help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2005-1012
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints, Permissions and Rights
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tran, T. T.
Right arrow Articles by Giacca, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tran, T. T.
Right arrow Articles by Giacca, A.
Endocrinology Vol. 147, No. 4 1830-1837
Copyright © 2006 by The Endocrine Society

Hyperinsulinemia, But Not Other Factors Associated with Insulin Resistance, Acutely Enhances Colorectal Epithelial Proliferation in Vivo

Thien T. Tran, Dinaz Naigamwalla, Andrei I. Oprescu, Loretta Lam, Gail McKeown-Eyssen, W. Robert Bruce and Adria Giacca

Departments of Nutritional Sciences (T.T.T., D.N., G.M.-E., W.R.B.), Physiology (A.I.O., L.L., A.G.), and Public Health Sciences (G.M.-E.), University of Toronto, Toronto, Canada M5S 1A8

Address all correspondence and requests for reprints to: Dr. Adria Giacca, Department of Physiology, Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada M5S 1A8. E-mail: adria.giacca{at}utoronto.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The similarity in risk factors for insulin resistance and colorectal cancer (CRC) led to the hypothesis that markers of insulin resistance, such as elevated circulating levels of insulin, glucose, fatty acids, and triglycerides, are energy sources and growth factors in the development of CRC. The objective was thus to examine the individual and combined effects of these circulating factors on colorectal epithelial proliferation in vivo. Rats were fasted overnight, randomized to six groups, infused iv with insulin, glucose, and/or Intralipid for 10 h, and assessed for 5-bromo-2-deoxyuridine labeling of replicating DNA in colorectal epithelial cells. Intravenous infusion of insulin, during a 10-h euglycemic clamp, increased colorectal epithelial proliferation in a dose-dependent manner. The addition of hyperglycemia to hyperinsulinemia did not further increase proliferation. Intralipid infusion alone did not affect proliferation; however, the combination of insulin, glucose, and Intralipid infusion resulted in greater hyperinsulinemia than the infusion of insulin alone and further increased proliferation. Insulin infusion during a 10-h euglycemic clamp decreased total IGF-I levels and did not affect insulin sensitivity. These results provide evidence for an acute role of insulin, at levels observed in insulin resistance, in the proliferation of colorectal epithelial cells in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SIMILAR LIFESTYLE factors are important in the pathogenesis of metabolic syndrome, type 2 diabetes and colorectal cancer (CRC) (1, 2). These lifestyle factors include diets high in calories and saturated fat and low levels of physical activity. A mechanism that links the development of CRC with the above lifestyle factors has been proposed. McKeown-Eyssen hypothesized that high-risk lifestyle factors often lead, possibly through insulin resistance, to elevated levels of insulin, triglycerides, nonesterified fatty acids (NEFA), and sometimes glucose, which act individually or in concert as growth factors and energy sources to promote colon carcinogenesis (3). Giovannucci proposed that the above high-risk lifestyles lead to elevated levels of insulin which promote the development of CRC (4). These hypotheses are supported by reports from case-control and prospective studies that showed associations between risk of CRC and elevated levels of plasma insulin, C-peptide, glucose, and triglycerides (5, 6, 7). In addition, there is evidence from studies in animal models that hyperinsulinemia/insulin resistance, whether diet-induced (8, 9) or genetic (10, 11), confers increased susceptibility to chemically induced carcinogenesis. We have shown that direct administration of insulin by sc injections in carcinogen-initiated rats increases the number of colorectal tumors (12). However, chronic insulin treatment also resulted in hypoglycemia and increased fasting triglyceride levels, which may have interfered with the effect of insulin.

The studies cited above add support to the insulin resistance-CRC hypothesis; however, several questions still remain, for example, whether diets that induce insulin resistance also increase CRC via hyperinsulinemia/insulin resistance or through a direct luminal mechanism, as suggested by intrarectal infusion of either beef fat, corn oil, or bile acids in rats and subsequent increases in colorectal epithelial proliferation (13). Proliferation is generally believed to be important in the induction and propagation of genetic mutations involved in cancer initiation and promotion respectively (14). Whether experimentally induced moderate elevation of circulating insulin, triglycerides, NEFA, and glucose levels, which are factors associated with insulin resistance, can affect colorectal epithelial proliferation has yet to be investigated. Insulin, glucose, diacylglycerol, and certain types of fatty acids can increase proliferation in CRC cell lines (15, 16, 17, 18). Direct insulin injection increased colonic epithelial proliferation in suckling (19) and adult mice (20); however, in these studies, the other metabolic variables were not controlled.

In the present study in rats, the hyperinsulinemic clamp technique allowed us to selectively elevate the plasma insulin levels at different target glucose levels, whereas a concomitant Intralipid infusion allowed us to elevate the plasma triglyceride and NEFA levels. The objective was to examine the individual and combined effects of intravascular elevation of insulin, glucose, and/or triglycerides/NEFA levels on colorectal epithelial proliferation in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male Fischer 344 rats weighing approximately 300 g were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN), and housed in the Department of Comparative Medicine at the University of Toronto. Care and treatment of the rats were in compliance with the guidelines of the Canadian Council on Animal Care, and the protocol was approved by the University of Toronto Animal Care Committee.

