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Endocrinology Vol. 140, No. 4 1841-1851
Copyright © 1999 by The Endocrine Society

ARTICLES

The Insulin-Sensitive Glucose Transporter (GLUT4) Is Involved in Early Bone Growth in Control and Diabetic Mice, But Is Regulated through the Insulin-Like Growth Factor I Receptor1

Gila Maor and Eddy Karnieli

Departments of Morphological Sciences (G.M.) and Endocrinology (E.K.), The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology; and Institute of Endocrinology, Diabetes and Metabolism (E.K.), Rambam Medical Center, 31096 Haifa, Israel

Address all correspondence and requests for reprints to: Dr. Eddy Karnieli, Institute of Endocrinology, Diabetes and Metabolism, Rambam Medical Center, P.O. Box 9602, 31096 Haifa, Israel. E-mail: eddy{at}rambam.health.gov.il


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Children with uncontrolled type I (insulin-dependent) diabetes mellitus are characterized by a slow growth rate, which improves upon adequate therapy. While skeletal growth is an energy-consuming process involving high glucose utilization, the role of glucose transporters (GLUT) and their regulation in the bone formation process are not yet fully understood. Thus, we studied both in vivo and in vitro early endochondral bone formation in control and streptozotocin-induced young diabetic mice. Using in situ hybridization and immunohistochemistry techniques, we demonstrated the novel existence of the insulin-sensitive glucose transporter (GLUT4), as well as GLUT1, in juvenile-derived murine mandibular condyles and in the humeral growth plate—two models for endochondral bone formation. Insulin-like growth factor (IGF) I receptors (IGF-I-R), but not insulin receptors (IR), were shown to have cellular distribution similar to GLUT4, being more abundant in mature chondrocytes. Further, in the skeletal growth centers of streptozotocin-induced diabetic mice, GLUT4, IGF-I, and IGF-I and insulin receptor levels, but not GLUT1, were markedly reduced. The decrease in GLUT4 and in IGF-I and insulin receptors was associated with severe histological changes in the mandibular condyles and humeral growth plate. Insulin therapy restored IR levels to normalcy, whereas IGF-I-R and GLUT4 levels were only partially recovered. Thus, GLUT4 and IGF-I-R have a potential role in early bone growth in mice. Further, during early bone growth GLUT4 may be regulated through the IGF-I receptor rather than via the insulin receptor. We propose that skeletal growth retardation in type I diabetes may be associated with reduced expression of the GLUT4 and IGF-I receptor in the bone growth center.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SOMATIC growth retardation that improves upon adequate therapy is characteristic of poorly controlled type 1 diabetes mellitus (IDDM) in children (1). Frequently, diminished growth velocity precedes IDDM (2). The skeletal growth process probably requires high glucose utilization rates because bone chondrocytes and cartilaginous growth centers accumulate large amounts of glycogen (3). While glucose transport is a rate-limiting step in adipose and muscle glucose metabolism, limited information exists about the role of glucose transporters in skeletal growth (4, 5).

In eukaryotic cells, glucose uptake is mediated by transmembrane glucose transporter (GLUT) proteins. Six closely related isoforms have been isolated and cloned, and their tissue specificity extensively characterized (6). While GLUT1 is the major isoform found in most cells, including immortalized and transformed cell lines, other GLUT isoforms show tissue-specific distribution (7). Normally, GLUT4 is exclusively expressed in insulin-responsive tissues, e.g. heart, skeletal muscle, and white and brown adipose tissues (8). In these tissues, insulin stimulates cellular glucose uptake by inducing the translocation of GLUT4 from an intracellular pool to the plasma membranes (9, 10, 11). Because they are involved in the first step of the glucose utilization cascade, GLUT4 proteins are highly regulated in physiological as well as pathophysiological states. Levels of regulation include gene transcription, protein synthesis, and degradation. Indeed, noninsulin-dependent diabetes mellitus and streptozotocin (STZ)-diabetic rats are associated with a decrease in cellular number and activity of GLUT4 (12, 13, 14, 15). The reduced gene expression of the GLUT4 isoform is probably regulated at the pretranslational level (16, 17).

In the preosteoblast cell lines, GLUT1 is regulated by insulin and is associated with an increase in 2-deoxyglucose uptake and alkaline phosphatase activity (4). However, there is no information about the involvement and regulation of GLUT4 (the insulin-sensitive glucose transporter) in the skeletal growth processes per se and in diabetes in particular. Thus, in the present study we examined GLUT1 and GLUT4 gene expression and regulation in juvenile-derived murine bone models, and their potential role in somatic growth retardation linked to diabetes.

