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Endocrinology Vol. 143, No. 6 2233-2241
Copyright © 2002 by The Endocrine Society

GROWTH FACTORS-CYTOKINES-ONCOGENES

Protein Calorie Restriction Affects Nonhepatic IGF-I Production and the Lymphoid System: Studies Using the Liver-Specific IGF-I Gene-Deleted Mouse Model

Wilson Mejia Naranjo, Shoshana Yakar, Myriam Sanchez-Gomez, Adriana Umana Perez, Jennifer Setser and Derek LERoith

Clinical Endocrinology Branch (S.Y., J.S., D.L.), National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-1758; and Department of Chemistry (W.M.N., M.S.-G., A.U.P.), National University of Colombia, Bogotá, Cundinamarca, Colombia

Address all correspondence and requests for reprints to: Derek LeRoith, M.D., Ph.D., Chief, Clinical Endocrinology Branch, Room 8D12, Building 10, National Institutes of Health, MSC 1758, Bethesda, Maryland 20892-1758. E-mail: . derek{at}helix.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nutritional status is a critical factor that modulates the responsiveness of the liver to GH and the resulting production of endocrine (mostly liver-derived) IGF-I. Using a conditional Cre/lox P system, we have established a liver-specific IGF-I-deficient mouse model. Despite the reduction in the circulating IGF-I (75%), the growth parameters are normal, except for the reduced spleen size, providing a unique model to study the effect of protein restriction on the autocrine/paracrine GH/IGF-I axis. To determine the effects of protein calorie malnutrition on the spleen, liver-specific IGF-I-deficient mice were assigned to one of four isocaloric diets, differing in the protein content (20, 12, 4, and 0%), for a period of 10 d. A low protein intake decreased the nonhepatic IGF-I secretion into the circulation, whereas it caused an increase in the level of circulating GH. This supports the view that nonhepatic IGF-I production contributes to circulating IGF-I levels. The lack of dietary protein led to an up-regulation of GH and IGF-I receptors expression in the spleen, whereas the IGF-I mRNA remained unchanged, as was demonstrated by flow cytometry and ribonuclease protection assay. B lymphocytes seem to be responsible for the up-regulated GH/IGF-I receptor expression. Northern blot analysis showed an up-regulation of IGF-binding protein-3 mRNA levels, which suggests that the protein deprivation may lead to an increased sequestration of circulating or locally synthesized IGF-I. These results support the hypothesis that the splenic GH/IGF-I axis responds to the nutritional stress caused by a low protein intake, to maintain the tissue homeostasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH AND IGF-I BOTH stimulate somatic growth and development and regulate various physiological functions (1). GH has both direct and indirect effects on body growth and metabolism. The direct effects of GH are mediated by the GH receptor (GHR) and are thought to be independent of IGF-I (2). The indirect actions of GH are mediated by IGF-I, which is produced in most tissues (3). Both circulating and locally produced GH (4) and IGF-I regulate the effects of IGF-I on growth (5, 6).

Under normal conditions, GH and IGF-I are bound in the circulation to specific binding proteins (7). GH is bound to a high-affinity GH-binding protein, and most of the circulating IGF-I is bound as a 150-kDa ternary complex that includes IGF-I, IGF-binding protein (IGFBP)-3, and an acid labile subunit. The remaining IGF-I circulates either free or bound in a binary complex with one of five other IGFBPs (8).

Nutritional status (i.e. dietary protein and/or energy intake) is a critical element in regulation of the GH/IGF-I system (9). In rats, dietary restriction of protein intake leads to a decrease in circulating levels of GH (10, 11), GH-binding protein (12, 13), IGF-I (14), IGFBP-3, and acid labile subunit (11, 15), whereas IGFBP-1 and IGFBP-2 levels are increased in response to reduced protein intake (16, 17). In contrast, in humans, protein deprivation causes a marked decrease in circulating IGF-I levels and an increase in GH secretion (18). These changes in protein levels are accompanied by similar changes in the corresponding hepatic mRNAs (19). Liver is the major site of IGF-I production and secretion into the circulation (20). In a previous study, we generated liver-specific IGF-I-deficient (LID) mice using a conditional Cre/lox P system (21). LID mice have markedly reduced serum levels of IGF-I (25% of IGF-I levels in control mice) (22). Despite this reduction in circulating IGF-I levels, postnatal growth and development in these animals is normal. Body weight and length measurements were not different between control and LID mice. Comparison of organ weight showed no difference between LID and control mice. However, splenic size was reduced by 20% in the LID mice, compared with controls (20, 22).

