundance in adipose tissue. To generate radiolabeled cRNA
for mouse resistin, RT-PCR was used to generated a 267-bp fragment of
mouse resistin corresponding to nucleotides 110-376 of the
published sequence (25). This fragment was subcloned into
pCR II vector (Invitrogen, San Diego, CA). Similarly, fragments of
mouse PPAR
corresponding to nucleotides 489-749
(6) and mouse RXR
corresponding to nucleotides
155-363 (5) were generated by RT-PCR and subcloned
into pCR II. The linearized plasmids were used as templates for RNA
probe synthesis. A human cRNA probe (15) was used to
compare transgene expression in skeletal muscle of 8-wk-old PGK-binding
protein (PGKBP)-3- and CMV binding protein (CMVBP)-3-Tg mice.
Radioimmunoassays. The fasting insulin concentrations in plasma were measured by radioimmunoassay (RIA; Pharmacia Upjohn Diagnostics, Uppsala, Sweden). The sensitivity of the assay was <2 µU/ml. Plasma leptin concentration in fasted and nonfasted animals was measured by an immunoradiometric assay specific for mouse leptin, purchased from Linco Research (St. Charles, MO). The sensitivity of the assay was 0.2 ng/ml. GH was measured by RIA with a sensitivity of 1.5 ng/ml by using reagents obtained from Amersham Pharmacia Biotech (Baie d'Urfe, QC, Canada). Resistin levels were measured in plasma from overnight-fasted mice by use of reagents obtained from Phoenix Pharmaceuticals (Belmont, CA). The sensitivity of the assay was 4 ng/ml. IGF-I was determined by using an IGF-I RIA kit purchased from Nichols Institute Diagnostics (San Juan Capistrano, CA). Sensitivity of the assay is 13.5 ng/ml, and intra-assay coefficient of variation was 6.3%. For total IGF-I measurement, IGF-I was separated from its binding proteins by acid-ethanol extraction. For separation of free IGF-I from serum, centrifugal ultrafiltration, under conditions approaching those in vivo, was used as described elsewhere (4). Briefly, 400 µl of serum were applied to Amicon YMT 30 membrane (Millipore, Bedford, MA), incubated for 30 min at 37°C at 5% CO2 atmosphere, and centrifuged (1,500 g at 30°C, 25 min). Serum-free IGF-I was determined directly in the ultrafiltrates. To minimize binding of IGF-I to plastic surfaces, filtrate cups were preincubated with 1 mg/ml RIA-grade bovine serum albumin (Sigma, St. Louis, MO) and washed twice with PBS before centrifugation.
Western blotting.
Epididymal fat tissues were homogenized in RIPA buffer (10 mM
Tris · HCl, pH 8.0, 10 mM EDTA, pH 8.0, 0.15 M NaCl, 1% NP-40, 0.5% SDS, 1 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl
fluoride). The homogenate was clarified by centrifugation at 15,000 g for 15 min at 4°C and separated by electrophoresis
through an 8% SDS-polyacrylamide gel. The separated proteins were
transferred to nitrocellulose membrane (MSI, Westborough, MA) at 200 mA
for 2 h. The blots were incubated overnight at 4°C with a 1:500
dilution of rabbit anti-PPAR or rabbit anti-RXR
antibody (Santa
Cruz Biotechnology, Santa Cruz, CA). After a washing with TBST buffer
(5 mM Tris · HCl, pH 7.4, 136 mM NaCl, and 0.05% Tween 20),
the blots were incubated for 2 h at room temperature with
horseradish peroxidase-linked goat anti-rabbit IgG (Santa Cruz
Biotechnology) at a dilution of 1:10,000. Detection of immune complexes
was achieved with the use of an enhanced chemiluminescence (ECL)
Western blotting kit (Amersham Pharmacia Biotech).
2-Deoxyglucose uptake.
Overnight-fasted mice were anesthetized with Avertin.