Insulin and IGF-I receptors share a high degree of similarity, as well as many common components of their signal transduction pathways (18). Both hormones stimulate GLUT4 translocation and regulate glucose uptake in insulin-responsive tissues (as adipose and muscle tissues) as well as in various cell lines (13, 14, 15, 16, 17). These hormones also modulate GLUT4 messenger RNA (mRNA) level (16, 17, 19). Furthermore, both of these hormones, but not GH, play a major role during the very early growth phase (5, 20, 21, 22). An interrelationship between insulin and IGF-I can be assumed, based on various data. For example, insulin stimulates the chondrogenic process (20) and concomitantly induces IGF-I production in the cartilage growth plate (11). IGF-I is also involved in the skeletal growth retardation observed in diabetics. In IDDM children, inadequate blood sugar control correlates with low plasma IGF-I levels and sluggish growth (23). Following correction of hyperglycemia, IGF-I receptor and hormone plasma levels normalize. In diabetic rats, circulating IGF-I levels are reduced, whereas the expression of IGF-I and IGF-II-receptor genes is increased in the kidney (24, 25). However, the regulation of the IGF-I receptor gene in the diabetic bone is not yet fully understood.

In diabetic mice, a significant growth retardation is expressed as severe malformations in both mandibular condyle and humeral growth plate. The pivotal role of GLUT4 in glucose homeostasis; the fact that bone growth can be modulated by insulin, IGF-I and diabetes; and the potential common origin of the bone and muscle (26) led us to examine the hypothesis that GLUT4 may have an important role in the bone growth process. We used the early bone growth model for this purpose. Indeed, our novel findings described below support the hypothesis that GLUT4 as well as the IGF-I receptor play an important role in growth of bone–a nonclassical insulin-sensitive tissue.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
STZ-induced diabetic mice and insulin replacement: in vivo studies
Six- to 8-day-old ICR mice were injected ip with either STZ, 120 mg/kg, or citrate buffer alone. Mice were kept with their mothers. All experimental groups (STZ and control) contained the same number of offspring per feeding mother. Blood sugar levels and weight were monitored daily. Only those mice that developed hyperglycemia > 12 mmol were included in the study. Forty-eight hours post STZ the diabetic mice were further subgrouped into nontreated and insulin-treated diabetic mice. The latter group was treated daily with sc 8 mU/g body weight of insulin (Humulin N: human recombinant insulin, Eli Lilly France S.A., Fegersheim, France) for additional 5 days aiming at achieving normoglycemia (< 7 mmol). Higher insulin doses induced severe hypoglycemia. Control and STZ-induced diabetic mice were killed 7, 10, and 15 days after STZ injection. Insulin-treated STZ animals were killed only at 7 days post STZ injection. Mandibular condyles and humeral bones were removed as previously described (20, 21) and processed according to the specific experimental aim. The protocol was approved by the Animal Protection Committee of the Technion-Israel Institute of Technology.

Tissue preparation
Mandibular condyles from ICR mice were prepared and processed by one of the following methods:

General morphological studies. Explants were fixed in buffered glutaraldehyde, postfin OsO4 in graduated ethanol, and embedded in Epon 812. Sections 1 µm thick were then stained with toluidine blue.

Morphology and morphometric studies. Paraffin sections (6 µm) were deparaffinized in xylene, hydrated in graduated ethanols, and stained in hematoxylin-eosin. Stained sections served for morphometric studies. Histomorphometric determinations of the length of various cellular cells layers were performed using an Olympus Corp. Cue-2 image analysis system with appropriate morphometry software (Olympus Corp., Lake Success, NY), a Zeiss Universal R photomicroscope fitted with a Panasonic WV-CD50 video camera, and an IBM-compatible PC.

In situ hybridization studies. Explants were immediately frozen in liquid nitrogen and kept at -70 C until reaction. Cryosections, 1 µm thick, were mounted on precleaned/polylysine-coated slides. Sections were air-dried and prefixed with 4% paraformaldehyde in PBS for 10 min at room temperature and immediately processed for in situ hybridization as described below.

Immunohistochemistry
Following standard protocols, 27 deparaffinized paraffin sections were reacted for 2 h at room temperature with specific antibody against either rabbit anti-GLUT1 and 4 (Chemicon International, Temecula, CA), rabbit anti-IGF-I receptor (anti-{alpha} subunit, catalog no. SC-712, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or rabbit antiinsulin receptor (anti-{alpha} subunit N-20, catalog no. SC-710, Santa Cruz Biotechnology, Inc.). Detection was checked by appropriate biotinylated second antibody with streptavidin-peroxidase conjugate and S-(2-aminoethyl)-L-cysteine (AEC) as substrate (Histostain-SP kit, Zymed Laboratories, Inc., San Francisco, CA). Counterstaining was done with hematoxylin.

Quantification of immunohistochemistry
Quantification of the immunohistochemistry results was performed using the same morphometric system described above and was expressed as the proportion of the positive-stained area from the overall cartilaginous size. We preferred to measure the positive area rather than to count positive cells since cellular populations are not evenly distributed throughout the growth center. Each point represents an average of 18 measurements obtained from: 3 sections of each tissue studied, from at least 2 animals of each experimental group (control, STZ and insulin treated STZ mice), derived from 3 separate experiments. Significance at P < 0.05 was determined using two tailed Student’s t test.

