Am J Physiol Gastrointest Liver Physiol 294: G679–G686, 2008.
First published January 3, 2008; doi:10.1152/ajpgi.00526.2007.
Reduced susceptibility of muscle-specific insulin receptor knockout mice
to colon carcinogenesis
Kafi N. Ealey,1 Suying Lu,1 Dominic Lau,1 and Michael C. Archer1,2
1
Department of Nutritional Sciences and 2Department of Medical Biophysics, Faculty of Medicine, University of Toronto,
Toronto, Canada
Submitted 13 November 2007; accepted in final form 22 December 2007
cause of Type 2 diabetes mellitus
(T2DM), is characterized by impaired biological response to
the action of insulin (31). The metabolic consequences of
insulin resistance include hyperinsulinemia, hyperglycemia,
and/or glucose intolerance, as well as elevated circulating
levels of triglycerides (TG) and free fatty acids (FFA) (6). Over
a decade ago, it was noted that the diet and lifestyle risk factors
for the development of colorectal cancer (CRC) are very
similar to those for insulin resistance (19, 30). On the basis of
these observations, it was hypothesized that hyperinsulinemia
or some factor(s) associated with insulin resistance promotes
colon carcinogenesis and stimulates the growth of colorectal
tumors (19, 30). Evidence from both epidemiological and
animal studies supports this association. Risk of CRC is increased threefold in men and women with elevated plasma
C-peptide, a surrogate measure of insulin secretion (22, 28).
T2DM was associated with a 40 –50% increase in the risk of
colon cancer in the Nurses’ Health Study and in another large
cohort of postmenopausal women (21, 26). Furthermore,
chronic injections of insulin in rats treated with azoxymethane
(AOM), a colon-specific carcinogen, promote the growth of
putative preneoplastic aberrant crypt foci (ACF) (11) and colon
tumors (46).
Obesity, particularly visceral adiposity, is an independent
risk factor for T2DM, insulin resistance, and hyperinsulinemia
(18, 48) and is associated with elevated risk of CRC (25, 39).
Excess abdominal tissue is associated with increased production of FFA, increased hepatic production of triglycerides
(TG), decreased glucose uptake, and impaired insulin signaling
(31). McKeown-Eyssen (30) hypothesized that serum TG and
glucose that are elevated by dietary and lifestyle risk factors for
CRC may themselves be positively associated with CRC. In
case-control studies, elevated circulating TG levels have been
associated with an increased risk of the development of colorectal adenomatous polyps (40, 41, 44) and carcinomas in situ
(52). In addition, higher serum TG levels were observed in
patients with familial adenomatous polyposis who developed
CRC compared with those who did not (34). Similar findings
were reported in ApcMin/⫹ mice that lack the adenomatous
polyposis coli (Apc) gene and spontaneously develop numerous polyps in the intestinal tract (33, 42). These mice have
significantly higher serum TG, FFA, and cholesterol compared
with wild-type mice because of a reduced expression of lipoprotein lipase (LPL) (36). Pharmacological activation of
LPL in Min mice resulted in suppression of serum lipid levels
and a significant reduction in the development of intestinal
polyps (35–37).
Studies that have investigated the relationship between insulin resistance and colon cancer have not separated the action
of insulin from the other effects caused by hyperinsulinemia
such as increased hepatic TG release and elevated circulating
FFA (11, 46). The objective of the present study was to test the
hypothesis that increased adiposity and elevated circulating
lipids commonly seen in insulin resistance promote colon
carcinogenesis independent of changes in insulin. We made
use of a tissue-specific knockout mouse model in which the
insulin receptor (IR) gene has been inactivated in the muscle using the Cre-loxP-mediated gene recombination technique
(43). MIRKO (muscle-specific insulin receptor knockout) mice
exhibit a ⬎95% reduction in IR content in muscle whereas IR
expression in other tissues is not affected (7). This results in
reduced insulin-stimulated signaling and glucose uptake into
skeletal muscle, giving rise to elevated serum TG and FFA and
Address for reprint requests and other correspondence: M. C. Archer, Dept.
of Nutritional Sciences, Univ. of Toronto, 150 College St., Toronto, Ontario,
Canada, M5S 3E2 (e-mail: [email protected]).
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.
colorectal cancer; adiposity; hyperlipidemia; insulin resistance
INSULIN RESISTANCE, A MAJOR
http://www.ajpgi.org
0193-1857/08 $8.00 Copyright © 2008 the American Physiological Society
G679
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Ealey KN, Lu S, Lau D, Archer MC. Reduced susceptibility of
muscle-specific insulin receptor knockout mice to colon carcinogenesis. Am J Physiol Gastrointest Liver Physiol 294: G679–G686, 2008.