2-Deoxy-[3H]glucose (2-DG; NEN Life Science Products) was
administered as a bolus via a tail vein. To achieve the same specific
activity immediately after the injection, individual mice received 0.1 µCi 2-DG · mmol1 · l
1 of
blood glucose. Blood was collected from retroorbital sinus before and 1, 5, 10, 20, 30, and 40 min after 2-DG injection for measurement of blood glucose and determination of blood glucose specific activity. After 40 min, animals were exsanguinated and tissues
were immediately frozen and stored at
70°C. For determination of
tissue 2-DG uptake, tissue samples were homogenized in distilled water
and centrifuged for 10 min at 10,000 g. These and subsequent steps were carried out at 4°C. Tissue supernatants were deproteinized with an equal volume of 7% perchloric acid, and, after 30 min on ice,
samples were centrifuged at 10,000 g for 10 min. Supernatant (600 µl) was then neutralized with 2.2 M KHCO3 (150 µl). An aliquot was taken for determination of radioactivity. For
measurement of plasma 2-DG radioactivity, 10 µl of plasma were
deproteinized as described above.
Statistical analysis. Data are expressed as means ± SE. Student's t-test was used for single comparisons between Tg and Wt mice. For determining statistical differences between multiple groups, an analysis of variance with repeated measures followed by Dunnett's t-test was used.
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RESULTS |
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Fasting blood glucose levels were significantly elevated in
8-wk-old CMVBP-3- and PGKBP-3-Tg mice compared with Wt mice (Fig. 1). Plasma insulin levels were
significantly elevated in CMVBP-3-Tg mice compared with Wt mice, but
the insulin levels were similar in Wt and PGKBP-3-Tg mice (Fig. 1).
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When the blood glucose response to an intraperitoneal glucose challenge
was assessed, more marked glycemic excursions were apparent in blood
from CMVBP-3- and PGKBP-3-Tg mice than from Wt mice (Fig.
2). PGKBP-3-Tg mice showed the largest
glycemic excursions. When the AUCglucose was
quantified, it was significantly greater in both CMVBP-3- and
PGKBP-3-Tg mice than in Wt mice (Fig. 2). The insulin response to the
glucose challenge, as measured as the area under the insulin curve
(AUCinsulin), was similar for PGKBP-3-Tg and Wt mice:
546 ± 23 vs. 608 ± 43, respectively (P = 0.34). In contrast, the AUCinsulin for CMVBP-3-Tg mice was significantly increased compared with Wt mice: 760 ± 57 vs.
608 ± 43 (P = 0.035).
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The effects of insulin and IGF-I on blood glucose were also examined in
Tg and Wt mice. The hypoglycemic effects of insulin were markedly
attenuated in CMVBP-3 mice and modestly reduced in PGKBP-3 mice (Fig.
3). The difference between the insulin
tolerance curve for CMVBP-3-Tg mice and Wt mice was significant
(P < 0.001, analysis of variance with repeated
measures), whereas the differences between curves for PGKBP-3-Tg and Wt
mice did not achieve statistical significance (P = 0.15). However, when the first 80 min were analyzed separately, there
was a statistical difference between the curves for
PGKBP-3-Tg and Wt mice (P = 0.04). Because the
starting basal blood glucose levels were lower in Wt mice than in
PGKBP-3 mice, a plateau in blood glucose was reached in Wt mice after
~1 h, possibly as a result of counterregulatory mechanisms. As a
consequence, the Wt and PGKBP-3 insulin tolerance curves overlapped in
the later time points.
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The effects of IGF-I on blood glucose were markedly attenuated in both CMVBP-3- and PGKBP-3-Tg mice compared with Wt mice (Fig. 3). The differences between CMVBP-3 and Wt mice and PGKBP-3 and Wt mice were significant (P = 0.002 and P = 0.021, respectively). However, there were no significant differences in the blood glucose response to IGF-I between CMVBP-3- and PGKBP-3-Tg mice.
Leptin levels were measured in both fasting and nonfasting 8-wk-old
male mice (Table 1). Leptin levels
were significantly increased in CMVBP-3 mice compared with Wt
mice but were similar in PGKBP-3-Tg mice and Wt mice. In all
groups of mice, food deprivation reduced plasma leptin levels by
~50%; however, leptin levels remained significantly higher in the
serum from fasted CMVBP-3-Tg mice than fasted Wt mice.
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Serum GH levels were undetectable in fasted mice, consistent with previous reports of fasting-induced suppression of GH secretion in rodents (26). In nonfasted mice, GH levels were similar in CMVBP-3-Tg and Wt mice. In contrast, GH levels were significantly increased twofold in PGKBP-3 mice (Table 1).
