help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2004-0323
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
145/11/5150    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
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 Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Metzger, S.
Right arrow Articles by Chajek-Shaul, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Metzger, S.
Right arrow Articles by Chajek-Shaul, T.
Endocrinology Vol. 145, No. 11 5150-5156
Copyright © 2004 by The Endocrine Society

Inhibition of Hepatic Gluconeogenesis and Enhanced Glucose Uptake Contribute to the Development of Hypoglycemia in Mice Bearing Interleukin-1ß- Secreting Tumor

Shulamit Metzger, Samir Nusair, David Planer, Varda Barash, Orit Pappo, Joel Shilyansky and Tova Chajek-Shaul

Department of Medicine (S.M., S.N., D.P., T.C.-S.), Hadassah University Hospital, Mount Scopus, and Departments of Biochemistry (V.B.) and Pathology (O.P.), Hadassah University Hospital, Jerusalem 91240, Israel; and Division of Pediatric Surgery (J.S.), Medical College of Wisconsin, Milwaukee, Wisconsin 53201

Address all correspondence and requests for reprints to: Tova Chajek-Shaul, M.D., Department of Medicine, Hadassah University Hospital, Mount Scopus, P.O. Box 24035, Jerusalem 91240, Israel. E-mail: chajek{at}hadassah.org.il.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mice bearing IL-1ß-secreting tumor were used to study the chronic effect of IL-1ß on glucose metabolism. Mice were injected with syngeneic tumor cells transduced with the human IL-1ß gene. Serum IL-1ß levels increased exponentially with time. Secretion of IL-1ß from the developed tumors was associated with decreased food consumption, reduced body weight, and reduced blood glucose levels. Body composition analysis revealed that IL-1ß caused a significant loss in fat tissue without affecting lean body mass and water content. Hepatic phosphoenolpyruvate carboxykinase and glucose-6-phosphatase activities and mRNA levels of these enzymes were reduced, and 2-deoxy-glucose uptake by peripheral tissues was enhanced. mRNA levels of glucose transporters (Gluts) in the liver were determined by real-time PCR analysis. Glut-3 mRNA levels were up-regulated by IL-1ß. Glut-1 and Glut-4 mRNA levels in IL-1ß mice were similar to mRNA levels in pair-fed mice bearing nonsecreting tumor. mRNA level of Glut-2, the major Glut of the liver, was down-regulated by IL-1ß. We concluded that both decreased glucose production by the liver and enhanced glucose disposal lead to the development of hypoglycemia in mice bearing IL-1ß-secreting tumor. The observed changes in expression of hepatic Gluts that are not dependent on insulin may contribute to the increased glucose uptake.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING INFLAMMATORY PROCESSES and in infectious and malignant diseases, there are changes in host energy homeostasis, resulting in anorexia and hypoglycemia (1). Proinflammatory cytokines, secreted from activated monocytes and lymphocytes, are known mediators of inflammation and lipopolysaccharide (LPS)-induced immune response. A primary role for IL-1{alpha} and IL-1ß in LPS-induced hypoglycemia in mice has been described (2). Although LPS stimulates the production of several proinflammatory cytokines, administration of recombinant IL-1ß receptor antagonist is capable of partially reversing LPS-induced hypoglycemia. IL-1{alpha}/ß double knockout mice are resistant to LPS-induced hypoglycemia but susceptible to other LPS responses, such as glucocorticoid secretion and production of fever. A single ip injection of mouse IL-1{alpha} reduced food intake and total body weight but increased liver weight as well as liver mRNA and protein content, probably due to the acute-phase response (3). A transient elevation of serum insulin, glucagon, and corticosterone and a decrease in glucose level (4) and in glycogen content in the liver are observed after IL-1ß administration (5). It is unlikely that this hypoglycemia is due to the induced anorexia because it occurs a very short time after IL-1ß administration before any effect on food intake is observed. In this model, hypoglycemia induced by fasting is further enhanced by IL-1ß administration. IL-1ß also causes hypoglycemia in adrenalectomized animals, where no hyperinsulinemia is measured, as well as in insulin-resistant diabetic mice (6). Because hypoglycemia induced by acute administration of IL-1ß is not insulin dependent and there is no glucose loss in the urine, it is probably due to enhanced glucose utilization, through IL-1ß-mediated induction of glucose transporters (Gluts). Several ex vivo and in vitro studies describe IL-1ß induction of insulin-independent glucose transport into organs and cells. A significant up-regulation of ovarian Glut-3 mRNA has been detected in rat ovary during ovulation, when intraovarian IL-1ß and its receptors are also upregulated (7). Direct effect of IL-1ß on Glut-1 and Glut-3 of whole ovarian dispersates has been demonstrated on the level of transcripts and proteins. In vitro studies showed a direct stimulatory insulin-independent effect of IL-1ß on glucose incorporation into adipocytes, fibroblasts (8), and astrocytes (9). Cytokines have also been shown to stimulate Glut-1 and Glut-3 expression in murine peritoneal macrophages (10). IL-1ß stimulates Glut-1 expression in human peritoneal mesothelial cells (11) and enhances glucose uptake in human articular chondrocytes via up-regulation of Glut-1 transcript and protein levels (12). In this study, we present a chronic model for the secretion of IL-1ß in mice. Mice bearing human IL-1ß-secreting tumors develop hypoglycemia, which is associated with depletion of glycogen and inhibited gluconeogenesis in the liver, a mild anorexia, and enhanced glucose consumption in peripheral tissues.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Retroviral vector and transduction
The tumor cell line, MCA102, is a methylcholanthrene-induced fibrosarcoma derived from female C57BL/6 mice. The human IL-1ß gene fused to the rat GH signal peptide was cloned into the retroviral vector Maloney murine leukemia virus, driven by the long terminal repeat promoter. The neomycin resistance gene was driven by the simian virus 40 promoter (13). Tumor cells (106) were incubated with retrovirus at a multiplicity of infection of 4:1 and polybrene (6 µg/ml; Sigma, St. Louis, MO). The cells were washed after 24 h and subjected to G418 selection (0.4 mg/ml; GIBCO Laboratories, Grand Island, NY) for 2 wk. Supernatants were examined by human IL-1ß Elisa kit (R&D, Minneapolis, MN). The high producer clone 4JK-hIL-1ß (>10,000 pg human IL-1ß/106 cells/ml) was used in this study.