First published January 3, 2008; doi:10.1152/ajpgi.00526.2007.—
Insulin resistance is a risk factor for colon cancer, but it is not clear
which of its metabolic sequelae are involved. The objective of this
study was to determine whether increased adiposity and elevated
circulating lipids commonly seen in insulin resistance promote colon
carcinogenesis independent of changes in insulin. We made use of
muscle-specific insulin receptor knockout (MIRKO) mice that exhibit
elevated serum triglycerides (TG), free fatty acids (FFA), and fat mass
but have similar body weights, circulating glucose, and insulin and
insulin sensitivity to their wild-type littermates used as controls.
Seven-week-old male MIRKO mice and controls received four
weekly intraperitoneal injections of either 5 mg/kg azoxymethane
(AOM) to induce aberrant crypt foci (ACF) or 10 mg/kg AOM to
induce tumors and were killed at 24 or 40 wk of age, respectively. The
MIRKO mice displayed hyperinsulinemia at 7 wk of age and reduced
insulin sensitivity at 16 wk of age compared with controls. The
previously reported MIRKO phenotype developed between 16 and 24
wk of age. By 40 wk of age, however, MIRKO mice were again
insulin resistant. ACF development did not differ between MIRKO
mice and controls, but MIRKO mice developed significantly fewer
colon tumors. Our results suggest that circulating TG and FFA are not
promoters of colon tumor development. Indeed, we show that the
cumulative effects of the metabolic changes that occur with knockout
of the insulin receptor in muscle are associated with reduced susceptibility to colon tumorigenesis.
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REDUCED SUSCEPTIBILITY TO COLON CANCER IN MIRKO MICE
increased fat mass (7). MIRKO mice, however, have been
reported to exhibit similar body weights, circulating levels of
glucose, and insulin and glucose tolerance compared with their
wild-type littermates (7). We show that MIRKO mice have a
reduced susceptibility to the development of colon tumors
compared with controls, and present evidence to suggest that
elevated adiposity in MIRKO mice may have inhibited carcinogenesis.
MATERIALS AND METHODS
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RESULTS
Serum parameters. We measured fasting serum levels of
TG, FFA, glucose, and insulin as well as body weights in
groups of untreated 7-, 16-, 24-, and 40-wk-old MIRKO mice
and littermate controls (n ⫽ 8 –10 per group) and in AOMtreated mice at the time they were killed for analysis of ACF
and tumors at 24 and 40 wk of age, respectively. As shown in
Table 1, at 7 wk of age, there were no significant differences in
TG, FFA, or glucose between MIRKO mice and controls,
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Animals. The generation of MIRKO mice that are homozygous for
the IR gene flanked by two loxP sites and also carry the MCK-Cre
transgene [IR(lox/lox): MCK-Cre (⫹/⫺)] has been described previously (7). A breeding pair of these mice was a generous gift from Dr.
Ronald Kahn (Joslin Diabetes Center and Harvard Medical School,
Boston, MA). All genotyping was performed by PCR using genomic
DNA isolated from the tails of weaned mice as previously described
(7). The animals were housed in the Department of Comparative
Medicine at the University of Toronto. All protocols for animal use
and treatment were reviewed and approved by the University of
Toronto Animal Care Committee and were in compliance with the
guidelines of the Canadian Council on Animal Care. Mice were
housed at 22°C and 50% humidity on a 12:12-h light-dark cycle and
fed a standard rodent chow diet and water ad libitum. Male MIRKO
mice were used in all of the studies described, and age-matched
littermates that were homozygous for the floxed IR gene [IR(lox/lox)]
but did not express Cre were used as controls. In all of the experiments, we used offspring from a number of individual breeding pairs
to obtain sufficient male MIRKO and control mice that were of the
same age. To minimize potential variability between litters from
different breeding pairs, MIRKO mice and controls from each of the
breeding pairs were usually included in each experiment.
Analytical procedures. Glucose tolerance tests (GTT) and insulin
tolerance tests (ITT) were performed on animals at various ages
throughout the study. For the GTT, mice (n ⫽ 7–13 per group) were
injected with 2 g/kg body wt of dextrose after an overnight fast and
blood samples were obtained from the tail vein immediately before
and 15, 30, 60, and 120 min after injection as previously described (7).