Antisense RNA probes for in situ hybridization
Antisense RNA probes were prepared for mouse IGF-I receptors cloned in pBluescript SK+ amp+, and for GLUT1 and GLUT4: HepG2-GLUT1 clone J-12 (AMPr-pGEM3/2.5 kb BamHI insert), kindly provided by Dr. H. Lodish, as well as for rat skeletal muscle GLUT4 clone pSM1–1-1 (AMPr-pBluescript KS+/2.5 kb EcoR-I insert), kindly provided by Dr. M. Birnbaum. After linearization, antisense RNA was transcribed using (Sp6/T7) Dig-RNA labeling kit (Boehringer Mannheim, Mannheim, Germany), following the company’s instructions.

In situ hybridization
Eight-micrometer frozen sections were loaded on precleaned poly-L-lysine-coated slides, prefixed for 5 min. in 4% paraformaldehyde (in 1 M PBS, pH 7.4), and acetylated in 0.1 M triethanolamine (pH 8.0) in 0.25% acetic anhydride. Prehybridization was performed by 10 min in 2 x SSC followed by 1 h in hybridization buffer composed of 50% formamide, 0.5 mg/ml salmon sperm DNA, 4 x SSC, and 1 x Denhardt’s reagent. Hybridization was conducted overnight (18 h) at 42 C in maximal humidity with 2–5 ng/µl digoxygenin < Dig >-labeled probe. Slides were covered with siliconized coverslips. At the end of incubation, slides were rinsed in SSC at increasing stringency conditions and then with 0.1 Tris and 0.15 M NaCl, pH 7.5. Hybrids were detected with anti-dig antibodies conjugated with fluorescein isothiocyanate (FITC) (Boehringer Mannheim).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Clinical characteristics
Compared with control ICR juvenile mice, ip injection of 120 mg/kg STZ resulted in stable plasma hyperglycemia (>12 mmol) and a significant reduction in body weight gain, as shown in Fig. 1Go. After 7 and 14 days the STZ-induced diabetic mice weighed approximately 75% and 60%, (P < 0.05) respectively, of the body weight of control mice. At the earlier days after STZ (days 2–4) only minimal changes were observed.



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  		Figure 1. A, Body weight of STZ-induced diabetic vs. control mouse. B, Appearance of diabetic mouse 14 days after administration of STZ. In comparison to the untreated control (bottom), the diabetic mouse (top) was much smaller, hairless, and had a distorted tail.

 
Morphological changes in diabetic skeletal growth centers
Skeletal growth centers were markedly affected in the STZ-induced diabetic mice. Compared with controls from the seventh day post STZ injection, 32% and 38% reduction in the overall sizes of the diabetic growth plate and the mandibular condyles, respectively, was observed (Fig. 2Go, P < 0.005 compared with control). This reduction in size was associated with severe histological changes. Insulin therapy partially improved the overall growth centers to 81% of the nondiabetic control length. It was still significantly lower than control (P < 0.05).



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  		Figure 2. Morphometric analysis of diabetic effect and insulin therapy on mouse humeral growth plate and mandibular condyle length. Six- to 8-day-old ICR mice were injected ip with either STZ, 120 mg/kg, or citrate buffer alone. Two days post STZ the mice were treated with or without insulin 8 mU/g body weight, for additional 5 days. Each point represents the mean ± SD of 18 measurements from at least three different experiments. Compared with control, length differences before and after insulin therapy were statistically significant at *P < 0.05 and **P < 0.005 level, respectively.

 
Growth plate of 7-day-old control mice (Fig. 3AGo) was composed of a crowded chondroblastic zone followed by large layers of chondrocytic and hypertrophic cells organized in rows. In the diabetic mice this chondrocytic zone was much narrower (Fig. 3BGo), containing sparse and unorganized dispersed cells. Similar changes were apparent in the diabetic mouse condyles (Fig. 3DGo). Furthermore, the mandibular condyle of 7-day-old control mice (Fig. 3CGo) was characterized by a typical developmental gradient of cells starting with thick upper progenitor cells followed by chondroblasts and chondrocytes that were embedded in calcified matrix and eventually gave rise to young trabecular bone. In the diabetic condyle (Fig. 3DGo) this normal cellular gradient was impeded. In both bone types, diabetes primarily affected the chondrocytic layer. Young chondrocytes almost disappeared, and hypertrophic cells were abnormally adjacent to the proliferative zone.



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  		Figure 3. Morphology of growth plates (A, B) and mandibular condyles (C, D) in control (A, C) and in diabetic mice 7 days post STZ injection (B, D). The most profound impact was observed in the mature chondrocytes, leading to a very sparse chondrocytic population in the growth plate (B) and to abnormal localization of hypertrophic cells adjacent to proliferative cells in the condyle (D). ch, Chondrocytes; hy, hypertrophic cells; pr, proliferative cells. Original magnification, x240.

 
GLUT 4 distribution in the normal and diabetic skeletal growth centers
The morphological studies indicated a specific suppressive effect on skeletal growth centers beyond the inhibitory effect on overall growth of the diabetic mouse. This could be due to specific sensitivity of growth centers to diabetes-induced hyperglycemia. To test this assumption, we looked at the existence of the "housekeeping" glucose transporter GLUT1 as well as the insulin-sensitive isoform GLUT4. Using an immunohistochemistry technique we examined the presence of GLUT1 and GLUT4 within the normal and diabetic skeletal growth centers. GLUT1 was present in control and diabetic animals in both bone growth centers. Compared with controls, no change in quantity and distribution was observed in diabetic mice (data not shown). However, regarding the presence of GLUT4, the insulin-sensitive glucose transporter, the picture was totally different. In both control growth centers, the epiphyseal growth plate and the mandibular condyle (Fig. 4Go, A and C), GLUT4 appeared present in abundance. GLUT4 was present in the young and mature chondrocytes and to a lesser degree in the chondroprogenitor and young chondroblastic zones. This distribution was similar within these two growth centers.