It has been established that protein malnutrition modulates the responsiveness of the liver to GH and the resulting production of endocrine (liver-derived) IGF-I (23, 24). In the present study, we have focused on the effects of protein-calorie malnutrition on the autocrine/paracrine somatotropic IGF-I axis. The LID mouse provides a unique model to study the effects of dietary protein restriction on the local GH/IGF-I axis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and dietary protocol
Control and LID mice were generated using the Cre/loxP system, as described previously (21, 22). In brief, both control and LID mice express the exon 4 of the igf-1 gene flanked by two lox/P sites. In addition, the LID mice also express the cre recombinase transgene exclusively in the liver, driven by the albumin enhancer/promoter sequence. Genotyping was carried out by PCR, as reported previously (21). Mice were weaned at postnatal d 21 and given a diet containing 12%-protein (i.e. 12 g crude protein per 100 g food pellets; ICN Biomedicals, Inc., Aurora, OH) for 1 wk. After this period, control and LID mice were randomly divided into four groups, with diets including either 0, 4, 12, or 20% protein for a period of 10 d (ICN Biomedicals, Inc.). These four diets are isocaloric, and each provides 3.7 kcal/g. Mice were housed three per cage and subjected to a 12-h light, 12-h dark cycle at a constant temperature (23 C). The animals had free access to water. Body weight was measured every third day. At the end of the study, mice were anesthetized using 100 µl of 2.5% Avertin, bled via the retroorbital vein, and the spleen was quickly removed. The animal protocol was approved by the Animal Care and Use Committee of the NIDDK, NIH.

Flow cytometric analysis
Spleens were removed from exsanguinated mice as described above. A single cell suspension of splenocytes was prepared in HBSS. The cells were washed, and red blood cells were lysed using PharM Lyse lysis solution (BD PharMingen, San Diego, CA). Splenocytes were resuspended in PBS containing 0.3% BSA and 0.1% sodium azide. Cell viability was determined by trypan blue exclusion using a hemocytometer. The following monoclonal antibodies (mAb) were purchased from BD PharMingen and used in these studies: phycoerythrin (PE)-conjugated rat antimouse CD4, B220, an IgG1 isotype control; Cy-chrome-rat antimouse CD8a (Ly-2); and an IgG2a isotype control. The CD4 mAb was used to identify helper T cells, the CD8 mAb was used to identify suppressor/cytotoxic T cells, and the B220 mAb was used to identify B cells.