Mice and tumor creation
Male C57BL/6 mice weighed 20–25 g and were maintained in a 12-h light, 12-h dark cycle. MCA 102 NeoR hIL-1ß (IL-1) or MCA 102 NeoR (Neo) cells (5 x 106) were injected sc into the flank area. Food consumption was measured daily and Neo mice were pair fed (NPF) in accordance with the food intake of the IL-1ß mice on the previous day. IL-1ß and NPF mice were killed at different time points after tumor inoculation as indicated in figures and tables. NPF mice were killed 1 d after IL-1ß mice to achieve the same food intake after pair feeding. Body weight and tumor weight were measured. Blood was drawn from the retroorbital plexus for analytical procedures. This study was approved by the Faculty Committee for Animal Care and Ethics of the Hebrew University.

Body composition analysis
After removal of internal organs and tumors, animals were dehydrated in an oven at 90 C for 4–7 d until a constant mass was achieved, and water content was calculated. The dried carcass was homogenized, and lipids were extracted from 1-g aliquots with chloroform-methanol (1:1) mixture. The extracted aliquots and the chloroform-methanol supernatants were dried and weighed and represented the lean body mass and the fat mass, respectively.

Analytical procedure
Blood glucose and insulin levels were determined as described (14). Mouse leptin and C-peptide levels were determined using RIA kits (Linco Research, Inc., St. Charles, MO). Serum human IL-1ß, mouse IL-6, and interferon-{gamma} levels were determined using an ELISA kit (Endogen Inc., Boston, MA).

Insulin tolerance test
Mice were injected ip with insulin (1 U/kg body weight; Humulin; Lilly, Indianapolis, IN), and tail vein glucose was measured at 0, 15, 30, and 60 min after injection using Glucometer Elite (Bayer, Elkhart, IN).

Preparation of liver homogenates for glycogen determination and enzymatic assays
Liver (200 mg) was removed and immediately digested with 2 ml of 33% KOH for glycogen determination. The glycogen was isolated by ethanol precipitation and subsequently hydrolyzed to glucose by amyloglucosidase (15). Liver homogenates (20%, wt/vol) were prepared in sucrose-Tris buffer (10 mM Tris HCl, pH 7.4; 0.25 M sucrose, and 0.5 mM EDTA) at 5 C. The homogenates were centrifuged at 12,000 x g for 20 min, and the resulting supernatants were recentrifuged for 45 min at 105,000 x g. The supernatants were used for determination of phosphoenolpyruvate carboxykinase (PEPCK) activity by measuring exchange between 14C-labeled KHCO3 and unlabeled oxaloacetate (16). The pellets were used for the determination of glucose-6-phosphatase (G6Pase) activity according to Burchell et al. (17). G6Pase activity assay was performed using intact or disrupted microsomes. Disrupted microsomes were obtained by incubation of microsomal fraction with 0.25% sodium deoxycholate for 15 min at 5 C. This procedure results in fully disrupted microsomes.

2-Deoxy-[3H]glucose uptakes
2-Deoxy-[3H]glucose (3 µCi) was injected iv into mice. Blood (30–40 µl) was drawn from the retroorbital plexus at 1, 20, and 60 min after injection for glucose, and radioactivity was determination. At 60 min, the mice were killed, and the organs (liver, spleen, heart, gastrocnemius muscle, kidney, lung, small intestine, skin, brain, and tumor) were excised. The amount of 2-deoxy-[3H]glucose phosphate in each tissue was determined as described by Ferre et al. (18).