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  		Figure 4. Localization of GLUT4 by immunohistochemistry. Growth plates (A, B) and mandibular condyles (C, D) were studied in control (A, C), STZ-induced diabetic (B, D) and insulin-treated diabetic mice (E), 7 days post STZ. Paraffin sections were reacted with relevant primary antibody, then with biotinylated second antibody, streptavidin-peroxidase conjugate, and AEC substrate. GLUT4 is present in young and mature chondrocytes (arrows) in both control growth plate and mandibular condyle (A, C) and is markedly reduced in diabetic mice (B, D). The effect of insulin replacement on GLUT4 level in the condyle is depicted in section E. Original magnification, x190. Morphometric analysis is brought in section F. **, Difference from control is significant at P < 0.005 level.

 
Induction of diabetes resulted in major diminution of GLUT4 protein concentration in both the growth plate (Fig. 4BGo) and the mandibular condyle (Fig. 4DGo). Compared with the diabetic state, insulin therapy of the diabetic mice induced a small increase in the GLUT4 presence, mainly in the condyle (Fig. 4EGo). The increase was predominant in the young chondrocytes population. However, GLUT4 level remained significantly lower compared with nondiabetic state. Morphometric analysis based on the immunohistochemistry studies (Fig. 4FGo) quantify these findings. Compared with normal control, diabetes is associated with significant 74% and 82% reduction (P < 0.005) of GLUT4 levels in the mandibular condyle and the humerus growth plate, respectively. Upon insulin therapy, GLUT4 levels inspite of almost 2-fold increase compared with diabetic state (P < 0.05 in the condyle), remained 50% reduced compared with normal control level (P < 0.05).

Distribution of IGF-I receptors in the mouse growth centers
Immunolocalization of IGF-I receptors using anti-IGF-I-receptor antibodies is shown (see Fig. 5Go). In the growth plate (Fig. 5AGo) IGF-I receptors were present mainly in the hypertrophic cells and to a lesser degree in the chondroprogenitor cells, while they were absent in the diabetic growth plate (Fig. 5BGo). This distribution of IGF-I receptors in both normal and diabetic condyles was similar to that of GLUT4 protein. In the normal condyles the IGF-I receptors were also observed in the young and mature chondrocytes (Fig. 5CGo). Again, except for a faint staining in the hypertrophic cells, most of the positive staining disappeared from the diabetic condyles (Fig. 5DGo). Similarly to GLUT4 population, compared with diabetic state, insulin therapy of the diabetic mice resulted in only small increase in the IGF-I-R level mainly in the young chondrocytes (Fig. 5EGo). However, as assessed by morphometry (Fig. 5FGo), it is still significantly lower, being 23% and 16% of control levels (P < 0.005), in condyle and humerus growth plate, respectively. Upon insulin therapy, IGF-I-R levels while increasing almost 2-fold compared with diabetic state, remained 56% of the nondiabetic level (P < 0.005). These changes are similar to GLUT4 modulation but not to IR (see below).



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  		Figure 5. Localization of IGF-I receptors by immunohistochemistry. Growth plates (A, B) and mandibular condyles (C, D) were studied in control (A, C) STZ-induced diabetic (B, D) and insulin-treated diabetic mice (E) 7 days post STZ. Paraffin sections were reacted with relevant primary antibody, then with biotinylated second antibody, streptavidin-peroxidase conjugate, and AEC substrate. IGF-I receptors are present in young and mature chondrocytes (arrows) in both control growth plate and mandibular condyle (A, C) and are markedly reduced in STZ mice (B, D). The effect of insulin replacement on IGF-I receptor level in the condyle is depicted in section E. Original magnification, x190. Morphometric analysis is brought in section F. **, Difference from control is significant at P < 0.005 level.

 
Distribution of insulin receptors in the mouse growth centers
Because insulin regulates GLUT4 in adipose and muscle tissues (8), we looked for the presence of insulin receptors within the control and diabetic growth centers and examined whether they colocalize within the same cell types as the GLUT4. We found that in the growth plate of the untreated mouse (Fig. 6AGo) insulin receptors were present in both the reserve and young chondroblastic cells and, to a lesser degree, in the hypertrophic cells. In the control condyle IRs were distributed in the chondroprogenitor and young chondrocytes zones (Fig. 6CGo). Compared with control, diabetes induction was associated with major reduction in cellular IR content in both growth centers, i.e. the growth plate and mandibular condyle (Fig. 6Go, B and D, respectively). Insulin therapy fully restored IR content through most of the growth centers (Fig. 6EGo). Using morphometric analysis (Fig. 6FGo) we found only 6% of IR cellular content in the diabetic state compared with control (P < 0.005). Insulin therapy restored these levels to normalcy. IR cellular distribution in the skeletal growth centers in the control, diabetic and insulin-treated diabetic mice are clearly distinct from that of GLUT4 and IGF-I-R.