Total and free IGF-I levels are shown in Table 1. Total IGF-I and free IGF-I levels were significantly elevated in both CMVBP-3- and PGKBP-3-Tg mice compared with Wt mice. The ratio of free to total IGF-I was not significantly different in any of the groups of mice.
Tritiated 2-DG uptake was assessed under basal conditions in Tg and Wt
mice. The disappearance curves for Tg and Wt mice are shown in Fig.
4. The clearance of 2-DG from the
circulation was significantly reduced in CMVBP-3-Tg mice compared with
Wt mice, and the uptake into skeletal muscle and adipose tissue was
also reduced in these Tg mice. The clearance of 2-DG from the
circulation of PGKBP-3-Tg mice was also significantly slower than that
of Wt mice, and there was a significant reduction in uptake of 2-DG in
the quadriceps muscle in PGKBP-3-Tg mice. There was no significant difference between Wt and Tg mice in tritiated 2-DG uptake in the
liver, spleen, or kidney (Table 2).
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Previously, we have demonstrated that adipose tissue from CMVBP-3- and
PGKBP-3-Tg mice had similar levels of transgene expression (15). However, we had not previously examined the levels
of expression of the transgene in skeletal muscle from these mice. Human IGFBP-3 mRNA was significantly increased (~14-fold) in skeletal muscle from CMVBP-3 compared with PGKBP-3-Tg mice (Fig.
5). Interestingly, the absolute
weight of calf muscles of both PGKBP-3- and
CMVBP-3-Tg mice was significantly reduced compared with Wt mice:
0.320 ± 0.011 and 0.361 ± 0.012 vs. 0.395 ± 0.006 g
(P < 0.0001 and P < 0.02, respectively). The relative weight of the calf muscles was also
significantly decreased in the CMVBP-3-Tg mice compared with Wt mice
(0.994 ± 0.026 vs. 1.148 ± 0.023; P < 0.0001) but not significantly different in the PGKBP-3 mice, which were
proportionately smaller and lighter than that of Wt mice.
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Because IGFBP-3 has been shown to interact with the RXR
transcriptional regulator (14), which can form
heterodimers with PPAR
(11), we investigated the
hypothesis that the effects of IGFBP-3 overexpression may have been
mediated via perturbation in the PPAR
/RXR
/resistin pathway. There
was no significant difference in the abundance of resistin mRNA in
white adipose tissues in any of the groups of mice (Fig.
6). Fasting plasma resistin levels were
similar in all groups of mice (Table 1). Similarly, PPAR
mRNA
abundance did not differ significantly among the three groups of mice
(Fig. 7). Immunoblotting was used to
evaluate the levels of PPAR
and RXR
protein in adipose tissue.
The levels of both PPAR
and RXR
proteins were similar in all
groups of mice (Fig. 8).
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DISCUSSION |
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Insulin is the central hormone in regulating blood glucose levels; however, other hormonal and nonhormonal factors are clearly important in blood glucose homeostasis. The majority of the insulin-like activity present in serum is due to the IGFs rather than insulin itself (4). Because most of IGF-I and -II present in the circulation is bound to the IGFBPs, predominantly IGFBP-3, insulin and the opposing counterregulatory hormones are able to acutely regulate the blood glucose in response to glucose influx in the prandial state. However, free unbound and/or easily dissociable IGF-I and -II, which constitute a minor component of the total IGF present in plasma (8), may have some role in short-term glucose regulation. Several lines of evidence support this notion. These include the acute changes in IGFBP-1 expression that accompany food intake (17), the hyperglycemia that accompanies infusion of IGFBP-1 (13) and IGFBP-3 (29), the glucose intolerance observed in liver-specific IGF-I knockout mice (30), and the hyperglycemia observed in Tg mice overexpressing IGFBP-1 (20) and IGFBP-3 (this study).
Partitioning of IGF among the free unbound, the easily dissociable, and the tightly bound, slowly turning over IGF pools in the circulation may also be important in longer-term control of glucose homeostasis. However, the free or the more appropriately termed easily dissociable IGF-I was elevated in the PGKBP-3-Tg and CMVBP-3-Tg mice compared with Wt mice. This assay may not measure true in vivo free IGF-I levels, particularly under abnormal conditions such as the elevated IGFBP-3 levels observed in the Tg mice. The ratio of the measured "free IGF-I" to total IGF-I was similar in Tg and Wt mice. However, in PGKBP-3-Tg mice, GH levels were significantly increased compared with CMVBP-3-Tg and Wt mice, possibly indicating that in the PGKBP-3-Tg mice, free IGF-I levels, at least in the pituitary gland, were reduced.