For the ITT, fed animals (n ⫽ 7–12 per group) were injected with 0.75
U/kg of human regular insulin (Sigma Chemicals, Oakville, Ontario,
Canada) and blood was collected at 0, 15, 30, 60, and 90 min after the
injection as in previously described protocols (5, 29). Glucose values
were determined from whole venous blood via an automatic glucose
analyzer (Ascensia Elite XL, Bayer, Toronto, Ontario, Canada).
At the time of euthanasia, blood was obtained from mice that had
either been fasted or fed for the determination of serum FFA, TG,
insulin, leptin, and adiponectin. Epididymal fat pads were removed
and weighed as previously described (29). Insulin, leptin, and adiponectin were measured in serum by radioimmunoassay (Linco Research, St. Charles, MO). FFA and TG in serum were measured by
colorimetric enzyme assays (Wako Chemicals, Richmond, VA and
Teco Diagnostics, Anaheim, CA, respectively).
Carcinogenesis studies. For the induction of ACF, 7-wk-old male
MIRKO mice and littermate controls (n ⫽ 12 per group) received 4
weekly intraperitoneal injections of 5 mg/kg AOM (Midwest Research Institute, Kansas City, MO) according to a previously published protocol (2). Body weights were monitored weekly. One
hundred days after the last injection, the animals were killed and
colons were removed for the analysis of ACF. The entire colon was
flushed with phosphate-buffered saline, cut open longitudinally, and
fixed flat between filter papers in 10% phosphate-buffered formalin
for ⱖ24 h. Colons were stained with 0.05% methylene blue (Sigma
Chemicals, Oakville, Canada) and the mucosal surface was assessed
for ACF with a light microscope at ⫻40 magnification. ACF were
distinguished from normal crypts on the basis of their enlarged size
and pericryptal zone, their thicker epithelial lining, and the slitlike
shape of the lumen (3). ACF analysis was carried out by a single
operator who was blinded to the identity of the samples.
To assess tumorigenesis, 7-wk-old male MIRKO mice and controls
(n ⫽ 23–27 per group) were treated with four weekly intraperitoneal
injections of 10 mg/kg AOM (4). Most animals were killed 30 wk
after the last AOM injection (40 wk of age), although a few mice were
killed 26 –29 wk after the last AOM injection because of rectal
bleeding. Colons were removed from each animal, flushed with PBS,
and cut open longitudinally. The total number of tumors per colon,
their location along the length of the colon (mm from the anus), and
their size (largest diameter) were recorded. The tumors were fixed in
10% formalin, sectioned, and stained with hematoxylin and eosin for
histological examination. Slides were coded and read by a pathologist
blinded to the analysis.
Tissue lysis, Western blotting, and immunoprecipitation. Colonic
mucosal scrapings were homogenized in lysis buffer [20 mM Tris (pH
7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100,
2.5 mM sodium pyrophosphate, 1 mM ␤-glycerophosphate, 1 mM
Na3VO4, protease inhibitor cocktail (Sigma Chemicals)] with a handheld motorized tissue grinder. Tissue homogenates were centrifuged
at 26,000 rpm for 30 min in a Beckman ultracentrifuge at 4°C and the
resulting supernatants were centrifuged again at 26,000 rpm for 30
min. The supernatants were collected and stored at ⫺80°C. Protein
concentrations were determined by the Bio-Rad protein assay.
For Western blotting analysis, 12.5–25 ␮g of total mucosal protein
was solubilized in Laemmli buffer, heated for 5 min at 95°C, electrophoresed by SDS-PAGE on 10% gels, and transferred to polyvinylidene difluoride membranes. After blocking for 1 h at room
temperature with 5% nonfat milk in Tris-buffered saline containing
0.1% Tween-20 (TBS-T), membranes were probed overnight at 4°C
with antibodies to phosphorylated MAPK (pMAPK), phosphorylated
Akt (Ser473), phosphorylated AMPK␣ (pAMPK␣) (Cell Signaling
Technology, Beverly, MA), and peroxisome proliferator-activated
receptor-␥ (PPAR␥) (Santa Cruz Biotechnology, Santa Cruz, CA).
Membranes were washed in TBS-T and incubated for 1 h with
anti-rabbit or anti-mouse IgG conjugated with horseradish peroxidase
(Santa Cruz Biotechnology). Signals were detected by enhanced
chemiluminescence (Millipore, Billerica, MA) on Biomax MR Kodak
film by autoradiography and protein bands were quantified by densitometry. The expression pattern of ␤-actin was used to ensure equal
loading. For immunoprecipitation, 500 ␮g of protein from colonic
mucosal lysates were immunoprecipitated overnight at 4°C with
anti-insulin receptor substrate-1 (IRS-1) (Cell Signaling Technology).