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  		Figure 6. Localization of insulin receptors by immunohistochemistry. Growth plates (A, B) and mandibular condyles (C, D) were studied in control (A, C) STZ-induced diabetic (B, D) and insulin-treated diabetic mice (E) 7 days post STZ. Paraffin sections were reacted with relevant primary antibody, then with biotinylated second antibody, streptavidin-peroxidase conjugate, and AEC substrate. Insulin receptors are in the reserve and chondroblastic cells of the humerus growth plate (A) and in the chondroprogenitor and young chondrocytes of the mandibular condyle (C) (arrows)—and are almost absent in both growth centers of the diabetic mice (B, D). The effect of insulin replacement on insulin receptors level in the condyle is depicted in section E. Original magnification, x190. Morphometric analysis is brought in section F. **, Difference from control is significant at P < 0.005 level.

 
Expression of GLUT4 and IGF-I receptors in the normal and diabetic growth centers
Using an in situ hybridization technique we examined GLUT4 and IGF-I-receptor gene expression to ascertain their parallel distribution in both the normal and diabetic bone growth centers. Indeed, GLUT4 and IGF-I-receptor mRNA (Figs. 7Go, A and C, respectively) were expressed mainly in the chondroprogenitor cells and young chondrocytes of the condyle and to a lesser degree in the chondrocytes. In the diabetic condyles most of the GLUT4 and IGF-I-receptor expression was reduced (Fig. 7Go, B and D, respectively). GLUT1 mRNA, which was expressed throughout the entire normal condyle, was not affected in the diabetic mouse (data not shown).



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  		Figure 7. Distribution of GLUT4 (A, B) and IGF-I-receptor (C, D) mRNAs in mandibular condyles of control (A, C) and diabetic mice 7 days post STZ injection (B, D). Frozen sections were reacted with either GLUT4 or IGF-I-receptor digoxygenin-labeled antisense RNA, then with FITC-conjugated anti-digoxygenin antibody. In control mandibular condyles mRNA for both GLUT4 and IGF-I-R is expressed in the chondroprogenitor cells and young chondrocytes. Original magnification, x240.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Bone in general is not considered a major player in overall glucose homeostasis. In the present study, we found the novel presence of GLUT4 mRNA and protein in chondroblastic and hypertrophic cells of mouse bone growth centers. In these cells, GLUT4 seems to be preferentially regulated through the IGF-I-receptor signaling cascade rather than with the insulin-receptor suggesting a potential regulation through the former signaling pathway. Furthermore, induction of diabetes by STZ markedly affected the skeletal growth centers. The diminution of GLUT4, IGF-I receptors in the chondrocytes might have been associated with the severe histological changes that led to the reduction in size of the skeletal growth centers.

As glucose uptake is a rate-limiting step for glucose metabolism, the decrease in cellular GLUT4 levels indicates a defect in the overall glucose metabolism of the growing bone, as has been shown for adipose cells from STZ-diabetic rats (12, 28). Our data suggest that defects in the glucose transport system—mainly GLUT4—are associated with morphological changes and slower bone growth, which is in accordance with Kelley et al. (29). The latter investigators suggested that impairment in glucose metabolism at the tissue level may play a role in IDDM-induced skeletal growth disturbances. Changes in glucose uptake were also associated with the development of the asymmetrical small-for-gestational-age (SGA) model in mice, whereas treatment of control mice with insulin and IGF-I increased glucose uptake and GLUT1 levels (protein and mRNA) in lung and muscle tissues, no change was observed in SGA mice (30). Similarly, in our model GLUT1 plays only minor role in the observed bone growth defects since GLUT1 mRNA and protein did not change during the diabetes state (data not shown). Further, while insulin receptors were restored to normalcy during insulin therapy of the diabetic mice, only partial recovery of bone length was observed.

The significant growth retardation observed in the present study in diabetic mice can be explained by the severe malformations in both mandibular condyle and humeral growth plate. The defects in the skeletal growth centers were more pronounced than would be expected from the overall 25% weight reduction of the diabetic mice, which suggests that the bone growth centers were specifically influenced. Moreover, within the growth centers the mature chondrocytes were the most affected cells (Figs. 2Go and 3Go). Normal skeletal growth depends on coupling between proliferation and differentiation. The morphological changes in the diabetic growth centers indicate a specific insult in the chondrocytic differentiative process. In our model, the young chondrocytic population almost disappeared and the growth center was occupied by hypertrophic cells, sparing the chondroprogenitor area, which suggests uncoupling between the proliferative and differentiative processes, although the proliferative process was intact. These results are in agreement with the clinical observations of bone mass reduction in type 1 diabetic children (31, 32).