RNA isolation, Northern blotting, and RT-PCR
RNA was extracted from liver using the acid guanidinium thiocyanate-phenol-chloroform extraction method (19). RNA (10 µg) was electrophoresed on 1% agarose gel containing 2% formaldehyde in 3[N-morholino]propanesulfonic acid buffer. RNA was transferred to Amersham Hybond nylon membranes (Amersham, Piscataway, NJ) and subjected to UV cross-linking and hybridization. Probes were radiolabeled using a Random Primed DNA labeling kit (Roche Molecular Biochemicals, Basel, Switzerland). DNA probes used for Northern blot analysis were 1.6-kb cDNA fragment of rat PEPCK (a gift from Professor L. Reshef, Hebrew University, Jerusalem, Israel) and 283-bp PCR product of mouse G6Pase sequence derived from exon 1. The primers for the amplification of the mouse G6Pase probe were 5'-GCTTGGACTCACTGCAC-3' (sense) and 5'-GAAGACGAG- GTTGAACC-3' (antisense) (20). PCR product was confirmed by sequencing. Reverse-transcriptase reaction was performed using 1 µg of hepatic RNA, 1 µg of oligo dT as a primer, and 200 U of Maloney murine leukemia virus reverse transcriptase (Promega, Madison, WI).

Real-time PCR reaction
Real-time quantitative PCR analysis was performed with an automated sequence detection system (ABI Prism 7000; Applied Biosystems, Weiterstadt, Germany). The PCR mix (20 µl) was composed of 10 µl Syber Universal PCR Master Mix (ABI, Warrington, UK), 1 µl cDNA (each sample in a triplicate), and a final concentration of 300 nM from each primer. The following primers were used: mouse L19, 5'-GAATGGCTCAACAGGTAAACA-3' (sense) and 5'-GGGTTCCAGAGTCAAGTTCAG-3' (antisense); Glut-1, 5'-AGTGTATCCTGTTGCCCTTCT-3' (sense) and 5'-CATCGGCTGTCCCTCGAAGC-3' (antisense); Glut-2, 5'-CTGGGTCTGCAATTTTGTCA-3' (sense) and 5'-TGTAAACAGGGTGAAGACCA-3' (antisense); Glut-3, 5'-GACTGCTTCTGAGTGCTGCTA-3' (sense) and 5'-CATTGGCGATCTGGTCAACC-3' (antisense); and Glut-4, 5'-GTCCTCCTGCTTGGCTTCTT-3' (sense) and 5'-AGCTGAGATCTGGTCAAACG-3' (antisense). mRNA quantification was performed as described (21).

Statistical analysis
Experimental values represent the means ± SE. Data were analyzed using ANOVA, followed by Dunnett’s test for comparing control mean values with the mean values of each group. Statistical significance was set at P < 0.05 (22).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tumor induction and IL-1ß production
C57BL/6 mice were injected sc with 5 x 106 MCA 102 NeoR IL-1ß or MCA 102 NeoR cells. Serum levels of IL-1ß in mice bearing IL-1ß-secreting tumor increased exponentially with time to 0.7 ng/ml on d 9 and 10-fold higher to 8.3 ng/ml on d 21 after tumor inoculation (Fig. 1Go). An elevation in serum IL-6 levels was measured in IL-1ß mice on d 9 and 18 (0.4 ± 0.1 and 0.6 ± 0.1 ng/ml, respectively). Serum IL-6 levels in NPF mice were undetected (Table 1Go). Serum interferon-{gamma} levels were undetected.



View larger version (10K):
[in this window]
[in a new window]
  		FIG. 1. IL-1ß levels in serum. Mice were inoculated with tumor cells expressing human IL-1ß gene. Blood was drawn on d 9, 12, 17, and 21 after tumor inoculation, and serum IL-1ß levels were determined. Values represent mean ± SE of eight mice.

 

View this table:
[in this window]
[in a new window]
  		TABLE 1. Effect of IL-1ß on serum levels of glucose, insulin, C-peptide, leptin, and IL-6

 
The effect of chronic IL-1ß secretion on food consumption
The profile of the effect of IL-1ß on food consumption was a multiphasic response (Fig. 2Go). Mice bearing IL-1ß-secreting tumor maintained their food intake until d 5 of the experiment, with no significant difference between IL-1ß and Neo mice (3.6 ± 0.2 and 3.2 ± 0.1 g/mouse·d, respectively). From d 6, a gradual decrease in food intake was observed in IL-1ß mice, which reached its nadir on d 9 (1.7 ± 0.1 g/mouse·d). Thereafter, food intake increased and plateaued at approximately 75% of food consumption of mice bearing nonsecreting tumor. This pattern was reproducible in many recurrent experiments, and further analysis was performed on d 9 and 10 when food consumption was minimal and on d 18 and 19 after tumor inoculation. NPF mice were fed in accordance with food consumption of IL-1ß mice on the previous day.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 2. Food intake of mice bearing IL-1ß-secreting tumors. Mice were inoculated with tumor cells secreting IL-1ß, and food intake was recorded daily. Values represent mean ± SE of 20 mice. Cont, Control.