The majority of circulating IGF is present in the ternary complex bound to IGFBP-3 (22). Unlike IGF present in binary complexes, IGF present in the ternary complex has a much slower turnover (10). Binary complexes of IGFBP-3-IGF-I or -II may exist, but because acid-labile subunit (ALS) is usually present in molar excess of IGFBP-3, this type of binary complex is not abundant under normal circumstances (12). In both strains of IGFBP-3-Tg mice, the majority of IGF-I is present in the ternary complex, indicating that human IGFBP-3 is able to bind to mouse ALS (Ref. 15 and unpublished observations). Unlike IGFBP-1, IGFBP-3 has a long half-life in the circulation (1) and does not show acute changes in response to nutrition. Thus IGFBP-3-associated IGF-I or IGF-II represents a relatively slowly turning over circulating reservoir of IGF that constitutes the majority of the IGF present in the circulation.
The functional significance of the ternary complex IGF reservoir has recently been questioned with the demonstration that normal postnatal growth can occur in mice with conditional nullification of hepatic IGF-I expression (23, 31). These mice have low circulating total IGF-I levels, although free IGF-I levels appear to be normal (30). Interestingly, these mice demonstrate mild glucose intolerance and skeletal muscle insulin resistance, although fasting glycemia is normal. The insulin resistance in these mice may be due in part to elevated GH levels (30).
The data reported here, using two different strains of Tg mice generated with different transgene constructs, demonstrate that overexpression of IGFBP-3 is associated with disturbances in glucose homeostasis and reduced insulin sensitivity. In both of these Tg mouse strains, there is a significant increase in the circulating concentrations of IGFBP-3, IGF-I, and ternary complex (15). Serum hIGFBP-3 levels were ~5 µg/ml, with no apparent reduction in endogenous mouse IGFBP-3 expression (15). Total circulating IGF-I, the majority of which was present as ternary complex, was increased ~1.5-fold compared with Wt mice. In contrast to the findings of Yakar et al. (30) in liver-specific IGF-I knockout mice, GH was not elevated in the CMVBP-3-Tg mice, which demonstrated a more marked degree of insulin resistance than the PGKBP-3 mice. The GH levels were elevated in PGKBP-3-Tg mice. This is consistent with the greater growth retardation observed in PGKBP-3-Tg mice compared with CMVBP-3 mice (15).
The actual free IGF-I level measured in Wt mice (~30 ng/ml) is consistent with that reported by Frystyk et al. (8) in fasted rats but is higher than that reported by Yakar et al. (30) in normal mice (~5 ng/ml). Furthermore, the actual percentage of the total IGF-I measured as free IGF-I in our hands (~12%) was higher than that reported for normal mice (~2%) or rats (~5%) (8, 30). The reason for this discrepancy is not immediately apparent, since a similar technique was used to measure free IGF-I in all reports.
In CMVBP-3- and PGKBP-3-Tg mice, both the fasting blood glucose and the glycemic response to a glucose challenge were significantly increased compared with Wt mice. CMVBP-3-Tg mice, unlike PGKBP-3-Tg mice, are characterized by increased adiposity. In the former but not the latter Tg mice, the fasting plasma insulin levels were significantly increased. However, although the fasting insulin levels were not elevated in the PGKBP-3-Tg mice, insulin resistance was demonstrable in these mice during the insulin tolerance test. In both strains of IGFBP-3-Tg mice, the hypoglycemic response to insulin was attenuated, although this was more marked in CMVBP-3-Tg mice. In contrast, the hypoglycemic response to IGF-I was equally reduced in both Tg mouse strains. Because circulating free IGF-I levels and the ratio of free to total IGF-I were similar in PGKBP-3- and CMVBP-3-Tg mice despite higher insulin levels in CMVBP-3-Tg mice, these parameters do not appear to correlate with the insulin resistance observed in the CMVBP-3-Tg mice. It is possible that the measurement of these parameters in the circulation does not reflect free IGF-I levels in skeletal muscle or other tissue beds important in insulin-sensitive glucose uptake, where abnormal production of IGFBP-3 has been generated by transgene expression. In this regard, it is of interest that expression of the transgene was markedly elevated in skeletal muscle from CMVBP-3-Tg mice compared with PGKBP-3-Tg mice. However, 2-DG uptake into skeletal muscle was also reduced in PGKBP-3-Tg mice.