Fluorescein labeling.
Recombinant rat (rr)GH was obtained from the National Hormone and Peptide Program, NIDDK (Torrance, CA). Recombinant human (rh)IGF-I was obtained from Genentech, Inc. (South San Francisco, CA). rrGH or rhIGF-I was conjugated to Fluos [5(6)-carboxyfluorescein-N-hydrosuccinimide ester] using a fluorescein labeling kit (Roche Molecular Biochemicals, Mannheim, Germany). Briefly, 1 mg hormone was dissolved in 1 ml 100-mM sodium bicarbonate buffer at a pH of 8.5. Fluos was dissolved in dimethylsulfoxide at 20 mg/ml, and 10 µl of this solution was added to the dissolved hormone. The resulting solution was gently stirred for 2 h, at room temperature, in the dark. Free Fluos was removed by gel filtration with Sephadex G-25 (Amersham Pharmacia Biotech, Uppsala, Sweden) in PBS with 0.1% sodium azide. The molar ratio of Fluos:protein was approximately 10:1. This ratio results in the incorporation of approximately 3–5 molecules of Fluos per molecule rhGH or rhIGF-I. BSA-Fluos was used as a control, with a similar molar ratio of Fluos:protein. The hormone-Fluos conjugate was mixed 1:1 with glycerol and stored at -20 C. The specificity of Fluos-rhIGF-I and Fluos-rrGH binding to the IGF-I receptor (IGF-IR) and GHR, respectively, was confirmed using a fibroblast cell line (NIH-3T3, B3) that overexpresses the IGF-IR and the IM-9 lymphocyte cell line that expresses the GHR. The total binding of Fluos-rhIGF-I to B3 cells was 98%, and that of Fluos-rrGH to the IM-9 cells corresponded to 85%. A 100-fold excess of unlabeled rhIGF-I inhibited the binding of the Fluos-rhIGF-I to B3 cells by 75%. A 100-fold excess of unlabeled rrGH inhibited the binding of Fluos-rrGH by 80% to IM-9 cells. A 100-fold excess of both hormones added to splenic lymphocytes cells inhibited the binding of both ligands by 80%. Blocking of the Fc with rat antimouse CD16/CD32 was performed by incubating 30 min before immunostaining. Splenocytes (1 x 106 cells) were colabeled with either PE-anti CD4, Cy-chrome CD8, or the corresponding isotype control antibody and with either Fluos-rGH, Fluos-rhIGF-I, or Fluos-BSA in PBS containing 0.3% BSA and 0.1% sodium azide. Hormones and BSA were used at a concentration of 1 µg/tube. Dual color staining was performed in the presence of PE-conjugated-anti-B220 or the corresponding isotype control antibody and with Fluos-rGH, Fluos-rhIGF-I, or Fluos-BSA. Cells were then washed twice and resuspended in PBS containing 1% paraformaldehyde. Cell acquisition was performed in a fluorescence-activated cell-sorter scan (FACS) flow cytometer (FACScan, Becton Dickinson and Co., Mountain View, CA), and a minimum of 50,000 events were acquired for each test. Data were analyzed using CellQuest software (Becton Dickinson and Co.).

Hormone determinations
Blood was collected from the retroorbital sinus at the end of the study. Serum IGF-I and GH levels were measured by RIA (rat/mouse IGF-I assay system and GH system, National Hormone and Pituitary Program, Harbor-UCLA Medical Center, Torrance, CA, kindly provided by Dr. A. F. Parlow). Serum insulin levels were determined using a highly sensitive rat insulin RIA kit (Linco Research, Inc., St. Charles, MO).

SDS-PAGE and Western ligand binding assay
Serum IGFBP levels were measured after 10 d on the diet. Samples containing 2 µl serum were mixed with 2 µl 2x nonreducing SDS protein gel loading solution (Quality Biological Inc., Gaithersburg, MD). Samples were subjected to SDS-PAGE on 4–20% gradient gels (Novex, San Diego, CA). Proteins were transferred to nitrocellulose membranes (0.2 µm) using standard electroblotting methods. Membranes were blocked with TBS containing 1% BSA and incubated with 1.5 x 106 cpm [125I]-IGF-I (Amersham Pharmacia Biotech, Chicago, IL) overnight, at 4 C, in TBS with 0.1% Tween-20. Membranes were washed three times with TBS containing 0.1% Tween-20 and exposed to PhosphorImager screens (Fuji Photo Film Co., Ltd., Kanagawa, Japan). Levels of IGFBP-3 and IGFBP-1 were determined by quantifying the levels of [125I]-IGF-I incorporated into bands corresponding to 40–45 kDa and 25–28 kDa, respectively.

Ribonuclease (RNase) protection assay
Tissues were homogenized with a polytron homogenizer (Brinkmann Instruments, Inc., Wesbury, NY) in RNAzol reagent (Tel-Test, Friendswood, TX), and total RNA was isolated according to the manufacturer’s instructions. Samples containing 50 µg total RNA were hybridized with 32P-labeled IGF-I exon 4 (21), IGF-IR exon 3, GHR exon 4 (BamHI/Ava fragment) (25), and ß-actin (Ambion, Inc., Austin, TX) riboprobes. Protected bands were separated on 8% polyacrylamide gels and exposed to OX-omat AR film (Eastman Kodak, Rochester, NY) overnight. The relative signals of the protected bands were quantified using a Fuji Photo Film Co., Ltd. PhosphorImager and normalized to the levels of 32P incorporated into the ß-actin-protected bands.