 
The effect of chronic IL-1ß secretion on body weight and body composition
IL-1ß mice were killed on d 9 or 18, and NPF mice were killed on d 10 or 19 after tumor inoculation. Mice bearing IL-1ß-secreting tumor produced smaller tumors than NPF mice (Table 2Go). On d 9 and 10, there was no significant difference between tumor size of IL-1ß and NPF mice (0.3 ± 0.1 and 0.5 ± 0.2 g, respectively). A significant difference in tumor size was observed on d 18 and 19 (1.0 ± 0.2 g in IL-1ß mice and 3.7 ± 0.4 g in NPF mice, P < 0.01). On d 18 and 19, there was no significant difference in body weight loss (difference between initial weight and final weight without tumor) between IL-1ß and NPF mice (Table 2Go). However, liver of IL-1ß mice weighed 1.7-fold more than liver of NPF mice, and spleen of IL-1ß mice weighed 10-fold more than spleen of NPF mice. Eviscerated body weight of IL-1ß mice was significantly reduced compared with NPF mice (Table 2Go). This effect was observed already 9 and 10 d after tumor injection, when food consumption was minimal. Body composition analysis revealed that reduced eviscerated body weight in IL-1ß mice was mainly due to loss of fat. No change was observed in percent water and lean body mass. Percent fat in IL-1ß mice was significantly reduced compared with NPF mice on d 9 and 10 (2.2 ± 0.6% and 4.1 ± 0.6%, respectively; P < 0.05) and on d 18 and 19 (3.8 ± 0.4% and 5.2 ± 1.0%, respectively; P < 0.05). Fat constituted 10 ± 1% of body composition in control-fed mice.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Effect of IL-1 secretion on body and organ weight

 
Serum glucose, insulin, C-peptide, and leptin levels in mice bearing IL-1ß-secreting tumor
Serum glucose level of control mice (without tumor and fed ad libitum) was 175 ± 4.5 mg/dl. In mice bearing IL-1ß-secreting tumor, blood glucose decreased with time. Glucose levels were determined 9 and 18 d after injection of IL-1ß-secreting tumor and were 90 ± 2.7 and 72 ± 2.5 mg/dl, respectively (Table 1Go). Mice bearing nonsecreting tumor had blood glucose levels of 141 ± 5.1 mg/dl on d 10 and 128 ± 6.4 mg/dl on d 19. In all measurements taken during the experiment, glucose levels of IL-1ß mice were significantly lower than glucose levels of NPF mice. There was a significant difference between serum insulin levels of control mice without tumor, fed ad libitum (34 ± 0.5 mU/ml), and NPF mice because of semi-starvation. On d 9 and 10, after tumor inoculation, serum insulin levels were 44 ± 3 mU/ml in IL-1ß mice and 21 ± 1.8 mU/ml in NPF mice. On d 18 and 19, there was no difference between insulin levels of IL-1ß (23 ± 2.0 mU/ml) and NPF mice (19 ± 4.9 mU/ml). On d 9 and 10, there was also an increase in serum C-peptide concentrations (718 ± 158 pM in IL-1ß mice and 291 ± 40 pM in NPF mice), which was compatible with the observed increase in serum insulin. No difference between C-peptide levels of IL-1ß and NPF mice was measured on d 18 and 19 (Table 1Go). A 25% increase was measured in leptin concentration in IL-1ß mice on d 18 compared with NPF mice on d 19 (3.6 ± 0.2 and 2.7 ± 0.3 ng/ml, respectively).

Liver glycogen content, enzymatic activity, and mRNA levels of G6Pase and PEPCK in mice bearing IL-1ß- secreting tumor
Liver glycogen content of control mice (without tumor and fed ad libitum) was 59 ± 6.7 mg/g. In both IL-1ß and NPF mice, liver glycogen content was reduced by 85%, mainly due to semi-starvation (Table 3Go).


View this table:
[in this window]
[in a new window]
  		TABLE 3. Effect of IL-1ß on liver glycogen, G6Pase, and PEPCK activities

 
PEPCK and G6Pase are key enzymes in the gluconeogenic pathway. PEPCK activity increased about 2-fold in NPF mice on d 10 and 19 compared with control mice (without tumor and fed ad libitum). In IL-1ß mice, no increase in PEPCK activity was observed compared with control mice. On d 9, it might have been due to elevated insulin levels in IL-1ß mice, but on d 18, serum insulin levels were low. G6Pase activity increased 1.4-fold in NPF mice compared with control mice (Table 3Go). A significant 2.5-fold decrease in G6Pase activity, both in intact and disrupted microsomes, was observed in IL-1ß mice compared with NPF mice in both early and late phases of the experiment. mRNA levels of PEPCK and G6Pase were determined using Northern blot analysis. It is evident that the regulation of IL-1ß on PEPCK and G6Pase activities is due to changes in mRNA levels of these enzymes (Fig. 3Go). Both mRNA levels were elevated in NPF mice due to food deprivation, and this effect was blocked by IL-1ß on d 18 and 19 as shown in Fig. 3Go (the same results were obtained on d 8 and 9, data not shown). PEPCK mRNA levels of IL-1ß mice were similar to mRNA levels of control-fed mice. G6Pase mRNA levels of IL-1ß mice were barely detectable and beneath control levels.



View larger version (51K):
[in this window]
[in a new window]
  		FIG. 3. Effect of IL-1ß secretion on G6Pase and PEPCK mRNA levels. Liver RNA was extracted from control mice fed ad libitum and from NPF and IL-1ß mice on d 19 and 18 after tumor inoculation, respectively. mRNA was detected by RNA blot hybridization using radiolabeled G6Pase and PEPCK DNAs as probes. ß-Actin mRNA expression was used as a control for mRNA content.