Although these were surprising findings, several potential explanations may exist for the presence of GLUT4 in the bone, a nonclassical insulin-sensitive tissue. First, skeletal and muscular tissues have a common embryonic origin arising from the mesodermal somites (26). Muscle cells, under the stimulation of demineralized bone matrix, can undergo transformation and become bone-originating cells (33). Second, while the GLUT4 expression in muscle cells, adipocytes, and 3T3-L1 cells are driven by different transcription factors (activators and/or repressors) (17, 34), they may share some common ones. For example, the promoters of GLUT4 and IGF-I receptor genes, contain DNA-binding sites for Sp1 (34, 35), known to be important in regulating osteoblast differentiation; it has been especially shown to be involved in the regulation of osteocalcin and bone sialoprotein (36, 37). It has also been shown to be important in modulating IGF-I-receptor gene expression (35). Furthermore, the PAX3 gene product, which is important in early differentiation of the muscle cells can stimulate the promoter of the GLUT4 gene as well (Armoni, M., D. Shapiro, M. Quon, and E. Karnieli, submitted for publication). These DNA-binding motifs and transcription proteins may be relevant in our system by regulating both IGF-I-receptor and GLUT4 gene expression. However, these and other assumptions should be further investigated.

In muscle and adipose cells GLUT4 is regulated mainly through the insulin receptor. Although we did not measure in vitro glucose uptake as a function of insulin or IGF-I in these bone cells, the cellular distribution and coexistence of GLUT4 with IGF-I receptors (Figs. 4Go and 5Go, respectively), but not with the insulin receptors, suggest their regulation by the former and not by the latter. The coregulation of the IGF-I receptors and GLUT4 is also supported by the similar decrease in their cell content in the diabetic state. In parallel, this notion is also supported by the concomitant and similar recovery of GLUT4 and IGF-I-R during insulin therapy. While IR were fully restored, GLUT4 and IGF-I-R were only partially recovered to 50–56% of their normal prediabetic levels. Also, we did not treat the diabetic mice with IGF-I as it is uncommon in the clinical practice, it seems that IGF-I is the major regulator of growth and glucose metabolism in the bone. Indeed, in different cell systems, IGF-I has been shown to modulate GLUT4 gene expression and induce its translocation from the intracellular pool to the plasma membrane (38, 39). However, these assumptions and whether GLUT4 in bone is structurally and functionally different await further studies.

Insulin receptor levels have been shown to be up-regulated or normal in insulin-sensitive tissues from STZ-induced diabetics (40). This was not the case in our model of bone growth centers (Fig. 6Go). Yet our data are in agreement with Kaplan (41), who showed that insulin receptors are usually down-regulated in fetal and juvenile hyperglycemic animals. Using in situ hybridization (Fig. 7Go) and immunohistochemistry, we found that IGF-I-receptor mRNA and protein were also decreased in the diabetic bone growth centers. This is in divergence with their up-regulation in rat diabetic renal cells (24, 25). Although at present the mechanism is still unclear, the decrease in IGF-I receptor strengthens the possibility that in the bone GLUT4 is regulated through the IGF-I receptors rather than through the insulin receptors. As mentioned above these assumptions need further investigations.

As IGF-I-receptor signaling shares a similar PI3K pathway with insulin, its reduction might lead to decrease of the GLUT4 translocation and/or protein in diabetes. Another possibility might exist, as the promoters of both GLUT4 and IGF-I-receptor genes are regulated by similar transcription factors (42). These data suggest a potential tissue-specific modulation of gene expression of the GLUT4 and IGF-I receptors. If endochondral bone formation is stimulated by both insulin and IGF-I through the IGF-I receptor, then reduction of the latter at the bone level will lead to a significant impairment in bone growth. Reversal, by insulin therapy, of IR and IGF-I-R to 96% and 56% of their prediabetic level, respectively, provides another indirect support to this hypothesis.

IGF-I and its receptor are also important for glucose metabolism. IGF-I stimulates glucose metabolism as well as GLUT4 mRNA (16). Using transgenic diabetic mouse models, Olson and Pessin (34) showed differential regulation of various regions of the human GLUT4 promoter by diabetes. In addition, the GLUT4 promoter contains several DNA-binding sites for transcription factors (39), some of which, like Sp1, are present in osteoblasts and are also important in regulating the IGF-I-receptor promoter (35). These DNA-binding motifs and transcription proteins may be relevant in our system by regulating both IGF-I-receptor and GLUT4 genes. Taken together, we further hypothesize that in diabetes, both the IGF-I receptors and GLUT4 are repressed by a similar transcription factor(s). This would result in a decrease of the IGF-I-receptor effect on various cellular components and a reduction in cellular glucose metabolism. Both events would lead to defects in bone growth process. Alternatively, if GLUT4 promoter is regulated through the IGF-I-receptor signal-transduction pathway, reduced expression of the IGF-I receptors may result in decreased expression of the GLUT4 gene. More studies are needed to clarify this issue.

Our study establishes the novel involvement of GLUT4 in early skeletal growth and suggests its importance to glucose metabolism in the growing bone. The specific cellular interaction between the IGF-I receptor and GLUT4 suggests the potential of GLUT4 via the IGF-I receptor rather than via the insulin receptor pathway. Furthermore, if shown in human, it would seem that impaired bone growth in type I diabetic children might be associated with reduced expression of GLUT4 and IGF-I-receptor genes. While further studies are needed, our observations contribute to better understanding of the mechanisms underlying the skeletal malformations associated with type 1 diabetes.