Immunocomplexes were captured by incubation with protein A agarose beads for 1 h at 4°C. Washed immunoprecipitates were resuspended in Laemmli buffer, boiled at 95°C, and subjected to Western
blotting on a 7.5% gel as described above. Membranes were probed with
a rabbit polyclonal antibody to the p85␣ regulatory subunit of phosphatidylinositol 3-kinase (Upstate Biotechnology, Lake Placid, NY).
Statistical analyses. Analysis of the incidence of early deaths,
overall tumor incidence, and incidence of adenocarcinomas and adenomas were compared by the Fisher’s exact probability test. All other
data were analyzed by unpaired Student’s t-test. Results are given as
means ⫾ SE. Statistical significance was accepted at P ⬍ 0.05.
REDUCED SUSCEPTIBILITY TO COLON CANCER IN MIRKO MICE
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Table 1. Body weights and serum parameters in MIRKO mice and controls
Age, wk
Body Weight, g
TG, mg/dl
FFA, mEq/l
Glucose, mg/dl
Insulin, ng/ml
Control
MIRKO
16
Control
MIRKO
24
Control
MIRKO
40
Control
MIRKO
25.4⫾0.4
24.3⫾0.6
104.4⫾9.2
106.4⫾16.5
1.24⫾0.12
1.46⫾0.22
157.4⫾22
189.3⫾15.2
0.29⫾0.04
0.53⫾0.15*
26.6⫾0.7
24.2⫾0.4*
60.9⫾7.0
89.6⫾11.9
1.11⫾0.22
1.32⫾0.08
105.8⫾16.3
95.2⫾16.3
0.43⫾0.09
0.37⫾0.08
30.3⫾0.9
27.3⫾0.8*
87.0⫾6.0
124.7⫾9.4*
1.00⫾0.08
2.22⫾0.45*
132.3⫾15
109.6⫾16.7
0.23⫾0.04
0.22⫾0.03
30.0⫾1.0
27.9⫾0.7
131.1⫾52.8
80.3⫾19.3
0.77⫾0.04
1.44⫾0.2*
127.1⫾35.3
71.3⫾13.0
0.53⫾0.30
0.61⫾0.30
7
although insulin was significantly elevated in MIRKO mice.
Furthermore, the 7-wk-old MIRKO mice and controls did not
differ in overall body weight (Table 1). There were no significant differences in any of the serum parameters in 16-wk-old
MIRKO mice compared with controls, although TG levels
were somewhat higher in the MIRKO mice but did not reach
statistical significance (P ⫽ 0.07, Table 1). Sixteen-week-old
MIRKO mice had a small but significantly lower body weight
compared with controls (Table 1).
By 24 wk of age, MIRKO mice had a distinctly different
metabolic phenotype compared with controls. This MIRKO
phenotype was very similar to that reported by others for these
mice at this age (29). Serum levels of TG and FFA were
significantly higher in MIRKO mice and there were no
differences in insulin or glucose compared with controls
(Table 1). MIRKO mice had a small but significantly lower
overall body weight compared with control littermates (Table 1).
Interestingly, by 40 wk of age, with the exception of FFA,
there were no significant differences in any of the serum
parameters between fasted MIRKO mice and their controls
(Table 1) and no significant differences in body weight between the two groups. We also measured serum parameters in
a subset of animals that were not fasted when killed and found
no differences between the two groups (data not shown). The
differences in serum parameters that we observed between
MIRKO mice and controls at 24 and 40 wk of age were not
affected by AOM treatment.
GTT and ITT. To determine whether MIRKO mice displayed changes in insulin resistance as they aged, we assessed
glucose tolerance in non-AOM treated MIRKO mice and
controls at 7, 16, 24, and 40 wk of age and insulin tolerance in
the three older groups. Figure 1A shows that 7-wk-old MIRKO
mice displayed similar glucose tolerance curves to controls
with no significant differences in the area under the curve
(AUC), although glucose levels were significantly lower in
MIRKO mice 120 min after the glucose challenge. MIRKO
mice and controls exhibited similar levels of glucose tolerance
and AUCs following injection of glucose (Fig. 1, B and C) at
both 16 and 24 wk of age. During the ITT, however, 16-wk-old
MIRKO mice initially had reduced sensitivity to injected
insulin, resulting in significantly higher blood glucose levels
after 15 min (Fig. 2A). By 24 wk of age, MIRKO mice were
equally sensitive to injected insulin and there were no differAJP-Gastrointest Liver Physiol • VOL
ences in glucose levels throughout the ITT (Fig. 2B). We did
not measure glucose at 90 min after insulin injection in 24wk-old mice because they were exhibiting signs of hypoglycemia.