 
The effect of IL-1 on glucose uptake of peripheral tissues
Glucose utilization of IL-1ß mice by peripheral tissues was evaluated by measuring 2-deoxy-glucose uptake, a nondegradable analog of glucose. Nine days after tumor injection, when insulin levels were higher in IL-1ß mice, there was a slight elevation in 2-deoxy-glucose uptake by IL-1ß tissues (Table 4Go). Significant changes (2- to 4-fold) were measured only in spleen, lung, heart, and tumor. However, 18 d after injection (late phase, when insulin levels are low), there was a significant elevation (5- to 10-fold) in 2-deoxy-glucose uptake in almost every tissue examined (Table 4Go). Glucose uptake in adipose tissue was not determined due to lack of fat depots.


View this table:
[in this window]
[in a new window]
  		TABLE 4. Effect of IL-1ß secretion on 2-deoxy-glucose uptake

 
The effect of IL-1ß on insulin-induced blood glucose reduction
The effect of IL-1ß on blood glucose decrease was examined by insulin bolus test. There was no significant difference in the decrease of blood glucose between IL-1ß and NPF mice (Fig. 4Go).



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 4. Effect of IL-1ß secretion on insulin-induced reduction of blood glucose. Mice were injected ip with insulin (1 U/kg body weight) on d 18 and 19. Tail vein glucose was measured at 0, 15, 30, and 60 min after injection. Results represent mean ± SE of six mice. *, P < 0.05.

 
The effect of IL-1ß on hepatic expression of Gluts
Relative mRNA levels of Gluts in the liver were determined by RT-PCR analysis on d 18 and 19 after tumor injection. Quantitative real-time PCR analysis demonstrated a 20-fold reduction in cDNA levels of Glut-2, the major Glut of the liver, in IL-1ß mice (Fig. 5Go). Glut-1 and Glut-3 are more efficient Gluts, with lower Michaelis-Menten constants (Km) for glucose than Glut-2 (23). Glut-1 is abundant in most tissues, and hepatic Glut-3, the most efficient among the three transporters, is abundant mainly in neuronal tissue. Glut-3 cDNA levels were 8-fold higher in IL-1ß mice. Glut-1 and Glut-4 cDNA levels in IL-1ß mice were similar to cDNA levels in NPF mice.



View larger version (11K):
[in this window]
[in a new window]
  		FIG. 5. Effect of IL-1ß secretion on hepatic expression of Gluts. Hepatic RNA was extracted from IL-1ß and NPF mice on d 18 and 19, respectively, after tumor injection. cDNA synthesis was performed by RT-PCR. Quantification of cDNA levels of Glut-1, Glut-2, Glut-3, Glut-4, and L19 (control) were performed by real-time PCR. Results represent mean ± SE of four individual mice. Amplification reactions were performed in triplicates. Results are presented as relative cDNA levels in IL-1ß mice compared with cDNA levels in NPF mice; *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study describes a murine model for chronic secretion of IL-1ß. Mice injected with cells transduced with the human IL-1ß gene develop tumors and elevated IL-1ß levels in their serum. The serum IL-1ß concentration increases to 0.7 and 8 ng/ml 9 and 18 d, respectively, after tumor inoculation. Humans suffering from infection or inflammatory conditions have elevated levels of plasma IL-1ß (24). During bacterial infection and shock, serum IL-1ß levels up to 0.9 and 2.5 ng/ml have been reported (25, 26). Patients with systemic juvenile rheumatoid arthritis had plasma IL-1ß levels of 0.1 ng/ml (27). Mice injected with IL-1ß-secreting cells develop smaller tumors than mice injected with nonsecreting cells. IL-1ß inhibits tumor cell growth in culture and induces tumor regression in methylcholanthrene-induced tumors; and it may promote the release of tumoricidal substances that mediate tumor rejection (24).