Experimental design
The protocol is outlined in Fig. 1Go. After 1 wk of acclimatization on rodent chow (Purina 5001), the rats had their jugular vein and carotid artery cannulated for infusion and sampling, respectively, and were allowed to recover from surgery for 1 wk (8). They were then fasted overnight and randomized into six groups (with group sizes 13, 9, 10, 12, 8, and 11 rats). Each group was then given 10-h iv infusions with one of the following: 1) saline (SAL); 2) insulin at an intermediate dose (12 mU/kg·min) with glucose clamped at fasting levels (6 mM) (I.INS); 3) insulin at a high dose (16.5 mU/kg·min) with glucose clamped at fasting levels (6 mM) (H.INS); 4) insulin at a high dose (16.5 mU/kg·min) with glucose clamped at postprandial levels (10 mM) (HIHG); 5) Intralipid (a 20% triglyceride emulsion; 6 µl/min) (LIP); or 6) Intralipid plus intermediate dose of insulin with glucose clamped at postprandial levels (6 µl/min, 12 mU/kg·min, 10 mM, respectively) (COMB). The high insulin dose was designed to achieve levels that can be found in obese fa/fa Zucker rats (21), and the intermediate insulin dose was designed to achieve insulin levels that were approximately half of those in the high insulin groups. The hyperglycemia achieved in the HIHG group matched peak levels of postprandial glucose (21). The NEFA and triglyceride levels achieved by Intralipid infusion are within the range observed in poorly controlled diabetes. Somatostatin was not used to inhibit insulin secretion during glucose or Intralipid infusion because somatostatin can suppress IGF-I and thus interfere with the possible effect of insulin on colorectal epithelial proliferation via IGF-I. To assess proliferation of the colorectal epithelial cells, a bolus of 5-bromo-2-deoxyuridine (BrdU) was given iv 8 h after the infusions were started. This time was chosen because results from colorectal cell cultures indicate that 8-h incubation was needed to demonstrate increased proliferation by insulin (15). Two hours later, colons were removed for the BrdU assay to measure DNA replication.


Figure 1
View larger version (28K):
[in this window]
[in a new window]
  		FIG. 1. Summary of protocol. (Diagram is not to scale.) After acclimatization and cannulation of vessels, rats were fasted overnight, then iv infused for 10 h with six different combinations of insulin, glucose, and/or Intralipid. Proliferation of the colorectal epithelial cells was determined by in vivo incorporation of BrdU into replicating DNA.

 
Experimental procedures
Saline was infused at a rate that matched that of the other infusion groups. Insulin was prepared by mixing Iletin II regular pork insulin (Eli Lilly, Indianapolis, IN) in 0.1% BSA (Sigma Chemicals, St. Louis, MO) in saline. During insulin infusion in the I.INS, H.INS, HIHG, and COMB groups, glucose was measured every 5 min for the first 2 h, and subsequently every 10 min for the remaining 8 h to adjust the rate of a variable glucose infusion that allowed attainment of euglycemia (~6 mM) or hyperglycemia (~10 mM). Intralipid (Pharmacia Inc., Mississauga, Ontario, Canada) was infused at a constant rate of 6 µl/min in the LIP and COMB groups. All six groups had 600 µl of blood taken every 2 h for a total of 10 h for measurements of plasma insulin, glucose, NEFA, and triglyceride levels. Red blood cells were immediately reinfused.

After the 8-h blood sample was taken, BrdU (50 mg/kg body weight in approximately 1 ml of PBS) was injected via the carotid catheter. All infusions were still delivered until the end of the 10 h when the last blood samples were taken. Rats were then euthanized with 1.0 ml of ketamine:xylazine:acepromazine (20:2:1).

Colons were removed, cleaned with PBS, slit open longitudinally, laid flat between filter paper, fixed in 10% phosphate-buffered formalin for 12 h, then transferred to 70% ethanol. Colons were then rolled, with the mucosal surface inside, from the rectal to the cecal end and embedded in paraffin.

Immunohistochemistry
Four-micrometer sections were cut perpendicular to the mucosal surface of the colons. BrdU visualization was based on an immunoperoxidase staining method (22). After slides were deparaffinized with xylene and rehydrated with ethanol, endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 20 min. Colons were digested with 20 µg/ml proteinase K in 10 mM Tris-HCl at pH 8.0 for 15 min, then hydrolyzed in 4 N HCl for 20 min at room temperature to denature DNA and expose the incorporated BrdU. Colon sections were incubated with the primary antibody (mouse anti-BrdU antibody, 1:40 dilution in PBS; Clone Bu20a; Dako, Mississauga, Ontario, Canada) in a humidified chamber placed at 4 C overnight. The secondary antibody (antimouse biotinylated antibody, 1:40 dilution; Labeled Strept-Avidin Biotin 2 kit with peroxidase; Dako) was added and sections were incubated for 60 min at room temperature. Next, the labeling complex (horseradish peroxidase conjugated streptavidin complex, 1:40 dilution; Labeled Strept-Avidin Biotin 2 kit with peroxidase) was added and sections were incubated for 60 min at room temperature. Visualization of the BrdU complex was achieved by staining with 0.05% 3,3'-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) in 0.05 M Tris-HCl at pH 7.6 in which 0.01% hydrogen peroxide was added immediately before staining for 10 min at room temperature. Sections were counterstained with Carazzi’s hematoxylin solution for 3 min.

Labeling index (LI)
All slides were coded so that the infusion groups were unknown to the scorer. Light microscopy at x100 magnification was used to examine the colorectal epithelial cells. Colorectal crypts were selected for analysis if a continuous column of colorectal epithelial cells extended from the basal muscularis mucosa to the luminal surface, and the cavity of the crypt opened up to the luminal surface. Each U-shaped crypt was analyzed as two crypt columns. BrdU-labeled cells were identified by the presence of brown-stained nuclei, and the position of BrdU cells along the crypt column was recorded. Crypts were scored for the following parameters: 1) crypt height (total number of cells in a crypt column extending from the luminal surface to the base of the crypt); 2) total LI (number of BrdU-labeled cells in a crypt column divided by crypt height); and 3) distribution of the LI in each of five equal compartments along the crypt column. These parameters were examined in 20 crypt columns in each animal.

Plasma assays and assessment of insulin sensitivity
Plasma glucose was measured by the glucose oxidase method with a Beckman Glucose Analyzer II (Beckman Instruments Co., Fullerton, CA). Plasma insulin was assessed with a RIA kit specific for rat insulin with 100% cross-reactivity for porcine insulin used for infusions (Linco Research Inc., St. Charles, MO). Plasma NEFA and triglycerides were measured with colorimetric kits (Wako Chemicals USA, Inc., Richmond, VA; and Roche Molecular Biochemicals Co., Indianapolis, IN, respectively). Plasma total IGF-I was determined by a RIA kit specific for rat IGF-I (Diagnostic Systems Laboratories, Inc., Webster, TX). Insulin sensitivity was assessed by calculating the glucose infusion rate (8) necessary to maintain plasma glucose either at fasting levels (I.INS and H.INS groups) or at hyperglycemic levels (HIHG and COMB groups) during insulin infusion.