At 40 wk of age, MIRKO mice showed evidence of impaired
glucose tolerance and reduced insulin sensitivity compared
with controls. During the GTT, MIRKO mice had significantly
higher blood glucose values at 15, 30, and 60 min, and the
AUCs were significantly higher (19.54 ⫾ 1.9 vs. 12.3 ⫾ 1.3
mg䡠dl⫺1 䡠min⫺1 䡠1,000⫺1, MIRKO vs. control, P ⬍ 0.05),
suggesting reduced ability to clear a glucose load (Fig. 1D).
This was confirmed by the reduced insulin sensitivity following the ITT resulting in significantly higher glucose levels 15,
30, and 90 min after insulin injection (Fig. 2C).
Adiposity, adiponectin, and leptin. We measured epididymal
fat pad weight as a percentage of body weight in groups of
untreated MIRKO mice and controls at 16, 24, and 40 wk
of age (n ⫽ 8 –23 per group). In addition, circulating levels of
adiponectin were assessed in 16- and 24-wk-old MIRKO mice
and controls. There were no significant differences in epididymal fat pad weight between MIRKO mice and controls at 16
and 24 wk of age, but at 40 wk of age MIRKO mice had a
significantly greater amount of epididymal fat compared with
the control group (Table 2). We observed no significant differences in adiponectin between 16-wk-old MIRKO mice and
controls but significantly higher adiponectin levels in 24wk-old MIRKO mice relative to controls, concomitant with the
development of the MIRKO phenotype (Table 2). Furthermore,
24-wk-old control mice had significantly lower adiponectin
levels than the controls at 16 wk of age, whereas this decrease
in adiponectin levels was not observed between 16- and 24wk-old MIRKO mice. In addition, there were no significant
differences in serum leptin between 24-wk-old MIRKO mice
and controls (0.79 ⫾ 0.1 vs. 0.63 ⫾ 0.2 ng/ml, MIRKO vs.
controls).
ACF study. For analysis of ACF, animals were killed at 24
wk of age, 100 days after treatment with four weekly doses of
5 mg/kg AOM. The average number of ACF per colon did not
differ between MIRKO mice and controls (8.2 ⫾ 1.0 vs. 7.7 ⫾
0.8, MIRKO vs. control). Similarly, there was no significant
difference in crypt multiplicity (number of crypts per focus)
between the two groups (1.38 ⫾ 0.1 vs. 1.46 ⫾ 0.1, MIRKO
vs. control).
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Values are means ⫾ SE. Metabolic measures were obtained from untreated 7- and 16-wk-old mice and from azoxymethane-treated mice at 24 and 40 wk of
age that were part of the carcinogenesis studies. MIRKO, muscle-specific insulin receptor knockout. *P ⬍ 0.05 vs. control.
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REDUCED SUSCEPTIBILITY TO COLON CANCER IN MIRKO MICE
AOM (40 wk of age). A few mice were killed 26 –29 wk after
AOM because of rectal bleeding. Furthermore, a number of
mice particularly in the control group developed rectal prolapse
between 18 and 23 wk after AOM and had to be killed or they
died prematurely. These animals were not included in any of
the analyses.
All macroscopic polypoid lesions were excised for histological examination. Although the difference in tumor incidence
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Fig. 1. Glucose tolerance tests (GTT) in muscle-specific insulin receptor
knockout (MIRKO) mice and controls. GTTs were performed on 7- to
8-wk-old (A), 16-wk-old (B), 24-wk-old (C), and 40-wk-old (D) male control
(F) and MIRKO (䊐) mice that were not treated with azoxymethane (AOM).
Fasting mice were injected with 2 g/kg body wt ip of dextrose, and blood
glucose was measured at various time points. Data are expressed as means ⫾
SE, *P ⬍ 0.05.
Tumor study. Colon tumorigenesis was initiated in 7-wk-old
male MIRKO and control mice by four weekly injections of 10
mg/kg AOM according to previously published protocols (4).