Northern blot analysis
Samples containing 50 µg RNA were separated by electrophoresis in a 0.8% (wt/vol) agarose gel containing 5% formaldehyde, transferred to a 0.2-µm nylon Nytran membrane (Schleicher \|[amp ]\| Schuell, Inc., Keene, NH). The membranes were prehybridized for 2 h at 42 C, and messenger RNA expression was analyzed by hybridization of Northern blots with [{alpha}32P]-deoxycytidine triphosphate-labeled DNA probes specific for IGFBP-1, -2, -3, -4, -5, and -6 (kindly provided by Dr. John Pintar) and with [{alpha} 32P]deoxyuridine 5-triphosphate-labeled ß-actin riboprobe (Ambion, Inc.). The quantification of the hybridization signals were performed using a Fuji Photo Film Co., Ltd. PhosphoImager, normalized to the levels of the 2.1-kb 32P incorporated into the ß-actin band.

Statistics
Statistical analysis was performed using the two-tailed t test (SigmaStat 2.03; Access Softek, Inc., San Rafael, CA). Values were considered to be statistically significantly different when P values were less than 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Body weight and food intake
The effect of 10 d of restricted dietary protein content on body weight was similar in LID and control mice. Thus, both LID and control mice fed 0- and 4%-protein diets each lost approximately 4 g and 1.5 g of body weight, respectively (Fig. 1AGo). Fed 12- and 20%-protein diets, both LID and control mice gained approximately 4 g and 5 g of body weight, respectively (Fig. 1Go). Mice fed a 0- or 4%-protein diet consumed significantly less food than did mice fed a 12- or 20%-protein diet (Table 1Go). However, the animals that received the 0%-protein diet ate significantly more food per gram of body weight than did animals that received 12- and 20%-protein diets (Table 1Go). Nevertheless, this relative increase in energy intake did not support normal growth (Fig. 1AGo). Thus, the 0- and 4%-protein diets induced protein deficiency, but not energy deficiency, because these mice consumed an equal or greater number of calories than did mice on 12- and 20%-protein diets.



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  		Figure 1. Effect of dietary protein restriction on body weight and splenic weight. Control mice (open bars) and LID mice (black bars) were fed diets consisting of either 0, 4, 12, or 20% protein for 10 d, as indicated. A, Data for body weight are expressed as average body weight gain ± SEM, in grams, from n = 6 mice in each group. B, Splenic weight data are expressed as average spleen weight/body weight ± SEM, from n = 6 mice in each group. a, LID mice vs. control mice, P < 0.01.

 

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  		Table 1. Nutritional status of LID and control mice

 
Spleen size
The spleen weights in control and LID mice were correlated with the relative protein content in their diets. In control mice, spleen weights fell from 60.6 ± 0.8 mg in mice fed 20%-protein diets, to 19.6 ± 7.4 mg in mice fed 0%-protein diets. A similar trend was seen in LID mice. Spleen weights fell from 39.5 mg ± 7.7 in mice fed 20%-protein diets, to 13.2 ± 3.4 mg in mice fed 0%-protein diets. When normalized to body weight, the spleen/body weight ratio was consistently significantly lower in LID mice than in control mice, across all four dietary conditions, as shown in Fig. 1BGo. This difference most likely reflects the lower circulating IGF-I levels in LID mice, compared with control mice, as previously described (22).

Serum IGF-I levels
Serum IGF-I levels decreased in response to decreasing the dietary protein content in both control and LID mice, as shown in Fig. 2AGo. IGF-I levels were significantly lower in LID mice fed 4-, 12-, and 20%-protein diets, compared with control mice that received these diets. There was no significant difference in serum IGF-I levels between control and LID mice fed 0%-protein diets; and in these animals, IGF-I levels were very low (<5 ng/ml). Of particular note was the reduction in serum IGF-I levels in LID mice fed 0- and 4%-protein diets.