Statistical analysis
Data are reported as mean ± SEM. ANOVA with three factors (infusion group, rat, and time) was performed to compare the infusion groups in terms of plasma levels of metabolites and glucose infusion rate (index of insulin sensitivity). ANOVA with three factors (infusion group, rat, crypt within each colon) was also carried out to compare the groups in terms of crypt height, total LI, and LI in each crypt compartment. When a significant difference (P < 0.05) was detected, Tukey’s t test was used to localize the difference between pairs of groups. Next, to separate the effect of hyperinsulinemia from that of insulin resistance and its associated metabolic variables on colorectal epithelial proliferation, univariate and stepwise multivariate regression analyses were performed with mean plasma levels of insulin over 10 h, change in insulin sensitivity [percentage change between glucose infusion rate (GIR) at 8–10 h and at 2–4 h], and mean plasma levels of metabolites as the independent variables and total LI as the dependent variable. In cases of unequal variance, variables were logarithmically transformed. All statistical analyses were performed with the SAS software program, version 8.00 (SAS Institute, Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Body weights
Body weights at the start of the 10-h infusions were not significantly different in the SAL, I.INS, H.INS, HIHG, LIP, and COMB groups (257 ± 4, 264 ± 4, 264 ± 4, 264 ± 1, 251 ± 4, and 261 ± 8 g, respectively).

Plasma levels
Figure 2Go shows plasma glucose, insulin, NEFA, and triglyceride levels during the 10-h infusions and clamps. Glucose levels were maintained at fasting levels in the SAL, I.INS, H.INS, and LIP groups. Glucose levels were maintained at hyperglycemic levels in the HIHG and COMB groups. Mean insulin levels over 10 h remained at fasting levels in the SAL group, tended to be higher but were not significantly different in the LIP group, were higher in the I.INS group, and highest in the H.INS, HIHG, and COMB groups. Mean NEFA and triglycerides levels remained at fasting levels in the SAL group, decreased in the I.INS, H.INS, and HIHG groups, increased in the COMB group, and, increased further in the LIP group when compared with the saline group. Figure 3Go shows total plasma IGF-I levels that were not significantly different at time 0 h, but after prolonged fasting, were significantly decreased in all groups at 10 h, with levels lowest in the H.INS and COMB groups.


Figure 2
View larger version (26K):
[in this window]
[in a new window]
  		FIG. 2. Circulating levels of glucose, insulin, NEFA, and triglycerides every 2 h during the 10-h infusions. Data are mean ± SEM. Mean levels with different letters (A–C) are significantly different between infusion groups at P < 0.05 as tested with ANOVA with three factors (infusion group, rat, time) and Tukey’s t test. §, Overlap between infusion groups.

 

Figure 3
View larger version (27K):
[in this window]
[in a new window]
  		FIG. 3. Total plasma IGF-I at 0 and 10 h for the six infusion groups. Data are mean ± SEM. Total IGF-I levels with different letters (A–D) are significantly different at the P < 0.05 as tested with an ANOVA for interactions between infusion group and time, and followed by Tukey’s t test.

 
Insulin sensitivity
Figure 4Go shows the steady-state GIR necessary to clamp the plasma glucose at the beginning and at the end of the infusions (2–4 h and 8–10 h, respectively) for the four groups that received insulin (I.INS, H.INS, HIHG, COMB). GIR at 2–4 h was not significantly different between the two groups that received insulin and were clamped at fasting glucose levels (I.INS and H.INS), presumably because of maximal stimulation of glucose disposal by insulin. GIR was higher in the two groups that were clamped at a higher glucose levels (HIHG and COMB), as expected. The GIR was lower at 8–10 h than at 2–4 h only in the COMB group, which received Intralipid, but not in the other groups. This indicates that Intralipid infusion induced insulin resistance whereas insulin per se did not affect insulin action within this time frame. Moderate hyperglycemia also did not appear to affect insulin action within this time frame.


Figure 4
View larger version (23K):
[in this window]
[in a new window]
  		FIG. 4. Insulin sensitivity. Glucose infusion rate (GIR, mean ± SEM) at the beginning (2–4 h) and end (8–10 h) of the infusions for the groups that received iv glucose and insulin. GIR during hyperinsulinemic clamps indicates insulin sensitivity when glucose and insulin levels are matched (e.g. within the same group over time).

 
LI of colorectal epithelial cells
The mean height of the colorectal crypts was 30 ± 1, 29 ± 1, 31 ± 1, 30 ± 1, 32 ± 1, and 32 ± 1 cells for the SAL, I.INS, H.INS, HIHG, LIP, and COMB groups respectively, and was not significantly different between the groups. Figure 5Go shows representative illustrations of BrdU labeling under light microscopy in the six groups. In Fig. 6AGo, the mean total LI for the six groups showed several differences. First, total LI was higher in the I.INS group than in the SAL group, and higher in the H.INS group than in the I.INS group. These results show a dose-dependent effect of insulin on colorectal epithelial proliferation, in the presence of euglycemia and low levels of NEFA and triglycerides. Second, total LI was similar between the H.INS and HIHG groups. Hence, in the presence of high insulin levels, high glucose levels did not affect total LI. Third, the LIP group had a similar total LI as the SAL group, which indicates that Intralipid infusion alone did not affect total LI significantly. Fourth, both the COMB and I.INS groups received the same intermediate dose of insulin infusion, but circulating insulin levels and total LI were significantly higher in the COMB than the I.INS group. It should be noted that the H.INS, HIHG, and COMB groups that had the highest (and similar) circulating insulin levels also had the highest (and similar) total LI, whereas I.INS had lower circulating insulin levels and lower total LI. Figure 6BGo shows the location of the BrdU-labeled cells along the longitudinal axis of the crypt for the six infusion groups. The crypts were analyzed in terms of five equal longitudinal compartments from the base to the luminal surface. The most notable differences are in compartments 2 and 3 near the base of the crypt, where LI, in general, were greatest in the high insulin groups (H.INS, HIHG, and COMB), lowest in the low insulin groups (SAL and LIP), and between the high and low insulin groups in the intermediate dose of insulin group (I.INS). A similar though less evident pattern was observed in compartment 1, and no significant pattern was observed in compartments 4 or 5.