Most of the mice were killed 30 wk after the last injection of
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Fig. 2. Insulin tolerance tests (ITT) in MIRKO mice and controls. ITTs were
performed on 16-wk-old (A), 24-wk-old (B), and 40-wk-old (C) male control
(F or Œ) and MIRKO (䊐) mice that were not treated with AOM. Fed mice were
injected intraperitoneally with 0.75 U/kg of human regular insulin, and blood
glucose was measured at various time points. Data are expressed as means ⫾
SE, *P ⬍ 0.05.
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REDUCED SUSCEPTIBILITY TO COLON CANCER IN MIRKO MICE
Table 2. Epididymal fat pad weight and serum adiponectin
in MIRKO mice and controls
Age, wk
Epididymal Fat
Adiponectin, ␮g/ml
0.71⫾0.4
0.56⫾0.1
3.6⫾0.4
4.6⫾1.0
0.55⫾0.1
0.71⫾0.1
1.9⫾0.3†
3.4⫾0.5*
0.69⫾0.1
1.09⫾0.1*
ND
16
Control
MIRKO
24
Control
MIRKO
40
Control
MIRKO
Values are means ⫾ SE. Epididymal fat pad weight is expressed as % of
body weight. ND, not done. *P ⬍ 0.05 vs. control, †P ⬍ 0.05 vs. 16-wk-old
control mice.
did not reach statistical significance, there was a large, highly
significant difference in tumor multiplicity (Table 3), with the
controls having three times more tumors per mouse than
MIRKO mice. Analysis of tumor histology showed that the
control group had a higher ratio of adenocarcinomas to adenomas (2.5) than MIRKO mice (1.6). The adenocarcinomas in
the MIRKO mice, however, were significantly larger than
those in the controls. Figure 3 illustrates that the difference in
overall tumor multiplicity shown in Table 3 was primarily due
to the significantly fewer carcinomas per mouse in the MIRKO
mice compared with controls.
Immunoblot analysis. Expression levels of key intermediates
of the adiponectin and insulin signaling pathways were analyzed by Western blot in colon mucosal cell lysates of untreated 24-wk-old MIRKO mice and controls. There were no
significant differences in the levels of pAMPK, p44/42
pMAPK, pAkt, or IRS-1 bound to p85 between MIRKO mice
and controls (data not shown). Furthermore, levels of PPAR␥
were also not different between the two groups.
but there were no differences in other serum parameters compared with controls. Elevated serum insulin may indicate that
these animals were insulin resistant, although they had normal
glucose tolerance and lower glucose levels by the end of the
GTT. By 16 wk of age, the MIRKO mice still did not display
the reported MIRKO phenotype. There were no differences in
serum parameters and they displayed evidence of insulin resistance compared with controls. At 24 wk of age when groups
were killed for ACF analysis, we observed the previously
reported MIRKO phenotype of significantly elevated FFA and
TG with insulin and glucose levels and glucose tolerance and
insulin sensitivity similar to controls. These phenotypic characteristics may have developed earlier, however, since we did
not make any measurements between 16 and 24 wk of age. By
40 wk of age, when the remaining mice were killed for analysis
of tumors, the MIRKO mice displayed impaired glucose and
insulin tolerance compared with controls. FFA levels were
significantly higher in MIRKO mice at this time point but there
were no differences in any other serum parameters compared
with controls.
Our observations differ from those of Bruning et al. (7), who
initially described the phenotype of the MIRKO mouse at 16
wk of age. Some studies, however, suggest that the age at
which the full MIRKO phenotype is expressed may vary
between 8 and 24 wk (7, 24, 29, 50). It is possible that the
changing phenotype of these mice results from changes in
insulin sensitivity that occur with age. Furthermore, there may
also be age-specific compensatory mechanisms in other tissues
that allow the mice to adapt to metabolic changes resulting
from reduced expression of insulin receptor in muscle, as has
DISCUSSION
The objective of this study was to determine whether increased adiposity and elevated circulating lipids commonly
seen in insulin resistance promote colon carcinogenesis, independent of changes in insulin. The rationale for choosing to
assess susceptibility to colon carcinogenesis in MIRKO mice
was on the basis of the initial report by Bruning et al. (7)
characterizing these mice as having elevated FFA, TG, and fat
mass but no differences in body weight, blood glucose, and
insulin as well as no impairments in glucose tolerance or
insulin sensitivity compared with littermate controls. Unexpectedly, however, we observed that the MIRKO phenotype
was not stable and changed with age. Circulating insulin levels
were significantly elevated in fasting 7-wk-old MIRKO mice
Table 3. Characteristics of tumors in MIRKO mice and controls
Tumor Type
Controls
MIRKO
Tumor Size, mm
n
Incidence
Multiplicity
Ad
Ac
Ad
Ac
Ac-to-Ad Ratio
14
23
11/14 (79%)
12/23 (52%)
2.29⫾0.6
0.78⫾0.2†
9 (28%)
7 (39%)
23 (72%)
11 (61%)
5.2⫾1.3
3.9⫾0.6
4.9⫾0.5
6.8⫾0.8*
2.5
1.6
Multiplicity values are means ⫾ SE of total number of tumors per mouse; tumor type values are total number of adenomas (Ad) and adenocarcinomas (Ac)
in surviving mice at approximately 40 wk of age as described in the text; tumor size values are means ⫾ SE of largest diameter of tumor (in mm). *P ⬍ 0.05,
†P ⫽ 0.005.