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  		Figure 2. Effect of dietary protein restriction on serum IGF-I (A), GH (B), insulin (C), IGFBP-3 (D), and IGFBP-1 (E) levels. Control mice (open bars and symbols) and LID mice (closed bars and symbols) were fed diets consisting of 0, 4, 12, or 20% protein for 10 d, as indicated. In A and C, data are expressed as average serum IGF-I levels or serum insulin levels ± SEM (ng/ml) from n = 10–12 mice in each group. In B, the data points indicate individual serum GH levels (ng/ml) from n = 12–18 mice in each group; 2.3, 5.4, 6.4, and 84 ng/ml are the mean values for control mice fed 20-, 12-, 4-, and 0%-protein diets, respectively; 8.7, 14.2, 33, and 73 ng/ml are the mean values for LID mice fed 20-, 12-, 4-, and 0%-protein diets, respectively. IGFBP-3 levels (D) or IGFBP-1 levels (E) are expressed as average fold increase over control ± SEM (ng/ml) from n = 9–13 mice in each group. a, Control vs. LID mice with the same diet, P < 0.01; b, control mice fed 20%-protein diet vs. control mice fed 12-, 4-, or 0%-protein diets, P < 0.01; c, LID mice fed 20%-protein diets vs. LID mice fed 12-, 4-, or 0%-protein diets, P < 0.001.

 
Serum GH levels
Serum GH levels increased in response to decreasing the dietary protein content in both control and LID mice, as shown in Fig. 2BGo. In the groups that received the 4-, 12-, and 20%-protein diets, serum GH levels were significantly higher in LID mice than in control mice. However, no statistical differences were observed between control and LID groups in the mice that received 0%, possibly because of the large variance in serum GH levels between individual mice under protein deprivation.

Serum insulin levels
Similar to IGF-I levels, serum insulin levels decreased in response to decreasing the dietary protein content, in both control and LID mice, as shown in Fig. 2CGo. However, serum insulin levels did not differ significantly between control and LID mice in the groups that received 0%-protein diets. Insulin levels were significantly higher in the LID mice fed 4-, 12-, and 20-protein diets, compared with control mice fed the same protein diets (Fig. 2CGo). Thus, protein restriction caused a reduction in serum insulin levels both in LID and control mice.

Serum IGFBP-3 and IGFBP-1 levels
Figure 2DGo shows the effect of dietary protein restriction on serum IGFBP-3 levels. Control mice exhibited a decrease in serum IGFBP-3 levels as dietary protein content was reduced from 20 to 0%. IGFBP-3 levels also generally decreased in LID mice as the dietary protein content decreased. In mice that were given 20-, 12-, 4-, and 0%-protein diets, IGFBP-3 levels were significantly lower in LID mice than in control mice. Fig. 2EGo shows the levels of IGFBP-1 in serum. IGFBP-1 levels were generally decreased in LID mice, compared with control mice, and increased in response to decreasing the dietary protein content in control mice. Mice that were fed the 0%-protein diet were an exception to this tendency (i.e. control and LID mice exhibited similar levels of IGFBP-1 on this diet).

Splenic IGFBP’s mRNA expression
IGFBP-3 mRNA expression increased in response to a reduction in the dietary protein content in both control and LID mice, as shown in Fig. 3AGo. IGFBP-3 mRNA levels were significantly higher in LID mice fed 0%-protein diet, compared with control mice fed 0%-protein diets and compared with LID mice fed 12- and 20%-protein diets. Similarly, IGFBP-3 mRNA levels were significantly lower in control mice fed 20%-protein diets, compared with control mice fed 4- and 0%-protein diets. In contrast, IGFBP-2 mRNA expression was significantly lower in LID and control mice fed 0%-protein diet, compared with LID or control mice fed 4-, 12-, or 20%-protein diets. There were no significant differences between LID and control groups fed the same content of protein diets. IGFBP-4 mRNA levels were lower in LID mice, compared with controls (P < 0.05), and increased in both groups on a 0%-protein diet (data not shown). IGFBP-6 levels were extremely low, but higher in control mice than in LID mice, at all levels of dietary protein (data not shown). IGFBP-1 and IGFBP-5 mRNA expression was not detected in the spleen.



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  		Figure 3. IGFBPs expression in spleen. Control mice (open bars) and LID mice (black bars) were fed diets consisting of either 0, 4, 12, or 20% protein for 10 d, as indicated. Expression of splenic IGFBP-3 is increased in response to protein malnutrition (A). Expression of IGFBP-2 is decreased in response to protein deprivation (B). mRNA levels were analyzed by Northern blot and normalized to the levels of ß-actin mRNA. Data are expressed as average ± SEM, from n = 6–8 mice in each group. a, Control vs. LID mice with the same diet, P < 0.01; b, control mice fed 20%-protein diet vs. control mice fed 12-, 4-, or 0%-protein diets, P < 0.01; c, LID mice fed 20%-protein diets vs. LID mice fed 12-, 4-, or 0%-protein diets, P < 0.001.