In PGKBP-3 mice, GH levels were increased, whereas this was not the case in CMVBP-3 mice. Elevated GH levels, insulin resistance, and a "lean" phenotype have been documented in liver-specific conditional IGF-I knockout mice (24). However, in the CMVBP-3-Tg mice, insulin resistance is present despite normal GH levels.
In both CMVBP-3- and PGKBP-3-Tg mice, 2-DG clearance from the circulation was delayed compared with Wt mice. We also calculated the decline in specific activity of the circulating glucose pool during the disappearance of 2-DG from the circulation. Because 2-DG is not recirculated from nonhepatic tissues, the decline in specific activity reflects the appearance of new glucose in the circulation, that is, gluconeogenesis. The decline in specific activity was not enhanced in CMVBP-3- or PGKBP-3-Tg mice, indicating that, in these Tg mice models, unlike the PGKBP-1-Tg mice (20), hepatic insulin resistance is unlikely. However, reduced 2-DG uptake was observed in both skeletal muscle and adipose tissue of the CMVBP-3- and PGKBP-3-Tg mice, consistent with some degree of insulin resistance in these tissues.
Recently, RXR has been identified as a binding partner for IGFBP-3
in a yeast two-hybrid screen. This observation, together with the
previous reports of nuclear localization of IGFBP-3 (14, 22), indicates that IGFBP-3 may have a role in modulating
nuclear transcription of various genes involved in growth and
metabolism. In this regard, it is of note that the nuclear
transcription factor PPAR
is also a binding partner for RXR
(11). PPAR
is involved in the regulation of genes that
control differentiation of preadipocytes (16). In terms of
adipose tissue mass, CMVBP-3- and PGKBP-3-Tg mice have markedly
different phenotypes (30). The CMVBP-3-Tg mice have marked
obesity, whereas in PGKBP-3-Tg mice adipose tissue mass is similar to
that in Wt mice. We have attributed this difference to subtle
differences in the timing or level of expression of the transgene
in the two mouse strains. In this regard, the increased expression of
the transgene in skeletal muscle from CMVBP-3-Tg mice is of interest.
Increased expression of the transgene in CMVBP-3-Tg mice could result
in enhanced insulin resistance and, as a consequence, increased
shunting of nutrients to adipose tissue. Despite differences in
adiposity and insulin sensitivity, expression of resistin, RXR
, and
PPAR
were similar in both strains of IGFBP-3-Tg mice and were not
significantly different from Wt mice. Thus direct interaction of
IGFBP-3 with RXR
, if this indeed occurs in vivo, does not appear to
result in a disturbance in expression of RXR
, its binding partner
PPAR
, or the downstream effector resistin.
The data reported here clearly demonstrate that overexpression of IGFBP-3 results in hyperglycemia, glucose intolerance, and insulin resistance. These effects cannot be explained by disturbances in either GH secretion, adiposity, or circulating "free" IGF-I levels.
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ACKNOWLEDGEMENTS |
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This research was supported by a grant from the Canadian Institutes for Health Research. L. J. Murphy is a recipient of an endowed Research Professorship in Metabolic Diseases.
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. J. Murphy, Dept. of Physiology, Univ. of Manitoba, Winnipeg R3E 0W3, Canada (E-mail: ljmurph{at}cc.umanitoba.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpendo.00014.2002
Received 15 January 2002; accepted in final form 27 June 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Arany, E,
Zabel P,
Freeman D,
and
Hill DJ.
Elimination of radiolabelled recombinant human insulin-like growth factor binding protein-3 from the circulation, and its distribution amongst organs and tissues in adult male rats.
Regul Pept
48:
133-143,
1993[ISI][Medline].
2.
Barrett, JR,
Plewe G,
Fagin KD,
and
Sherwin RS.