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Fig. 3. Total number of adenomas (Ad) and adenocarcinomas (Ac) per mouse
colon from AOM-treated MIRKO mice and controls. Sections of the tumors
from each animal were processed for hematoxylin and eosin staining and
classified histologically as Ac or Ad. Data are expressed as means ⫾ SE for the
total number of Ac or Ad per animal colon, **P ⫽ 0.003. WT, wild type,
controls.
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REDUCED SUSCEPTIBILITY TO COLON CANCER IN MIRKO MICE
AJP-Gastrointest Liver Physiol • VOL
promotion of ACF into tumors occurred. In contrast to these
observations, a positive association between increased adiposity and CRC has been clearly established from human and
animal studies (12, 25). It has been proposed that this association results in part from obesity-induced insulin resistance
and/or increased secretion of proinflammatory cytokines from
adipose tissue (20, 39). The morphology and biology of adipose tissue in MIRKO mice, however, differ from other rodent
models of obesity and/or insulin resistance (9). MIRKO mice
exhibit increased adipose-specific insulin sensitivity as a result
of increased differentiation of small, insulin-sensitive adipocytes. In contrast, enlarged insulin-resistant cells often seen in
diet-induced obesity are associated with reduced insulin responsiveness (5, 9). It is possible, therefore, that the increased
levels of insulin-sensitive adipose tissue in MIRKO mice may
have protected them from colon tumor development.
Two recent studies give support to this notion. One study
reported that feeding a high-calcium diet to ApcMin/⫹ mice that
are highly susceptible to spontaneous intestinal tumorigenesis
resulted in a significant loss of adipose tissue and increased
tumor formation compared with ApcMin/⫹ mice on a lowcalcium diet (15). When the ApcMin/⫹ mice were crossed with
obesity-prone Ay/a lethal yellow agouti mice, the resulting
“fat” Ay/ApcMin/⫹ mice exhibited loss of adipose tissue when
fed the high-calcium diet but were protected from increased
intestinal tumorigenesis (15). The authors proposed that there
is a critical level of adipose tissue required to maintain a
protective effect against intestinal tumorigenesis and that this
protective effect may result from the secretion of adipokines
that directly or indirectly suppress tumor formation (15). Another study assessed cancer development in lipodystrophic
A-ZIP/F-1 mice that are diabetic and insulin resistant and have
elevated circulating levels of glucose, insulin, lipids, and proinflammatory cytokines but lack white adipose tissue and have
undetectable levels of circulating adipokines (32, 38). Compared with lean controls that have normal amounts of body fat,
the fatless A-ZIP/F-1 mice were shown to be more susceptible
to skin and mammary carcinogenesis (38). The A-ZIP/F-1
mice also developed more skin tumors than obese ob/ob mice
that are insulin resistant but have significant levels of circulating adiponectin (1). The authors suggested that adiposity or
some “carcinogenesis-suppressing factor” produced by adipose
tissue such as adiponectin may be anticarcinogenic (1). We
observed significantly higher serum adiponectin, a potent insulin sensitizer (10), in 24-wk-old MIRKO mice compared
with controls. Elevated circulating adiponectin has been associated with reduced risk for CRC in human studies (39, 49) and
is thought to act either indirectly by altering circulating levels
of cytokines and hormones associated with insulin resistance
(13, 23) or directly through effects on signaling pathways in
cancer cells (8, 14). However, we did not observe any differences in the levels of pAMPK or its downstream metabolites in
the colons of untreated 24-wk-old MIRKO mice and controls.
AMPK is a key signaling protein activated by adiponectin (53).