 
Relative distribution of splenic lymphocytes
FACS analysis was used to determine the cellular distribution of B cells, CD4+ T cells, and CD8+ T cells in the spleens of control and LID mice fed various diets (Table 2Go). The percentage of B cells in the spleens of mice fed 4-, 12-, or 20%-protein diets did not differ between LID and control groups. However, in mice that were fed 0%-protein diets, the percentage of B cells in the control mice was significantly less than that in the LID mice (31 vs. 39%, respectively). Furthermore, the average percentage of splenic B cells tended to increase as the amount of dietary protein was increased (Table 2Go). In contrast, the percentage of splenic CD4+ and CD8+ T cells tended to drop in both control and LID mice as the amount of dietary protein was increased. For 0%-protein diets, we observed significantly higher levels of CD4+ T cells in control mice vs. LID mice, although similar levels of CD4+ T cells were observed in control and LID mice that were fed 4-, 12-, or 20%-protein diets. The percentage of CD8+ T cells was similar in control and LID mice. In control mice fed 12- and 20%-protein diets, the percentage of CD8+ T cells was significantly lower than in control mice fed 0- and 4%-protein diets (P < 0.02).


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  		Table 2. Lymphocyte subpopulations distribution in the spleen

 
GHR, IGF-IR, and IGF-I expression in splenic lymphocytes
Among the different subsets of lymphocytes, B cells were the most responsive to changes in dietary protein in both LID and control mice. Figure 4AGo shows the percentage of B cells that were immunoreactive for GHR (GHR+). Control and LID mice fed a 0%-protein diet exhibited a 2.5- to 3-fold increase in the percentage of GHR+ B cells, compared with mice fed diets containing 4, 12, or 20% protein. Of the animals that received the 0%-protein diet, LID mice had a significantly higher percentage of GHR+ B cells in the spleen than did control mice. A similar effect of dietary protein restriction was observed on splenic GHR mRNA levels, as determined by RNase protection assay (Fig. 4CGo). GHR mRNA levels were highest in LID and control mice that received a 0%-protein diet; in mice fed the other diets, GHR mRNA levels were consistently about 60% lower. These findings are consistent with the hypothesis that a reduction in serum IGF-I levels, which directly correlate with dietary protein content, results in an increase in circulating GH and up-regulation of GHR expression in B cells. Similarly, a reduction in dietary protein content increased IGF-IR expression in B cells. Control and LID mice on 0%-protein diets expressed approximately 2-fold higher levels of IGF-IR immunoreactive (IGF-IR+) B cells than did mice on 4-, 12-, or 20%-protein diets (Fig. 4BGo). Similar results were seen with IGF-IR mRNA levels (Fig. 4EGo). Across all four diets, the levels of IGF-I mRNA were not different between LID and control mice (Fig. 4DGo). Thus, dietary protein does not significantly affect the expression of splenic IGF-I mRNA.



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  		Figure 4. Effect of dietary protein restriction on the relative number of B cells that express the IGF-IR and GHR. Splenocytes were isolated as described, and the number of B cells immunolabeled for the GHR or IGF-IR was determined by FACS (A and B). GHR (C), IGF-IR (D), and IGF-I (E) mRNA levels were determined by RNase protection assay normalized to the levels of ß-actin mRNA. Data are expressed as average ± SEM, from n = 6 mice in each group (a, vs. control mice with the same diet, P < 0.02; b, vs. control mice fed 12- or 20%-protein diets, P < 0.01; c, vs. LID mice fed 12- or 20%-protein diets, P < 0.001).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The aim of this study was to determine the effect of protein-calorie malnutrition on the autocrine/paracrine IGF-I system, using the LID mice model. The LID mice provide a unique physiological model, in which IGF-I production is abrogated specifically in the liver. This genetic manipulation results in a 75% reduction in circulating IGF-I levels, compared with control mice (20, 22). We have focused our investigations on splenic lymphocytes because the spleen was the only organ that exhibited a significant, albeit modest, size difference in LID mice, compared with controls.