Elevated IL-1ß levels in the circulation affect food intake, body weight, body composition, and serum glucose level. Food intake is gradually reduced from d 6–9 and then increases to 75% of food consumption of mice bearing nonsecreting tumors. The tolerance acquired, regarding the IL-1ß effect on food intake, might result from the interference of IL-1ß receptor antagonist with IL-1ß influence on appetite and food consumption. Ob mice that have a blunted production of IL-1ß receptor antagonist (28) didn’t regain appetite at the late phase of the experiment, and their daily food intake was decreased gradually from 5.8 g on d 5 to 1.6 g on d 9 and to 0.6 g on d 18 (Metzger, S., and T. Chajek-Shaul, unpublished data). The analysis of IL-1ß effect on body weight changes is complex in our model due to the enlargement of internal organs in IL-1ß mice. Liver of IL-1ß mice weighed 1.4- and 1.7-fold more than liver of NPF mice and spleen of IL-1ß mice weighed 3- and 10-fold more than spleen of NPF mice on d 9 and 18, respectively. Eviscerated body weight of IL-1ß mice was only 80% of the eviscerated body weight of NPF mice. Body composition analysis showed a significant reduction in fat content of IL-1ß mice. These data suggest that IL-1ß exhibits its effect on body weight and fat content beyond its effect on appetite and food consumption. Blood glucose levels of IL-1ß mice were reduced compared with control mice fed ad libitum or with NPF mice. Reduced glucose levels of NPF mice were due to semi-starvation as a result of pair feeding according to the food consumption of IL-1ß mice. IL-1ß induced a further reduction in glucose levels. Measured insulin levels of IL-1ß mice on d 9 were higher than insulin levels detected in serum of NPF mice, but no differences in insulin levels between IL-1ß and NPF mice were detected on d 18 and 19. It has been reported that IL-1ß inhibits proinsulin conversion to insulin in ß-cells (29). However, in our in vivo model, the increase in serum insulin levels on d 9 in IL-1ß mice was associated with a compatible increase in serum C-peptide concentration, indicating that an increase in insulin rather than in proinsulin occurs. Induction of insulin release by IL-1ß has been previously reported in rodent pancreatic islet in vivo (30). Our experimental data show a major effect of IL-1ß on carbohydrate metabolism. An 85% decrease in hepatic glycogen was observed in NPF and IL-1ß mice compared with glycogen content in control mice fed ad libitum. The two key regulatory enzymes of gluconeogenesis, G6Pase and PEPCK, were also affected by IL-1ß. Both enzymes were up-regulated due to semi-starvation in NPF compared with control-fed mice. IL-1ß prevented the stimulatory effect of fasting on PEPCK activity but inhibited G6Pase activity beyond the activity of fed controls. mRNAs of both enzymes (PEPCK and the catalytic unit of the G6Pase complex) were up-regulated in NPF mice compared with control-fed mice, and the different influence of IL-1ß on PEPCK and G6Pase activities was already demonstrated at the level of their gene expression. Depletion of glycogen and inhibition of gluconeogenesis might contribute to an inhibition of glucose secretion from the liver. Lately, it has been shown that inhibition of the G6Pase complex is associated with inhibition of the rate of glucose output from rat hepatocytes (31) and that the inhibition of hepatic gluconeogenic enzymes, PEPCK and G6Pase, is associated with an inhibited rate of endogenous glucose production in mice (32). In the IL-1ß-secreting mice, there was increased glucose utilization by various tissues as measured by 2-deoxy-glucose uptake. There was a tendency toward enhanced glucose uptake at the early phase in IL-1ß mice, with a significant elevation of glucose uptake in heart, spleen, lung, and tumor. At the late phase of the experiment, when serum insulin levels were very low, a significant increase in 2-deoxy-glucose uptake was observed in most tissues examined, compared with glucose uptake in NPF, control pair fed, and control-fed ad libitum mice. The major effect of IL-1ß on glucose uptake for gram tissue was detected in spleen and liver, and it was further enhanced considering the enlargement of these organs in IL-1ß mice compared with NPF mice. Enhanced glucose uptake may be due to increased insulin sensitivity induced by IL-1ß, but the insulin tolerance test in our in vivo model did not show that IL-1ß promotes an insulin-induced decrease in blood glucose. IL-1ß mice had a 2- to 3-fold increase in the insulin to glucose ratio. This may indicate the occurrence of insulin resistance, as described for TNF-{alpha} and IL-6, via insulin signaling (33); nevertheless, insulin levels are in the low physiological range. The mild increase in serum leptin in IL-1ß mice measured on d 18 and the known effects of leptin on hepatic and peripheral carbohydrate metabolism suggest that leptin is not a major mediator in our model (34). IL-1ß may induce secretion of other proinflammatory cytokines such as IL-6 and TNF. We have previously described a murine model of hypoglycemia induced by chronic secretion of IL-6 (35). Induction of hypoglycemia by IL-6 is due to inhibition of hepatic gluconeogenesis, depletion of liver glycogen content beyond the effect of reduced food intake, and a strong anorectic effect. Low concentrations of IL-6 have been detected in the serum of IL-1ß mice. However, according to the IL-6 model, the low serum IL-6 levels have a mild effect on liver glycogen content and on blood glucose level but do not affect food consumption. No effect of IL-6 on glucose uptake by peripheral tissues has been detected even at 100-fold higher IL-6 concentration. Yet, secretion of other cytokines may contribute to changes in metabolism, as observed in IL-1ß mice.

Our data show that the pattern of expression of Gluts in the liver is altered in IL-1ß mice. The direct effect of IL-1ß on insulin-independent Glut expression (mainly glut-1 and glut-3) has been demonstrated in ex vivo and in vitro experiments (5, 9, 10, 11, 12). Glycosylation of Glut-1 is associated with increased affinity to glucose in leukemic and thyroid anaplastic cells (36, 37). We intend to continue using this in vivo model of chronic IL-1ß secretion to further study the effect of IL-1ß on Glut expression and glycosylation in peripheral tissues. We conclude that chronic secretion of IL-1ß induces hypoglycemia in mice by a concerted action on reduced food intake, inhibited gluconeogenesis, and enhanced glucose utilization. The inhibition of glucose production by the liver and the increase in glucose uptake by various tissues represent a mirror image of type 2 diabetes. Further studies may elucidate the importance of this model in the development of treatment modalities for noninsulin-dependent diabetes mellitus.