   Acknowledgments
 
The authors wish to thank Ms. Irena Reiter for her excellent technical assistance, Ms. Margalit Levy for her excellent secretarial assistance and Ms. Ruth Singer for her help in editing this manuscript.


   Footnotes
 
1 These studies were supported in part by grants (to E.K.) from the L.R. Diamond Fund, the San Francisco Diabetes Research Fund, and the Technion-Israel Institute of Technology’s Vice President for Research Fund. Back

Received July 13, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Salardi S, Tonioli P, Tassoni P, Tellarini M, Mazzanti L, Cacciari E 1987 Growth and growth factors in diabetes mellitus. Arch Dis Child 62:57–62[Abstract]
  2. Catanese V, Sciavolino P, Lango M 1993 Discordant, organ-specific regulation of insulin-like growth factor-I messenger ribonucleic acid in insulin-deficient diabetes in rats. Endocrinology 132:496–503[Abstract]
  3. Shepard N, Mitchell N 1988 Periodic acid Schiff–p phenylenediamine staining of glycogen in chondrocytes. A new combination which improves both cellular detail and glycogen identification. Histochemistry 88:127–130[CrossRef][Medline]
  4. Thomas D, Hards D, Rogers S, Ng K, Best J 1996 Insulin receptor expression in bone. J Bone Miner Res 11:13212–11320
  5. de Pablo F, Scott L, Roth J 1990 Insulin and insulin-like growth factor I in early development: peptides, receptors and biological events. Endocr Rev 11:558–577[CrossRef][Medline]
  6. Mueckler M 1994 Facilitative glucose transporters. Eur J Biochem 219:713–725[Medline]
  7. Czech M, Clancy B, Pessino A, Woon C-W, Harrison S 1992 Complex regulation of simple sugar transport in insulin-responsive cells. Trends Biochem Sci 17:197–201[CrossRef][Medline]
  8. James D, Piper R, Slot J 1993 Targeting of mammalian glucose transporters. J Cell Sci 104:607–612[Medline]
  9. Karnieli E, Zarnowski M, Hissin P, Simpson I, Salans L, Cushman S 1981 Insulin-stimulated translocation of glucose transport systems in the isolated rat adipose cell: time-course, reversal, insulin concentration dependency, and relationship to glucose transport activity. J Biol Chem 256:4772–4777[Free Full Text]
  10. Cushman S, Wardzala L 1980 Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. Apparent translocation of intracellular transport systems to the plasma membrane. J Biol Chem 255:4758–4762[Free Full Text]
  11. Suzuki K, Kono T 1980 Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site. Proc Natl Acad Sci USA 77:2542–2545[Abstract/Free Full Text]
  12. Karnieli E, Hissin P, Simpson I, Salans L, Cushman S 1981 A possible mechanism of insulin resistance in the rat adipose cell in streptozotocin-induced diabetes mellitus. Depletion of intracellular glucose transport systems. J Clin Invest 68:811–814
  13. Karnieli E, Barzilai A, Rafaeloff R, Armoni M 1986 Distribution of glucose transporters in membrane fractions isolated from human adipose cells: Relation to cell size. J Clin Invest 78:1051–1055
  14. Kahn B, Cushman S 1987 Mechanism for markedly hyperresponsive insulin-stimulated glucose transport activity in adipose cells from insulin-treated streptozotocin diabetic rats. Evidence for increased glucose transporter intrinsic activity. J Biol Chem 262:5118–5124[Abstract/Free Full Text]
  15. Garvey W, Huecksteadt T, Matthaei S, Olefsky J 1988 Role of glucose transporters in the cellular insulin resistance of type II non-insulin-dependent diabetes mellitus. J Clin Invest 81:1528–1536
  16. Weiland M, Bahr F, Hohne M, Schurmann A, Ziehm D, Joost H 1991 The signaling potential of the receptors for insulin and insulin-like growth factor I (IGF-I) in 3T3–L1 adipocytes: comparison of glucose transport activity, induction of oncogene c-fos, glucose transporter mRNA, and DNA-synthesis. J Cell Physiol 149:428–435[CrossRef][Medline]
  17. MacDougald O, Lane M 1995 Transcriptional regulation of gene expression during adipocyte differentiation. Annu Rev Biochem 64:345–373[CrossRef][Medline]
  18. Chakravorty A, Joslyn M, Davis J 1993 Characterization of insulin and insulin-like growth factor-I actions in the bovine luteal cell: regulation of receptor tyrosine kinase activity, phosphatidylinositol-3-kinase, and deoxyribonucleic acid synthesis. Endocrinology 133:1331–1340[Abstract]
  19. Mora S, Kaliman P, Chillaron J, Testar X, Palacin M, Zorano A 1995 Insulin and insulin-like growth factor-I (IGF-I) stimulate GLUT4 glucose transporter translocation in Xenopus oocytes. Biochem J 311:59–65