Protein malnutrition (induced by feeding animals diets containing 0 and 4% protein) decreased serum IGF-I levels in both control and LID mice. Thus, dietary protein content can regulate the levels of circulating IGF-I in LID mice, which is presumably derived from nonhepatic tissues. Previous studies have demonstrated that nutritional status is a major determinant of IGF-I gene expression in both hepatic and nonhepatic tissues (26, 27, 28). Various tissues respond to nutritional alterations differently, possibly reflecting different needs for IGF-I or changes in other regulatory factors such as GH, insulin, and cortisol, which affect IGF-I gene expression (29, 30). One tissue that could contribute to the nonhepatic circulating IGF-I is skeletal muscle, which is sensitive to changes in nutritional status. A low-protein diet has been shown to significantly reduce IGF-I mRNA and protein levels in the gastrocnemius muscle (31). Other possible sources of circulating IGF-I from nonhepatic tissues include adipocytes that express high levels of IGF-I mRNA.

Changes in circulating levels of IGF-I are known to affect GH secretion from the pituitary and circulating GH levels (32, 33). Interestingly, in our studies, protein malnutrition had a significant effect on GH levels in both control and LID mice, compared with normal diets. In animals fed a 0%-protein diet, serum IGF-I levels were reduced, and GH levels were correspondingly elevated. Two hypothalamic neuropeptides (GHRH and its antagonist, somatostatin) regulate GH secretion from the pituitary (34). The profile of GH release in males is characterized by high amplitude secretory episodes every 3.3 h and low interpulse levels of GH (35). A lack of dietary protein has been shown to blunt spontaneous pulsatile GH release, attributable (in part) to an excess of somatostatin and a decrease in GHRH secretion (36). Dietary protein restriction also attenuates GH responsiveness to GHRH challenge and reduces pituitary size and GH content in the rat (37). Serum GH levels decline when food intake is decreased (11). In mice, however, the effect of food deprivation on GH secretion has not been extensively studied. According to the results of the present study, nutritional regulation of the GH/IGF-I axis in mice seems to be more closely related to that in humans, rather than in rats. Our previous studies in LID mice showed that these animals exhibited a significant increase in the levels of circulating GH (22). This effect might be attributable, in part, to the lack of an inhibitory feedback mechanism because of lower circulating IGF-I levels.

Suprisingly, control mice that were fed diets consisting of 4% protein exhibited lower serum IGF-I levels in the absence of increased serum GH levels, compared with control mice fed 12- and 20%-protein diets. This may reflect the fact that we made only single measurements of GH for each mouse, and a pulsatile pattern of GH secretion (vida supra) has been well established. On the other hand, LID mice fed 4%-protein diets did show elevated GH levels, presumably because of the reduction in IGF-I levels.

An equally important aim of this study was to determine the effect of circulating GH and IGF-I levels on the immune system. Numerous studies have shown that GH and IGF-I-induced proliferation and differentiation can be demonstrated in a wide variety of immune cells in culture (38). Similarly, in vivo studies have shown that rhGH and rhIGF-I have effects on the immune system (39). The weight of the spleen, relative to body weight, was modestly (but consistently) smaller in LID mice, compared with control mice, suggesting that circulating IGF-I is important for this effect. Furthermore, the elevated serum GH levels had no effect. As IGF-I levels decreased with reduced dietary protein content, the splenic weights also decreased, further suggesting a direct effect of circulating IGF-I on splenic size. In contrast, the relative proportion of lymphocytes within the spleen (i.e. the percentage of B or T cells) remained constant in mice that were fed 4-, 12-, or 20%-protein diets. On 0%-protein diets, mice exhibited a significant reduction in B cells and a proportional increase in the percentage of CD4+ T cells. This correlated with the extremely low levels of IGF-I and elevated GH levels in the circulation. To determine whether this effect was associated with changes in circulating hormone levels or with local changes, we also analyzed IGF-I, IGF-IR, IGFBPs, and GHR gene expression on splenic cells. The absence of change in splenic IGF-I mRNA levels suggested that the increased IGF-IR mRNA was a secondary response to reduced circulating levels of IGF-I, a finding consistent with previously reported relationships between serum IGF-I and tissue IGF-IR expression (26).