   Footnotes
 
Abbreviations: Glut, Glucose transporter; G6Pase, glucose-6-phosphatase; LPS, lipopolysaccharide; NPF, pair-fed Neo mice; PEPCK, phosphoenolpyruvate carboxykinase.

Received March 12, 2004.

Accepted for publication July 28, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Inui A 1999 Cancer anorexia-cachexia syndrome: are neuropeptides the key? Cancer Res 59:4493–4501[Abstract/Free Full Text]
  2. Oguri S, Motegi K, Iwakura Y, Endo Y 2002 Primary role of interleukin-1 {alpha} and interleukin-1 ß in lipopolysaccharide-induced hypoglycemia in mice. Clin Diagn Lab Immunol 9:1307–1312[Abstract/Free Full Text]
  3. Moldawer LL, Andersson C, Gelin J, Lundholm KG 1988 Regulation of food intake and hepatic protein synthesis by recombinant-derived cytokines. Am J Physiol 254:G450–G456
  4. del Rey A, Besedovsky H 1987 Interleukin 1 affects glucose homeostasis. Am J Physiol 253:R794–R798
  5. del Rey A, Monge-Arditi G, Besedovsky HO 1998 Central and peripheral mechanisms contribute to the hypoglycemia induced by interleukin-1. Ann NY Acad Sci 840:153–161[Abstract/Free Full Text]
  6. del Rey A, Besedovsky H 1989 Antidiabetic effects of interleukin 1. Proc Natl Acad Sci USA 86:5943–5947[Abstract/Free Full Text]
  7. Kol S, Ben-Shlomo I, Ruutiainen K, Ando M, Davies-Hill TM, Rohan RM, Simpson IA, Adashi EY 1997 The midcycle increase in ovarian glucose uptake is associated with enhanced expression of glucose transporter 3. Possible role for interleukin-1, a putative intermediary in the ovulatory process. J Clin Invest 99:2274–2283[Medline]
  8. Bird TA, Davies A, Baldwin SA, Saklatvala J 1990 Interleukin 1 stimulates hexose transport in fibroblasts by increasing the expression of glucose transporters. J Biol Chem 265:13578–13583[Abstract/Free Full Text]
  9. Vega C, Pellerin L, Dantzer R, Magistretti PJ 2002 Long-term modulation of glucose utilization by IL-1{alpha} and TNF-{alpha} in astrocytes: Na+ pump activity as a potential target via distinct signaling mechanisms. Glia 39:10–18[CrossRef][Medline]
  10. Fukuzumi M, Shinomiya H, Shimizu Y, Ohishi K, Utsumi S 1996 Endotoxin-induced enhancement of glucose influx into murine peritoneal macrophages via GLUT1. Infect Immun 64:108–112[Abstract]
  11. Fischereder M, Schroppel B, Wiese P, Fink M, Banas B, Schmidbauer S, Schlondorff D 2003 Regulation of glucose transporters in human peritoneal mesothelial cells. J Nephrol 16:103–109[CrossRef][Medline]
  12. Shikhman AR, Brinson DC, Valbracht J, Lotz MK 2001 Cytokine regulation of facilitated glucose transport in human articular chondrocytes. J Immunol 167:7001–7008[Abstract/Free Full Text]
  13. Brandt SJ, Bodine DM, Dunbar CE, Nienhuis AW 1990 Dysregulated interleukin 6 expression produces a syndrome resembling Castleman’s disease in mice. J Clin Invest 86:592–599
  14. Chajek-Shaul T, Barash V, Weidenfeld J, Friedman G, Ziv E, Shohami E, Shiloni E 1990 Lethal hypoglycemia and hypothermia induced by administration of low doses of tumor necrosis factor to adrenalectomized rats. Metabolism 39:242–250[CrossRef][Medline]
  15. Huijing F 1970 A rapid enzymic method for glycogen estimation in very small tissue samples. Clin Chim Acta 30:567–572[CrossRef][Medline]
  16. Chang HC, Lane MD 1966 The enzymatic carboxylation of phosphoenolpyruvate. II. Purification and properties of liver mitochondrial phosphoenolpyruvate carboxykinase. J Biol Chem 241:2413–2420[Abstract/Free Full Text]
  17. Burchell A, Hume R, Burchell B 1988 A new microtechnique for the analysis of the human hepatic microsomal glucose-6-phosphatase system. Clin Chim Acta 173:183–191[CrossRef][Medline]
  18. Ferre P, Leturque A, Burnol AF, Penicaud L, Girard J 1985 A method to quantify glucose utilization in vivo in skeletal muscle and white adipose tissue of the anaesthetized rat. Biochem J 228:103–110[Medline]
  19. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  20. Shelly LL, Lei KJ, Pan CJ, Sakata SF, Ruppert S, Schutz G, Chou JY 1993 Isolation of the gene for murine glucose-6-phosphatase, the enzyme deficient in glycogen storage disease type 1A. J Biol Chem 268:21482–21485[Abstract/Free Full Text]
  21. Yajima T, Yagihashi A, Kameshima H, Kobayashi D, Furuya D, Hirata K, Watanabe N 1998 Quantitative reverse transcription-PCR assay of the RNA component of human telomerase using the TaqMan fluorogenic detection system. Clin Chem 44:2441–2445[Abstract/Free Full Text]