Figure 5
View larger version (103K):
[in this window]
[in a new window]
  		FIG. 5. Representative illustrations of immunohistochemical BrdU labeling of colorectal epithelial cells under light microscopy for the six infusion groups.

 

Figure 6
View larger version (27K):
[in this window]
[in a new window]
  		FIG. 6. Panel A, Total BrdU LI of colorectal epithelial cells for each of the six infusion groups. Panel B, BrdU LI in the five compartments of the colorectal epithelium. Data are mean ± SEM. Means with different letters (A–C) are significantly different between infusion groups at the P < 0.05 as tested with an ANOVA with three factors and Tukey’s t test.

 
Regression analysis
To separate the effect of hyperinsulinemia from any effect of insulin resistance and its associated metabolic variables on colorectal epithelial proliferation, regression analysis was carried out with total LI as the dependent variable and the following independent variables: mean levels of plasma insulin, glucose, NEFA, and triglyceride over 10 h, and change in insulin sensitivity (% change between GIR at 8–10 h and at 2–4 h). In the univariate regression analyses, insulin was most strongly correlated with total LI, whereas glucose and NEFA showed small and borderline correlations with total LI, and triglycerides and change in insulin sensitivity were not correlated with total LI (Table 1Go). Stepwise multivariate regression analysis of insulin, glucose, NEFA, and triglyceride levels indicated that insulin levels were most strongly correlated with total LI, and accounted for about 35% (P < 0.001) of the variation in total LI. Glucose, NEFA, or triglyceride levels did not provide a significant additional contribution after insulin was entered into the model. Change in insulin sensitivity was left out of the multivariate regression model because its univariate r2 (Pearson’s correlation coefficient squared) was small, and its inclusion would have greatly decreased the sample size. It should be noted that change in insulin sensitivity was calculated only in the groups that received iv glucose and insulin (I.INS, H.INS, HIHG, and COMB).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Regression analyses of plasma insulin, glucose, NEFA, and triglyceride concentrations and change in insulin sensitivity with respect to colorectal epithelial proliferation as measured by total labeling index

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study examined the effects of a 10-h intravascular exposure to insulin, glucose, and/or Intralipid on colorectal epithelial proliferation in rats. The results demonstrate that iv infusion of insulin results in a dose-dependent increase in colorectal epithelial proliferation, as assessed by BrdU total LI of replicating DNA. Insulin’s effect in this in vivo model could not be accounted for by changes in glucose, NEFA, triglycerides, total IGF-I levels or by insulin-induced changes in insulin sensitivity. The insulin levels achieved in the present study were high, but still comparable to those found in Zucker fatty rats (21). These levels are relevant to states of obesity and insulin resistance.

The addition of hyperglycemia to hyperinsulinemia did not further increase total LI above that of hyperinsulinemia alone. Intralipid infusion alone did not affect total LI in colorectal epithelial cells when compared with the saline group. Intralipid, when coinfused with an intermediate dose of insulin and with glucose held at postprandial levels, increased plasma insulin to the levels observed in the group infused with a high dose of insulin. This was expected because we and other authors have shown that Intralipid infusion reduces insulin clearance (23, 24). Coinfusion of Intralipid, intermediate dose of insulin and glucose (COMB group) significantly increased total LI in comparison to the group with intermediate dose of insulin alone. Thus, intravascular Intralipid alone did not affect proliferation, but Intralipid together with insulin and glucose appears to further increase proliferation by increasing insulin levels.

In metabolic syndrome, hyperinsulinemia is associated with insulin resistance, elevated levels of NEFA, and triglycerides, and sometimes hyperglycemia. In the present study, stepwise multivariate regression analysis indicated that insulin levels were most strongly correlated with total LI, and accounted for 35% of the variation in total LI. In contrast, glucose, NEFA, triglyceride levels and insulin sensitivity did not provide any additional contribution after insulin was entered into the regression model. However, the present study did not allow us to address the in vivo effect on colorectal epithelial proliferation of: 1) elevated glucose levels at lower insulin levels; 2) chronic hyperglycemia; 3) chronic lipemia; and 4) saturated fatty acids, because Intralipid (commercially available emulsion for iv use) consists mainly of n-6 polyunsaturated fat, whereas beef tallow, which is predominantly composed of saturated fat, is known to promote CRC in animals (25).

In addition to measuring colorectal epithelial proliferation as total LI, proliferation was also assessed as the LI in each of the five compartments along the longitudinal axis of the colonic crypt. In normal colorectal epithelial cells, proliferation is usually highest near the base of the crypt where stem cells appear to reside, and proliferation decreases as cells mature, differentiate, and move up the crypt (26). It is generally hypothesized that dysregulation of stem cell proliferation is involved in the development of cancer (26). In the present study, LI in compartments 1 through 3 at the base of the crypt was greatest in the groups with high insulin levels. Thus, insulin could be affecting proliferation of the stem cells in normal colorectal cells.