The increase in GHR levels in B cells may also be a secondary response to the elevated GH levels in the circulation in the mice that received 0%-protein diets. A similar effect has been previously described for GHR expression in muscle (40). Thus, circulating hormone levels apparently play a role in gene expression within lymphoid tissue, at least in the spleen. The exact roles played by these hormones on immune function are still under investigation. It also remains to be determined whether these changes in IGF-IR and GHR levels occur as a compensatory response to the lower circulating IGF-I levels.

According to our working hypothesis, splenic immune function is not regulated by dietary protein restriction. We have considered two possible mechanisms that might be involved in maintaining splenic homeostasis during the nutritional stress induced by low protein intake. First, the increased levels of both GHR mRNA and GH binding capacity induced by high levels of circulating GH could help to stabilize immune function. Second, the high levels of IGF-I binding capacity and IGF-IR and IGFBP-3 mRNA expression in conditions where circulating IGF-I is reduced may reflect a local production of IGF-I that could account for local anabolic actions. A locally up-regulated IGF-I system has been previously described in a study investigating the mechanism responsible for the suppression of growth plate function during inadequate calorie intake. This study showed that GHR mRNA and IGF-I mRNA levels were each increased, in the growth plate, by dietary restriction (41).

In view of the complexity of the IGF system, the potential contribution of IGFBPs in nutritional regulation of the GH/IGF-I axis in LID mice needs to be considered. It has been well documented that a low protein intake is associated with changes in IGFBPs (42, 43). Several studies have shown that IGFBP-1 gene expression is inhibited by insulin both in vivo and in vitro. In the present study, the levels of circulating IGFBP-1 were lower, when compared with their respective controls, on each diet. It is reasonable to assume that, in mice fed a diet consisting of 20% protein, the increased levels of insulin affected the circulating levels of IGFBP-1. At lower levels of protein intake, other factors may account for the regulation of IGFBP-1. In fact, in vitro, amino acid deprivation causes an increase in the levels of IGFBP-1 mRNA (44). In vivo, protein restriction increases IGFBP-1 levels in the rat liver (45, 46). In contrast, GH is known to have an inhibitory effect on IGFBP-1 expression (47). LID mice have high levels of GH because of a reduction in circulating IGF-I levels. The high GH levels were demonstrated to antagonize insulin action in peripheral tissues, such as muscle, and cause insulin insensitivity (48). This might cause the elevation in insulin levels in serum. Both of these factors might account for the decrease in levels of circulating IGFBP-1, which is negatively correlated with insulin levels in LID mice. This effect is worsened, at lower levels of protein intake, by the high levels of serum GH.

In summary, we have demonstrated that low protein diets have profound effects on the GH-IGF-I axis. In view of the role played by the GH-IGF-I axis in immune function, these changes would be predicted to alter the immune system functions. In this study, we did not specifically evaluate the immune function per se. However, our data suggest that the increased splenic IGFBP-3 and IGF-IR expression during protein malnutrition may recruit the circulating or locally produced IGF-I as a way to compensate for chronically low levels of IGF-I. This increase could also affect the homeostasis of the lymphocyte population and preclude dietary protein restriction from exerting major effects on immune function. Finally, the changes in circulating IGF-I levels in LID mice that are fed low-protein diets further supports the notion that nonhepatic sources of IGF-I do contribute to circulating (endocrine) IGF-I levels.


   Acknowledgments
 
We thank Dr. German Iniguez and Bernice Samuels for their help with the techniques; and Dr. Parlow, UCLA/National Pituitary Agency, for help in setting up the GH and IGF-I immunoassays.


   Footnotes
 
This work was supported by Colciencias, Colombia and the International Program in the Chemical Sciences (to W.M.N. and M.S.-G.), Uppsala University, Sweden.

Abbreviations: FACS, Fluorescence-activated cell sorter scan; GHR, GH receptor; IGFBP, IGF-binding protein; IGF-IR, IGF-I receptor; LID, liver-specific IGF-I-deficient; mAb, monoclonal antibody(ies); PE, phycoerythrin; rh, recombinant human; rr, recombinant rat; RNase, ribonuclease.

Received June 12, 2001.

Accepted for publication February 19, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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