It is possible, however, that adiponectin affects cell signaling
pathways in preneoplastic but not normal colonic epithelial
cells, as has been observed for leptin (16, 17).
In conclusion, we observed that the MIRKO phenotype,
displaying elevated TG and FFA but no differences in insulin
and glucose levels and glucose tolerance and insulin sensitivity
compared with controls, developed between 16 and 24 wk of
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been reported in other tissue-specific knockout models of
insulin resistance and diabetes (27, 45).
To determine the susceptibility of MIRKO mice to the early
stages of colon carcinogenesis, we treated 7-wk-old mice with
AOM and analyzed ACF at 24 wk of age. There were no
significant differences in the total number of ACF or in ACF
size between MIRKO mice and controls. The changes in
phenotype that occurred in MIRKO mice between 7 and 24 wk
of age, however, limit the interpretation of this result. We were
concerned that hyperinsulinemia in the 7-wk-old MIRKO mice
may have interfered with metabolic activation of AOM, since
insulin has been shown to downregulate hepatic CYP2E1,
the major enzyme of AOM metabolism (51). We observed no
differences, however, in hepatic CYP2E1 activity between
MIRKO and controls at 7 wk of age (data not shown). Insulin
has also been shown to be a promoter of ACF development
(11), although its transient elevation between 7 and 16 wk of
age clearly produced no observable effect on ACF. In addition,
we observed that blood lipids were not elevated in the MIRKO
mice relative to controls until after 16 wk of age, which may
have been too late to see a possible promotional effect on ACF.
Although there were no differences in ACF formation between MIRKO mice and controls, by 40 wk of age the MIRKO
mice developed significantly fewer colon tumors, particularly
adenocarcinomas, compared with controls. As discussed
above, MIRKO mice had higher FFA and TG at 24 wk of age,
but by 40 wk of age only FFA remained elevated compared
with controls. This suggests that FFA are not promoters of
colon carcinogenesis, but because of the normalization of TG
between 24 and 40 wk of age in MIRKO mice, we cannot make
any conclusions about the role of TG in promoting the growth
of ACF into tumors. A study by Tran et al. (47), however,
provides indirect support for the notion that neither FFA nor
TG promote colon carcinogenesis. They observed in rats that
acute intravenous infusion of intralipid giving rise to elevated
circulating TG and FFA did not promote colonic epithelial cell
proliferation, whereas infusion with insulin led to a significant
dose-dependent increase in proliferation (47). Furthermore,
infusion of intralipid with insulin had no additional effects on
cell proliferation, suggesting that even within a hyperinsulinemic environment, the lipids play no role in colonocyte proliferation (47).
Although MIRKO mice developed significantly fewer tumors than controls, the size of the tumors, particularly adenocarcinomas, was larger in MIRKO mice. The reasons for this
are unclear but may relate to the poorer insulin sensitivity and
glucose tolerance of 40-wk-old MIRKO mice relative to controls. It is possible that the previously mentioned compensatory
mechanisms that occur in younger MIRKO mice gradually
overwhelm the tissues, eventually resulting in an insulinresistant metabolic milieu that promotes increased growth of
established tumors in the older mice.
Our results suggest that there is some factor(s) in MIRKO
mice that acts to inhibit the promotion of ACF into colonic
tumors. MIRKO mice have been reported to have significantly
larger fat depots than controls despite having no differences in
body weight (7, 9). This results from a shift in utilization of
energy substrates and a redistribution of these substrates to
adipose tissue. Indeed, we observed that MIRKO mice accumulated significantly more epididymal fat than controls between 24 and 40 wk of age, the time period during which
REDUCED SUSCEPTIBILITY TO COLON CANCER IN MIRKO MICE
age. ACF development did not differ between MIRKO mice
and controls. MIRKO mice, however, developed significantly
fewer colon tumors than controls, and this may be related to an
inhibitory effect of an increased accumulation of insulinsensitive adipose tissue on the growth of ACF into tumors in
MIRKO mice compared with controls. Our results suggest that
circulating TG and FFA are not promoters of colon tumor
development. Indeed, we show that the cumulative effects of
the metabolic changes that occur with knockout of the insulin
receptor in muscle are associated with reduced susceptibility to
colon tumorigenesis.
15.
16.
17.
18.
ACKNOWLEDGMENTS
We thank Dr. Alan Medline for histopathological analysis of tumors.
19.
20.
This research was supported by the Terry Fox Foundation of the National
Cancer Institute of Canada, grant number 15030.
21.
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