  22. Zar J 1974 Biostatistical analysis. Engelwood Cliffs, NJ: Prentice Hall
  23. Shepherd PR, Kahn BB 1999 Glucose transporters and insulin action–implications for insulin resistance and diabetes mellitus. N Engl J Med 341:248–257[Free Full Text]
  24. Dinarello CA 1996 Biologic basis for interleukin-1 in disease. Blood 87:2095–2147[Abstract/Free Full Text]
  25. Antunes G, Evans SA, Lordan JL, Frew AJ 2002 Systemic cytokine levels in community-acquired pneumonia and their association with disease severity. Eur Respir J 20:990–995[Abstract/Free Full Text]
  26. van Deuren M, van der Ven-Jongekrijg J, Vannier E, van Dalen R, Pesman G, Bartelink AK, Dinarello CA, van der Meer JW 1997 The pattern of interleukin-1ß (IL-1ß) and its modulating agents IL-1 receptor antagonist and IL-1 soluble receptor type II in acute meningococcal infections. Blood 90:1101–1108[Abstract/Free Full Text]
  27. Yilmaz M, Kendirli SG, Altintas D, Bingol G, Antmen B 2001 Cytokine levels in serum of patients with juvenile rheumatoid arthritis. Clin Rheumatol 20:30–35[CrossRef][Medline]
  28. Faggioni R, Fantuzzi G, Gabay C, Moser A, Dinarello CA, Feingold KR, Grunfeld C 1999 Leptin deficiency enhances sensitivity to endotoxin-induced lethality. Am J Physiol 276:R136–R142
  29. Zambre Y, Van Schravendijk C, Ling Z 2001 Interleukin-1ß inhibits proinsulin conversion in rat ß-cells via a nitric oxide-dependent pathway. Horm Metab Res 33:639–644[CrossRef][Medline]
  30. Yelich MR 1990 In vivo endotoxin and IL-1 potentiate insulin secretion in pancreatic islets. Am J Physiol 258:R1070–R1077
  31. Parker JC, VanVolkenburg MA, Levy CB, Martin WH, Burk SH, Kwon Y, Giragossian C, Gant TG, Carpino PA, McPherson RK, Vestergaard P, Treadway JL 1998 Plasma glucose levels are reduced in rats and mice treated with an inhibitor of glucose-6-phosphate translocase. Diabetes 47:1630–1636[Abstract]
  32. Combs TP, Berg AH, Obici S, Scherer PE, Rossetti L 2001 Endogenous glucose production is inhibited by the adipose-derived protein Acrp30. J Clin Invest 108:1875–1881[CrossRef][Medline]
  33. Ueki K, Kondo T, Kahn CR 2004 Suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3 cause insulin resistance through inhibition of tyrosine phosphorylation of insulin receptor substrate proteins by discrete mechanisms. Mol Cell Biol 24:5434–5446[Abstract/Free Full Text]
  34. Kamohara S, Burcelin R, Halaas JL, Friedman JM, Charron MJ 1997 Acute stimulation of glucose metabolism in mice by leptin treatment. Nature 389:374–377[CrossRef][Medline]
  35. Metzger S, Goldschmidt N, Barash V, Peretz T, Drize O, Shilyansky J, Shiloni E, Chajek-Shaul T 1997 Interleukin-6 secretion in mice is associated with reduced glucose-6-phosphatase and liver glycogen levels. Am J Physiol 273:E262–E267
  36. Samih N, Hovsepian S, Notel F, Prorok M, Zattara-Cannoni H, Mathieu S, Lombardo D, Fayet G, El-Battari A 2003 The impact of N- and O-glycosylation on the functions of Glut-1 transporter in human thyroid anaplastic cells. Biochim Biophys Acta 1621:92–101[Medline]
  37. Ahmed N, Berridge MV 1999 N-glycosylation of glucose transporter-1 (Glut-1) is associated with increased transporter affinity for glucose in human leukemic cells. Leuk Res 23:395–401[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
A. del Rey, E. Roggero, A. Randolf, C. Mahuad, S. McCann, V. Rettori, and H. O. Besedovsky
IL-1 resets glucose homeostasis at central levels
PNAS, October 24, 2006; 103(43): 16039 - 16044.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
E. Somm, E. Henrichot, A. Pernin, C. E. Juge-Aubry, P. Muzzin, J.-M. Dayer, M. J.H. Nicklin, and C. A. Meier
Decreased Fat Mass in Interleukin-1 Receptor Antagonist-Deficient Mice: Impact on Adipogenesis, Food Intake, and Energy Expenditure
Diabetes, December 1, 2005; 54(12): 3503 - 3509.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
145/11/5150    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
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 Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Metzger, S.
Right arrow Articles by Chajek-Shaul, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Metzger, S.
Right arrow Articles by Chajek-Shaul, T.

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