Acute effects of insulin-like growth factor I on glucose and amino acid metabolism in the awake fasted rat. Comparison with insulin.
J Clin Invest
83:
1717-1723,
1989[ISI][Medline].
3.
Baxter, RC.
Insulin-like growth factor binding proteins in the human circulation: a review.
Horm Res
42:
140-144,
1994[ISI][Medline].
4.
Burgi, H,
Muller WA,
Humbel RE,
Labhart A,
and
Froesch ER.
Non-suppressible insulin-like activity of human serum. I. Physiochemical properties, extraction and partial purification.
Biochim Biophys Acta
121:
349-359,
1966[ISI][Medline].
5.
Chambon, P,
Mader S,
Garnier JM,
Staub A,
Chen JY,
Zacharewsi T,
Saunders M,
Nakshatri H,
Lyons R,
Kastner P,
and
Leid M.
Purification, cloning, and RXR identity of the Hela cell factor with which RAR or TR heterodimerizes bind to target sequences efficiently.
Cell
68:
377-395,
1992[ISI][Medline].
6.
Chen, F,
Law SW,
and
O'Malley BW.
Identification of two mPPAR related receptors and evidence for existence of five subfamily members.
Biochem Biophys Res Commun
196:
671-677,
1993[ISI][Medline].
7.
Daughaday, WH,
and
Kapadia M.
Significance of abnormal serum binding of insulin-like growth factor II in the development of hypoglycemia in patients with non-islet-cell tumors.
Proc Natl Acad Sci USA
86:
6778-6782,
1989[Abstract].
8.
Frystyk, J,
Delhanty PJD,
Skjaerbaek C,
and
Baxter RC.
Changes in the circulating IGF system during short-term fasting and refeeding in rats.
Am J Physiol Endocrinol Metab
277:
E245-E252,
1999
9.
Furth, PA,
Hennighausen L,
Baker C,
Beatty B,
and
Woychick R.
The variability in activity of the universally expressed human cytomegalovirus major immediate early gene 1 enhancer/promoter in transgenic mice.
Nucleic Acids Res
19:
6205-6208,
1991[Abstract].
10.
Guler, HP,
Zapf J,
Schmid C,
and
Froesch ER.
Insulin-like growth factors I and II in healthy man. Estimations of half-lives and production rates.
Acta Endocrinol
121:
753-758,
1989[ISI][Medline].
11.
Juge-Aubry, C,
Pernin A,
Favez T,
Burger AG,
Wahli W,
Meier CA,
and
Desvergne B.
DNA binding properties of peroxisome proliferator-activated receptor subtypes on various natural peroxisome proliferator response elements.
J Biol Chem
272:
25252-25259,
1997
12.
Lee, CY,
and
Rechler MM.
Purified rat acid-labile subunit and recombinant human insulin-like growth factor (IGF)-binding protein-3 can form a 150-kilodalton binary complex in vitro in the absence of IGFs.
Endocrinology
136:
4982-4989,
1995[Abstract].
13.
Lewitt, MS,
Denyer GS,
Cooney GJ,
and
Baxter RC.
Insulin-like growth factor binding protein-1 modulates blood glucose levels.
Endocrinology
129:
2254-2256,
1991[Abstract].
14.
Liu, B,
Lee HY,
Weinzimer SA,
Powell DR,
Clifford JL,
Kurie JM,
and
Cohen P.
Direct functional interactions between insulin-like growth factor-binding protein-3 and retinoid x receptor- regulate transcriptional signaling and apoptosis.
J Biol Chem
275:
33607-33613,
2000
15.
Modric, T,
Silha J,
Shi Z,
Gui Y,
Suwanichkul A,
Durham SK,
Powell DR,
and
Murphy LJ.
Phenotypic manifestations of insulin-like growth factor binding protein-3 overexpression in transgenic mice.
Endocrinology
142:
1958-67,
2001
16.
Morrison, RF,
and
Farmer SR.
Hormonal signaling and transcriptional control of adipocyte differentiation.
J Nutr
130:
3116-3121,
2000.
17.
Murphy, LJ,
Luo J,
and
Seneviratne C.
Hormonal regulation of insulin-like growth factor binding protein-1 expression in the rat.
Adv Exp Med Biol
293:
149-161,
1991[Medline].
18.