Insulin’s effect on proliferation may be direct or mediated by other hormones and putative growth factors for the colon. IGF-I is a putative growth factor for colon carcinogenesis (5). In the present study, 10 h of insulin infusion, together with fasting or high glucose levels, decreased total circulating IGF-I levels in rats. Free IGF-I levels and IGF binding proteins were not measured in our study because of limited sample volume. Insulin can increase free IGF-I levels by decreasing synthesis of IGF binding proteins-1 and 2 (acute actions) (27, 28), and by increasing total IGF-I synthesis (long-term action) (29). However, during hyperinsulinemic-euglycemic clamps of 3–6 h, total IGF-I levels remained unchanged (30) or decreased (31, 32), presumably because of a negative feedback on GH initiated by the early increase in free IGF-I levels (31, 33). Whether insulin affects colorectal epithelial proliferation through an increase in free IGF-I in an in vivo model requires further investigation. Regarding possible effects mediated by insulin through other hormones, it is well known that, during hyperinsulinemic-euglycemic clamps, GH and glucagon levels decrease, and epinephrine, norepinephrine and glucocorticoid levels tend to increase. GH does not appear to affect colorectal epithelial proliferation directly (34), pharmacological levels of glucagon may increase it (35), pharmacologic levels of epinephrine may decrease it (36), and the acute effects of glucocorticoids are also mainly inhibitory (37). Thus, effects of hyperinsulinemia on colorectal epithelial proliferation in the present study are not likely to be mediated by these hormones.

The presence of circulating factors for colorectal epithelial cell growth is supported by the present study as well as by experiments involving colostomies in which intraluminal factors were diverted away from the colon but an increase in colorectal epithelial proliferation was still observed after feeding (38). Under normal physiological conditions, insulin could act as a sensor of dietary input and stimulate colonic epithelial proliferation to replace cells lost during the digestive process. In the present study, rats were fasted and not fed during the 10-h infusions to exclude effects of dietary components. Other luminal factors that may affect colorectal epithelial proliferation, such as bile acids, were not measured in the present study. However, it is unlikely that iv infusions of insulin in fasted animals affected total hepatic bile acid output into the colonic lumen. This is because insulin appears to increase bile flow but decrease bile acid concentration without changing total hepatic bile acid output (39).

Insulin may increase colorectal epithelial proliferation via several mechanisms. Insulin can bind insulin receptors and, to a lesser extent, IGF-I and insulin/IGF-I hybrid receptors, to stimulate several signaling transduction pathways in colorectal epithelial cells (40, 41, 42, 43). Activation of insulin receptor tyrosine kinase and, subsequently Ras, can lead to activation of several downstream targets such as MAPK, and thereby result in direct mitogenesis in colorectal epithelial cells (44, 45, 46, 47). Insulin-stimulated ras may also indirectly lead to mitogenesis; e.g. insulin appears to be required for the farnesylation of Ras to prime and enhance cellular response to other growth factors that may also use the Ras/MAPK pathway, such as IGF-I and epidermal growth factor (48). Another transduction pathway in colorectal epithelial cells downstream of the insulin receptor, which is mainly independent of Ras and can contribute to mitogenesis, is the phosphatidylinositol 3-kinase pathway (49, 50, 51). Through phosphatidylinositol 3-kinase-mediated inhibition of glycogen synthase kinase-3ß and through Ras/MAPK, insulin could also increase ß-catenin levels, which appear to be important in colorectal carcinogenesis (47, 52). Reactive oxygen species, which can increase colorectal epithelial proliferation (53), have been reported to be increased (53) or decreased (54) by insulin. Further studies such as examination of frozen colonic tissue sections (which could not be collected in the present study) are required to address the signaling mechanisms involved in the mitogenic effects of insulin in vivo. In addition to stimulating mitogenesis (15, 16), insulin can decrease apoptosis (55) in CRC cells in vitro. In the present study, apoptosis was not measured because any insulin-induced decreases in apoptosis are difficult to detect in normal colorectal cells with basal apoptosis rates that are already low (55).

In summary, we have demonstrated for the first time that circulating insulin, at levels seen in insulin resistance, acutely increases proliferation of normal colorectal epithelial cells in vivo in a dose-dependent manner. Further study is required to determine whether chronic exposure to this growth promoting effect of insulin directly enhances colon carcinogenesis in dysplastic mucosa, as well as to determine the effects of chronic exposure to elevated glucose and lipids. If chronic effects are similar to the acute effects we have observed, it would imply that in obesity and metabolic syndrome, hyperinsulinemia rather than lack of insulin action (i.e. insulin resistance) could be the main risk factor for colorectal cancer. This would be in contrast to the current thinking in respect to the risk for cardiovascular disease (56), which implicates lack of insulin action rather than hyperinsulinemia (56).


   Acknowledgments
 
We thank Dr. Jim Korkola for demonstrating the bromodeoxyuridine immunohistochemistry method, as well as Diana Hiesl, Karen Parisien, and An Jing Wang for assistance with animal care.


   Footnotes
 
This research was supported by a Strategic Grant in Nutrition and Cancer from The Cancer Research Society Inc., Canada.

T.T.T. is currently affiliated with the Joslin Diabetes Center, Harvard Medical School (Boston, MA).

Disclosure of Potential Conflicts of Interest: All authors have nothing to declare.

First Published Online January 12, 2006

Abbreviations: BrdU, 5-Bromo-2-deoxyuridine; COMB group, Intralipid plus intermediate dose of insulin with glucose clamped at postprandial levels group; CRC, colorectal cancer; GIR, glucose infusion rate; HIHG group, insulin at a high dose with glucose clamped at postprandial levels group; H.INS group, insulin at a high dose with glucose clamped at fasting levels group; I.INS group, insulin at an intermediate dose with glucose clamped at fasting levels group; LI, labeling index; LIP group, Intralipid group; NEFA, nonesterified fatty acids; SAL, saline group.

Received August 8, 2005.