  20. Maor G, Silbermann M, von der Mark K, Heingard D, Laron Z 1993 Insulin enhances the growth of cartilage in organ and tissue cultures of mouse neonatal mandibular condyle. Calcif Tissue Int 52:291–299[CrossRef][Medline]
  21. Maor G, Hochberg Z, Silbermann M 1993 Insulin-like growth factor I accelerates proliferation and differentiation of cartilage progenitor cells in cultures of neonatal mandibular condyles. Acta Endocrinol (Copenh) 128:56–64[Medline]
  22. Isaksson O, Lindahl A, Nilsson A, Isgaard J 1987 Mechanism of the stimulatory effect of growth hormone on longitudinal bone growth. Endocr Rev 8:426–438[CrossRef][Medline]
  23. Murata A, Yasuda T, Niimi H 1994 Two cases of insulin-dependent diabetes mellitus under insulin treatment with slow height velocity: relationship of growth hormone-binding protein, metabolic control and growth. Acta Paediatr Japn 36:272–275
  24. Bach L 1992 IGF-I and IGF binding proteins in diabetes-related kidney growth. Growth Regul 2:30–39[Medline]
  25. Werner H, Shen-Orr Z, Stannard B, Burguera B, Roberts Jr C, LeRoith D 1990 Experimental diabetes increases insulin-like growth factor I and II receptor concentration and gene expression in kidney. Diabetes 39:1490–1497[Abstract]
  26. Nimni M, Bernick S, Ertl D, Nishimoto S, Paule W, Strates B, Villanueva J 1988 Ectopic bone formation is enhanced in senescent animals implanted with embryonic cells. Clin Orthop 234:255–266
  27. Maor G, von der Mark K, Reddi A, Heinegard D, Franzen A, Silbermann M 1987 Acceleration of cartilage and bone differentiation on collagenous substrata. Coll Relat Res 7:351–370[Medline]
  28. Karnieli E, Armoni M, Cohen P, Kanter Y, Rafaeloff R 1987 Reversal of insulin resistance of diabetic rats by insulin therapy: restoration of the pool of glucose transporters and enhancement of glucose transport activity. Diabetes 36:925–931[Abstract]
  29. Kelly K, Russell S, Matteucci M, Nicoll C 1993 An insulin-like growth factor I-resistant state in cartilage of diabetic rats is ameliorated by hypophysectomy. Possible role of metabolism. Diabetes 42:463–469[Abstract]
  30. Simmons R, Flozak A, Ogata E 1993 The effect of insulin and insulin-like growth factor-I on gucose transport in normal and small for gestational age fetal rats. Endocrinology 133:1361–1368[Abstract]
  31. Hui S, Epstein S, Johnston Jr C 1985 A prospective study of bone mass in patients with type I diabetes. J Clin Endocrinol Metab 60:74–80[Abstract]
  32. Wiske P, Wentworth S, Norton Jr J, Epstein S, Johnston Jr C 1982 Evaluation of bone mass and growth in young diabetics. Metabolism 31:848–854[CrossRef][Medline]
  33. Volek-Smith H, Mr U 1996 Recombinant human bone morphogenetic protein (rhBMP) induced hetrotopic bone development in vivo and in vitro. Proc Soc Exp Biol Med 211:265–272[Abstract]
  34. Olson A, Pessin J 1995 Transcriptional regulation of the human GLUT4 gene promoter in diabetic transgenic mice. J Biol Chem 270:23491–23495[Abstract/Free Full Text]
  35. Beitner-Jonson D, Werner H, Roberts Jr C, LeRoith D 1995 Regulation of insulin-like growth factor I receptor gene expression by Sp1: physical and functional interactions of Sp1 at GC boxes and at a CT element. Mol Endocrinol 9:1147–1156[Abstract]
  36. Lian J, Stein G, Stein J, Van Wijnen A, McCabe L, Banerjee C, Hoffmann H 1996 The osteocalcin gene promoter provides a molecular blueprint for regulatory mechanisms controlling bone tissue formation: role of transcription factors involved in development. Connect Tissue Res 35:15–21[Medline]
  37. Sodek J, Li J, Kim R, Ogata Y, Yamauchi M 1996 Characterization of the bone sialoprotein (BSP) gene promoter. Connect Tissue Res 35:23–31[Medline]
  38. Lund S, Flyvbjerg A, Holman G, Larsen F, Pedersen O, Schmitz O 1994 Comparative effects of IGF-I and insulin on the glucose transporter system in rat muscle. Am J Physiol 267:E461–E466
  39. Wilson C, Mitsumoto Y, Maher F, Klip A 1995 Regulation of cell surface GLUT1, GLUT3, and GLUT4 by insulin and IGF-I in L6 myotubes. FEBS Lett 368:19–22[CrossRef][Medline]
  40. Kobayashi M, Olefsky J 1978 Long-term regulation of adipocyte glucose transport capacity by circulating insulin in rats. J Clin Invest 28:87–95
  41. Kaplan S 1984 The insulin receptor. J Pediatr 104:327–336[Medline]
  42. Reul B, Ongemba L, Pottier A, Henquin J, Brichard S 1997 Insulin and insulin-like growth factor-I antagonize the stimulation of ob gene expression by dexamethasone in cultured rat adipose tissue. Biochem J 324:605–610




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