Oh, Y,
Gucev Z,
Ng L,
Muller HL,
and
Rosenfeld RG.
Antiproliferative actions of insulin-like growth factor binding protein (IGFBP)-3 in human breast cancer cells.
Prog Growth Factor Res
6:
205-212,
1995.
19.
Oh, Y,
Muller HL,
Pham H,
and
Rosenfeld RG.
Demonstration of receptors for insulin-like growth factor binding protein-3 on Hs578T human breast cancer cells.
J Biol Chem
268:
26045-26048,
1993
20.
Rajkumar, K,
Krsek M,
Dheen ST,
and
Murphy LJ.
Impaired glucose homeostasis in insulin-like growth factor binding protein-1 transgenic mice.
J Clin Invest
98:
1818-1825,
1996
21.
Rennert, NJ,
Boulware SD,
Kerr D,
Caprio S,
Tamborlane WV,
and
Sherwin RS.
Metabolic effects of rhIGF-I in normal human subjects.
Adv Exp Med Biol
343:
311-318,
1993[Medline].
22.
Schedlich, LJ,
LePage SL,
Firth SM,
Briggs LJ,
Jans DA,
and
Baxter RC.
Nuclear import of insulin-like growth factor-binding protein-3 and -5 is mediated by the importin beta subunit.
J Biol Chem
275:
23462-70,
2000
23.
Sjogren, K,
Liu JL,
Blad K,
Skrtic S,
Vidal O,
Wallenius V,
LeRoith D,
Tornell J,
Isaksson OG,
Jansson JO,
and
Ohlisson C.
Liver-derived insulin-like growth factor-I (IGF-I) is the principal source of IGF-I in blood but is not required for postnatal body growth in mice.
Proc Natl Acad Sci USA
96:
7088-7092,
1999
24.
Sjogren, K,
Wallenius K,
Liu JL,
Bohlooly-YM,
Pacini G,
Svensson L,
Tornell J,
Isaksson OGP,
Ahren B,
Jansson JO,
and
Ohlsson C.
Liver-derived IGF-I is of importance for normal carbohydrate and lipid metabolism.
Diabetes
60:
1539-1545,
2001.
25.
Steppan, CM,
Bailey ST,
Bhat S,
Brown EJ,
Banerjee RR,
Wright CM,
Patel HR,
Ahima RS,
and
Lazar MA.
The hormone resistin links obesity to diabetes.
Nature
409:
307-312,
2001[ISI][Medline].
26.
Tannenbaum, GS,
Rorstad O,
and
Brazeau P.
Effects of prolonged food deprivation on the ultradian growth hormone rhythm and immunoreactive somatostatin tissue levels in the rat.
Endocrinology
104:
1733-1738,
1979[ISI][Medline].
27.
Tybulewicz, VLJ,
Crawford CE,
Jackson PK,
Bronson RT,
and
Mulligan RC.
Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene.
Cell
65:
1153-1163,
1991[ISI][Medline].
28.
Valentinis, B,
Bhala A,
DeAngelis T,
Baserga R,
and
Cohen P.
The human insulin-like growth factor (IF) binding protein-3 inhibits the growth of fibroblasts with a targeted disruption of the IGF-I receptor gene.
Mol Endocrinol
9:
361-367,
1995[Abstract].
29.
Vuguin, PM,
Shim ML,
Cohen P,
and
Barzilai N.
IGFBP-3 induces hepatic and peripheral insulin resistance in vivo (Abstract).
Diabetes
50 Suppl 2:
A268,
2001.
30.
Yakar, S,
Liu JL,
Fernandez AM,
Wu Y,
Schally AV,
Frystyk J,
Chernausek SD,
Mejia W,
and
Le Roith D.
Liver-specific igf-1 gene deletion leads to muscle insulin insensitivity.
Diabetes
50:
1110-1118,
2001
31.
Yakar, S,
Liu JL,
Stannard B,
Butler A,
Accili D,
Sauer B,
and
LeRoith D.
Normal growth and development in the absence of hepatic insulin-like growth factor I.
Proc Natl Acad Sci USA
96:
7324-7329,
1999
32.
Zapf, J.
Role of insulin-like growth factor II and IGF binding proteins in extrapancreatic tumor hypoglycemia.
Horm Res
42:
20-26,
1994[ISI][Medline].