Accepted for publication December 29, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. World Cancer Research Fund 1997 Food, nutrition and the prevention of cancer: a global perspective. Washington D.C.: American Institute for Cancer Research; 1–670
  2. Hu FB, Manson JE, Stampfer MJ, Colditz G, Liu S, Solomon CG, Willett WC 2001 Diet, lifestyle, and the risk of type 2 diabetes mellitus in women. N Engl J Med 345:790–797[Abstract/Free Full Text]
  3. McKeown-Eyssen G 1994 Epidemiology of colorectal cancer revisited: are serum triglycerides and/or plasma glucose associated with risk? Cancer Epidemiol Biomarkers Prev 3:687–695[Abstract]
  4. Giovannucci E 1995 Insulin and colon cancer. Cancer Causes Control 6:164–179[CrossRef][Medline]
  5. Giovannucci E 2001 Insulin, insulin-like growth factors and colon cancer: a review of the evidence. J Nutr 131:3109S–3120S
  6. Ma J, Giovannucci E, Pollak M, Leavitt A, Tao Y, Gaziano JM, Stampfer MJ 2004 A prospective study of plasma C-peptide and colorectal cancer risk in men. J Natl Cancer Inst 96:546–553[Abstract/Free Full Text]
  7. Wei EK, Ma J, Pollak MN, Rifai N, Fuchs CS, Hankinson SE, Giovannucci E 2005 A prospective study of C-peptide, insulin-like growth factor-I, insulin-like growth factor binding protein-1, and the risk of colorectal cancer in women. Cancer Epidemiol Biomarkers Prev 14:850–855[Abstract/Free Full Text]
  8. Tran TT, Gupta N, Goh T, Naigamwalla D, Chia MC, Koohestani N, Mehrotra S, McKeown-Eyssen G, Giacca A, Bruce WR 2003 Direct measure of insulin sensitivity with the hyperinsulinemic-euglycemic clamp and surrogate measures of insulin sensitivity with the oral glucose tolerance test: correlations with aberrant crypt foci promotion in rats. Cancer Epidemiol Biomarkers Prev 12:47–56[Abstract/Free Full Text]
  9. Koohestani N, Chia MC, Pham NA, Tran TT, Minkin S, Wolever TM, Bruce WR 1998 Aberrant crypt focus promotion and glucose intolerance: correlation in the rat across diets differing in fat, n-3 fatty acids and energy. Carcinogenesis 19:1679–1684[Abstract/Free Full Text]
  10. Lee WM, Lu S, Medline A, Archer MC 2001 Susceptibility of lean and obese Zucker rats to tumorigenesis induced by N-methyl-N-nitrosourea. Cancer Lett 162:155–160[CrossRef][Medline]
  11. Weber RV, Stein DE, Scholes J, Kral JG 2000 Obesity potentiates AOM-induced colon cancer. Dig Dis Sci 45:890–895[CrossRef][Medline]
  12. Tran TT, Medline A, Bruce WR 1996 Insulin promotion of colon tumors in rats. Cancer Epidemiol Biomarkers Prev 5:1013–1015[Abstract]
  13. Bull AW, Marnett LJ, Dawe EJ, Nigro ND 1983 Stimulation of deoxythymidine incorporation in the colon of rats treated intrarectally with bile acids and fats. Carcinogenesis 4:207–210[Abstract/Free Full Text]
  14. Lipkin M 1988 Biomarkers of increased susceptibility to gastrointestinal cancer: new application to studies of cancer prevention in human subjects. Cancer Res 48:235–245[Free Full Text]
  15. Cezard JP, Forgue-Lafitte ME, Chamblier MC, Rosselin GE 1981 Growth-promoting effect, biological activity, and binding of insulin in human intestinal cancer cells in culture. Cancer Res 41:1148–1153[Abstract/Free Full Text]
  16. Koenuma M, Yamori T, Tsuruo T 1989 Insulin and insulin-like growth factor 1 stimulate proliferation of metastatic variants of colon carcinoma 26. Jpn J Cancer Res 80:51–58[CrossRef]
  17. Smith TA, Titley JC, McCready VR 1998 Proliferation is associated with 2-deoxy-D-[1–3H]glucose uptake by T47D breast tumour and SW480 and SW620 colonic tumour cells. Nucl Med Biol 25:481–485[Medline]
  18. Taylor DD, Gercel-Taylor C, Weese JL 1996 Modulation of colon tumor oncogene expression by cancer patient-derived lipids. J Surg Oncol 63:46–51[Medline]
  19. Menard D, Dagenais P 1993 Stimulatory effects of insulin on DNA synthesis in suckling mouse colon. Biol Neonate 63:310–315[Medline]
  20. Scheving LA, Scheving LE, Tsai TH, Pauly JE 1982 Circadian stage-dependent effects of insulin and glucagon on incorporation of [3H]thymidine into deoxyribonucleic acid in the esophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, and spleen of the adult female mouse. Endocrinology 111:308–315[Abstract/Free Full Text]
  21. Qu X, Seale JP, Donnelly R 1999 Tissue and isoform-selective activation of protein kinase C in insulin-resistant obese Zucker rats—effects of feeding. J Endocrinol 162:207–214[Abstract]
  22. Morstyn G, Pyke K, Gardner J, Ashcroft R, de Fazio A, Bhathal P 1986 Immunohistochemical identification of proliferating cells in organ culture using bromodeoxyuridine and a monoclonal antibody. J Histochem Cytochem 34:697–701[Abstract]
  23. Wiesenthal SR, Sandhu H, McCall RH, Tchipashvili V, Yoshii H, Polonsky K, Shi ZQ, Lewis GF, Mari A, Giacca A 1999 Free fatty acids impair hepatic insulin extraction in vivo. Diabetes 48:766–774[Abstract]
  24. Hennes MM, Dua A, Kissebah AH 1997 Effects of free fatty acids and glucose on splanchnic insulin dynamics. Diabetes 46:57–62[Abstract]
  25. Nigro ND, Singh DV, Campbell RL, Sook M 1975 Effect of dietary beef fat on intestinal tumor formation by azoxymethane in rats. J Natl Cancer Inst 54:439–442[Medline]
  26. Bach SP, Renehan AG, Potten CS 2000 Stem cells: the intestinal stem cell as a paradigm. Carcinogenesis 21:469–476[Abstract/Free Full Text]
  27. Boni-Schnetzler M, Schmid C, Mary JL, Zimmerli B, Meier PJ, Zapf J, Schwander J, Froesch ER 1990 Insulin regulates the expression of the insulin-like growth factor binding protein 2 mRNA in rat hepatocytes. Mol Endocrinol 4:1320–1326[Abstract/Free Full Text]
  28. Powell DR, Suwanichkul A, Cubbage ML, DePaolis LA, Snuggs MB, Lee PD 1991 Insulin inhibits transcription of the human gene for insulin-like growth factor-binding protein-1. J Biol Chem 266:18868–18876[Abstract/Free Full Text]
  29. Salamon EA, Luo J, Murphy LJ 1989 The effect of acute and chronic insulin administration on insulin-like growth factor-I expression in the pituitary-intact and hypophysectomised rat. Diabetologia 32:348–353[CrossRef][Medline]
  30. Carmina E, Stanczyk FZ, Morris RS, Lee PD, Savjani G, Lobo RA 1995 Altered regulation of insulin-like growth factor binding protein-1 in patients with polycystic ovary syndrome. J Soc Gynecol Investig 2:743–747[CrossRef][Medline]
  31. Nygren J, Carlsson-Skwirut C, Brismar K, Thorell A, Ljungqvist O, Bang P 2001 Insulin infusion increases levels of free IGF-I and IGFBP-3 proteolytic activity in patients after surgery. Am J Physiol Endocrinol Metab 281:E736–E741
  32. Fisher SJ, Shi ZQ, Lickley HL, Efendic S, Vranic M, Giacca A 2001 Low-dose IGF-I has no selective advantage over insulin in regulating glucose metabolism in hyperglycemic depancreatized dogs. J Endocrinol 168:49–58[Abstract]
  33. Attia N, Tamborlane WV, Heptulla R, Maggs D, Grozman A, Sherwin RS, Caprio S 1998 The metabolic syndrome and insulin-like growth factor I regulation in adolescent obesity. J Clin Endocrinol Metab 83:1467–1471[Abstract/Free Full Text]
  34. Fiebig HH, Dengler W, Hendriks HR 2000 No evidence of tumor growth stimulation in human tumors in vitro following treatment with recombinant human growth hormone. Anticancer Drugs 11:659–664[Medline]
  35. Moyer MP, Aust JB, Dixon PS, Levine BA, Sirinek KR 1985 Glucagon enhances growth of cultured human colorectal cancer cells in vitro. Am J Surg 150:676–679[Medline]
  36. Kennedy MF, Tutton PJ, Barkla DH 1983 Adrenergic factors involved in the control of crypt cell proliferation in jejunum and descending colon of mouse. Clin Exp Pharmacol Physiol 10:577–586[Medline]
  37. Tutton PJ, Barkla DH 1981 Effects of glucocorticoid hormones on cell proliferation in dimethylhydrazine-induced tumours in rat colon. Virchows Arch B Cell Pathol Incl Mol Pathol 38:247–251[Medline]
  38. Haentjens P, Delvaux G, Chayvialle JA, Willems G 1986 Postprandial stimulation of epithelial cell proliferation in defunctioned colon of rats is not caused by gastrin. Gastroenterology 90:939–945[Medline]
  39. Sack J, Krusch DA, Hanks JB, Jones RS 1990 Effect of verapamil on insulin-stimulated choleresis. J Surg Res 49:88–91[Medline]
  40. Bergeron JJ, Rachubinski R, Searle N, Borts D, Sikstrom R, Posner BI 1980 Polypeptide hormone receptors in vivo: demonstration of insulin binding to adrenal gland and gastrointestinal epithelium by quantitative radioautography. J Histochem Cytochem 28:824–835[Abstract]
  41. Soos MA, Whittaker J, Lammers R, Ullrich A, Siddle K 1990 Receptors for insulin and insulin-like growth factor-I can form hybrid dimers. Characterisation of hybrid receptors in transfected cells. Biochem J 270:383–390[Medline]
  42. Van Wyk JJ, Graves DC, Casella SJ, Jacobs S 1985 Evidence from monoclonal antibody studies that insulin stimulates deoxyribonucleic acid synthesis through the type I somatomedin receptor. J Clin Endocrinol Metab 61:639–643[Abstract/Free Full Text]
  43. Wong M, Holdaway IM 1985 Insulin binding by normal and neoplastic colon tissue. Int J Cancer 35:335–341[Medline]
  44. Aliaga JC, Deschenes C, Beaulieu JF, Calvo EL, Rivard N 1999 Requirement of the MAP kinase cascade for cell cycle progression and differentiation of human intestinal cells. Am J Physiol 277:G631–G641
  45. Bissonnette M, Khare S, von Lintig FC, Wali RK, Nguyen L, Zhang Y, Hart J, Skarosi S, Varki N, Boss GR, Brasitus TA 2000 Mutational and nonmutational activation of p21ras in rat colonic azoxymethane-induced tumors: effects on mitogen-activated protein kinase, cyclooxygenase-2, and cyclin D1. Cancer Res 60:4602–4609[Abstract/Free Full Text]
  46. Corleta HE, Capp E, Corleta OC 1996 Insulin receptor tyrosine kinase activity in colon carcinoma. Braz J Med Biol Res 29:1593–1597[Medline]
  47. Takayama T, Ohi M, Hayashi T, Miyanishi K, Nobuoka A, Nakajima T, Satoh T, Takimoto R, Kato J, Sakamaki S, Niitsu Y 2001 Analysis of K-ras, APC, and beta-catenin in aberrant crypt foci in sporadic adenoma, cancer, and familial adenomatous polyposis. Gastroenterology 121:599–611[CrossRef][Medline]
  48. Goalstone ML, Leitner JW, Wall K, Dolgonos L, Rother KI, Accili D, Draznin B 1998 Effect of insulin on farnesyltransferase. Specificity of insulin action and potentiation of nuclear effects of insulin-like growth factor-1, epidermal growth factor, and platelet-derived growth factor. J Biol Chem 273:23892–23896[Abstract/Free Full Text]
  49. Phillips WA, St Clair F, Munday AD, Thomas RJ, Mitchell CA 1998 Increased levels of phosphatidylinositol 3-kinase activity in colorectal tumors. Cancer 83:41–47[CrossRef][Medline]
  50. Benistant C, Chapuis H, Roche S 2000 A specific function for phosphatidylinositol 3-kinase {alpha} (p85{alpha}-p110{alpha}) in cell survival and for phosphatidylinositol 3-kinase ß (p85{alpha}-p110ß) in de novo DNA synthesis of human colon carcinoma cells. Oncogene 19:5083–5090[CrossRef][Medline]
  51. Katso R, Okkenhaug K, Ahmadi K, White S, Timms J, Waterfield MD 2001 Cellular function of phosphoinositide 3-kinases: implications for development, homeostasis, and cancer. Annu Rev Cell Dev Biol 17:615–675[CrossRef][Medline]
  52. Desbois-Mouthon C, Cadoret A, Blivet-Van Eggelpoel MJ, Bertrand F, Cherqui G, Perret C, Capeau J 2001 Insulin and IGF-1 stimulate the ß-catenin pathway through two signalling cascades involving GSK-3ß inhibition and Ras activation. Oncogene 20:252–259[CrossRef][Medline]
  53. Giulivi C, Cadenas E 1998 Extracellular activation of fluorinated aziridinylbenzoquinone in HT29 cells EPR studies. Chem Biol Interact 113:191–204[CrossRef][Medline]
  54. Kang S, Song J, Kang H, Kim S, Lee Y, Park D 2003 Insulin can block apoptosis by decreasing oxidative stress via phosphatidylinositol 3-kinase- and extracellular signal-regulated protein kinase-dependent signaling pathways in HepG2 cells. Eur J Endocrinol 148:147–155[Abstract]
  55. Wu X, Fan Z, Masui H, Rosen N, Mendelsohn J 1995 Apoptosis induced by an anti-epidermal growth factor receptor monoclonal antibody in a human colorectal carcinoma cell line and its delay by insulin. J Clin Invest 95:1897–1905[Medline]
  56. Dandona P, Aljada A, Mohanty P 2002 The anti-inflammatory and potential anti-atherogenic effect of insulin: a new paradigm. Diabetologia 45:924–930[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Nutr.Home page
T. J. Hartman, P. S. Albert, Z. Zhang, D. Bagshaw, P. M. Kris-Etherton, J. Ulbrecht, C. K. Miller, G. Bobe, N. H. Colburn, and E. Lanza
Consumption of a Legume-Enriched, Low-Glycemic Index Diet Is Associated with Biomarkers of Insulin Resistance and Inflammation among Men at Risk for Colorectal Cancer
J. Nutr., January 1, 2010; 140(1): 60 - 67.
[Abstract] [Full Text] [PDF]

Home page
 GutHome page
H Endo, K Hosono, T Fujisawa, H Takahashi, M Sugiyama, K Yoneda, Y Nozaki, K Fujita, M Yoneda, M Inamori, et al.
Involvement of JNK pathway in the promotion of the early stage of colorectal carcinogenesis under high-fat dietary conditions
Gut, December 1, 2009; 58(12): 1637 - 1643.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Zhao, Z.-l. Wan, L. Whittaker, B. Xu, N. B. Phillips, P. G. Katsoyannis, F. Ismail-Beigi, J. Whittaker, and M. A. Weiss
Design of an Insulin Analog with Enhanced Receptor Binding Selectivity: RATIONALE, STRUCTURE, AND THERAPEUTIC IMPLICATIONS
J. Biol. Chem., November 13, 2009; 284(46): 32178 - 32187.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
R. L. Derr, X. Ye, M. U. Islas, S. Desideri, C. D. Saudek, and S. A. Grossman
Association Between Hyperglycemia and Survival in Patients With Newly Diagnosed Glioblastoma
J. Clin. Oncol., March 1, 2009; 27(7): 1082 - 1086.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
B. M. Wolpin, J. A. Meyerhardt, A. T. Chan, K. Ng, J. A. Chan, K. Wu, M. N. Pollak, E. L. Giovannucci, and C. S. Fuchs
Insulin, the Insulin-Like Growth Factor Axis, and Mortality in Patients With Nonmetastatic Colorectal Cancer
J. Clin. Oncol., January 10, 2009; 27(2): 176 - 185.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
K. N. Ealey, S. Lu, D. Lau, and M. C. Archer
Reduced susceptibility of muscle-specific insulin receptor knockout mice to colon carcinogenesis
Am J Physiol Gastrointest Liver Physiol, March 1, 2008; 294(3): G679 - G686.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. J. Gunter, D. R. Hoover, H. Yu, S. Wassertheil-Smoller, T. E. Rohan, J. E. Manson, B. V. Howard, J. Wylie-Rosett, G. L. Anderson, G. Y.F. Ho, et al.
Insulin, Insulin-like Growth Factor-I, Endogenous Estradiol, and Risk of Colorectal Cancer in Postmenopausal Women
Cancer Res., January 1, 2008; 68(1): 329 - 337.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
J. E. Drew, A. J. Farquharson, S. Padidar, G. G. Duthie, J. G. Mercer, J. R. Arthur, P. C. Morrice, and L. N. Barrera
Insulin, leptin, and adiponectin receptors in colon: regulation relative to differing body adiposity independent of diet and in response to dimethylhydrazine
Am J Physiol Gastrointest Liver Physiol, October 1, 2007; 293(4): G682 - G691.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Clin. Nutr.Home page
E. Giovannucci
Metabolic syndrome, hyperinsulinemia, and colon cancer: a review
Am. J. Clinical Nutrition, September 1, 2007; 86(3): 836S - 842S.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
P. T. Campbell, M. Cotterchio, E. Dicks, P. Parfrey, S. Gallinger, and J. R. McLaughlin
Excess Body Weight and Colorectal Cancer Risk in Canada: Associations in Subgroups of Clinically Defined Familial Risk of Cancer
Cancer Epidemiol. Biomarkers Prev., September 1, 2007; 16(9): 1735 - 1744.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints, Permissions and Rights
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tran, T. T.
Right arrow Articles by Giacca, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tran, T. T.
Right arrow Articles by Giacca, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals