Hyperglycemia diverts dividing osteoblastic precursor cells to an adipogenic pathway and induces synthesis of a hyaluronan matrix that is adhesive for monocytes

Abstract

Isolated rat bone marrow stromal cells cultured in osteogenic medium in which the normal 5.6 mm glucose is changed to hyperglycemic 25.6 mm glucose greatly increase lipid formation between 21-31 days of culture that is associated with decreased biomineralization, up-regulate expression of cyclin D3 and two adipogenic markers (CCAAT/enhancer binding protein α and peroxisome proliferator-activated receptor γ) within 5 days of culture, increase neutral and polar lipid synthesis within 5 days of culture, and form a monocyte-adhesive hyaluronan matrix through an endoplasmic reticulum stress-induced autophagic mechanism. Evidence is also provided that, by 4 weeks after diabetes onset in the streptozotocin-induced diabetic rat model, there is a large loss of trabecular bone mineral density without apparent proportional changes in underlying collagen matrices, a large accumulation of a hyaluronan matrix within the trabecular bone marrow, and adipocytes and macrophages embedded in this hyaluronan matrix. These results support the hypothesis that hyperglycemia in bone marrow diverts dividing osteoblastic precursor cells (bone marrow stromal cells) to a metabolically stressed adipogenic pathway that induces synthesis of a hyaluronan matrix that recruits inflammatory cells and establishes a chronic inflammatory process that demineralizes trabecular cancellous bone.

Keywords: Adipogenesis; Autophagy; Diabetes; ER Stress; Heparin; Inflammation; Monocyte Adhesive Hyaluronan Matrix; Osteogenesis; Osteopenia; Stromal Cell.

FIGURE 1.

Demineralization in trabecular bone in 4-week-old diabetic rat tibia. Micro-CT sagittal slices (A and B) and three-dimensional, segmented trabecular structure (C and D) show extensive demineralization in the diabetic cancellous bone (boxes) compared with the control. Autofluorescence of demineralized sections in the same trabecular bone region shows little difference (E and F).

FIGURE 2.

Adipocytes are present in the diabetic trabecular bone marrow. Demineralized sections of 4-week-old normal (A, C, and E) and diabetic (B, D, and F) trabecular bone regions were H&E-stained. The trabecular collagen bone matrix is similar in both (arrows). Adipocytes (asterisks) are prevalent in the diabetic bone (D) and absent in the normal bone (C). E and F, enlarged regions from C and D to provide better views of the adipocytes in F.

FIGURE 3.

Adipocytes embedded in the hyaluronan matrix of diabetic bone stain for cyclin D3, C/EBPα, and LC3. Demineralized sections of 4-week-old normal (A, C, and E) and diabetic (B, D, and F) trabecular bone regions were stained for hyaluronan (green) and cyclin D3 (red) (A and B) or hyaluronan (green) and C/EBPα (red) (C and D). E and F showed staining for cyclin D3 (red) and LC3 (green), and their colocalization in adipocytes in the diabetic bone section indicates the presence of autophagic responses. Condensations of cyclin D3 and C/EBPα (asterisks) that have structures characteristic of autophagosomes are associated with the adipocytes in the diabetic bone sections.

FIGURE 4.

Large numbers of CD44-positive mononuclear cells are embedded in the hyaluronan matrix in diabetic trabecular bone marrow. Demineralized sections of 4-week-old normal (A, merged) and diabetic trabecular bone regions were stained for hyaluronan (green, B) and CD44 (red, C) and merged (D). Numerous CD44-positive mononuclear cells are embedded in the hyaluronan matrix in the diabetic bone section. F–H, staining for hyaluronan (green) and macrophages (red) compared with the merged control (E). Numerous macrophages are embedded in the bone marrow, as shown by a red stain for MAC (G). Examples of adipocytes are shown in H (asterisks).

FIGURE 5.

Effects of hyperglycemic glucose on biomineralization (Alizarin Red S) and lipid (Oil Red O) formation in cultured RMSCs. Rat marrow stromal cells were stimulated in osteogenic medium in the presence of normal (5.6 mm) (Low) or hyperglycemic (25.6 mm) (High) glucose or a mannitol control (5.6 mm glucose + 20.0 mm mannitol) for 21–31 days. The biomineralization and lipid formation in the cultures were determined by Alizarin Red S (left panel) and Oil Red O staining (right panel).

FIGURE 6.

RMSCs increase synthesis of lipids, hyaluronan, and cyclin D3 when normal (5.6 mm glucose) osteogenic medium is increased to hyperglycemic (25.6 mm). Rat marrow stromal cells cultured for 5 days in osteogenic medium with normal (5.6 mm) glucose (A, D, and G), hyperglycemic (25.6 mm) glucose (B, E, and H), or normal glucose plus 20 mm mannitol (C, F, and I) were stained with Nile Red (A–C) or permeabilized and stained for cyclin D3 (red) and either hyaluronan (green, D–F) or LC3 (green, G–I).

FIGURE 7.

U937 monocytes adhere to RMSCs cultured in hyperglycemic osteogenic medium. U937 monocytes (refractive dots) were bound at 4 °C to rat bone marrow stromal cells cultured for 5 days in medium with normal glucose (Low), hyperglycemic glucose alone (High), 1 μg/ml heparin (Hep) (High + Heparin), 0.25 mm 4-MU-xyl (xyl), or with the PKC inhibitor (PKCi) 100 nm bisindolylmaleimide I (High + PKC inhibitor). The bar graph shows the number of bound U937 monocytes normalized to the area as described under “Experimental Procedures” for three cultures for each treatment (black bars) and for three cultures pretreated with Streptomyces hyaluronidase (HA'ase) before monocyte addition (open bars). Unpaired Student's t test was used to compare the means of two groups (*, p < 0.01).

FIGURE 8.

FACE analyses of HA in the RMSC cultures. The gel shows an example of a FACE analysis for a set of cultures treated as described in Fig. 7. The bar graph shows the results of FACE analyses for three cultures for each treatment. Unpaired Student's t test was used to compare the means of two groups (*, p < 0.01) normalized to the results of the low glucose-treated cultures. PKCi, PKC inhibitor; xyl, 4-MU-xyl; Hep, heparin; Std, disaccharide standards from chondroitin sulfates and hyaluronan.

FIGURE 9.

Analyses of 3T3-L1 cells stimulated to undergo adipogenesis. A, 3T3-L1 cells stimulated to divide in hyperglycemic medium routinely used to promote adipogenesis were permeabilized at 48 h and stained for hyaluronan (green), cyclin D3 (red), and nuclei (blue). The enlarged insets demonstrate what appears to be an autophagosome (top right) and an example of an aggresome (bottom left). The role of the extensive hyaluronan matrix for U937 monocyte adhesion (B) was demonstrated by pretreating a separate culture with Streptomyces hyaluronidase (specific for hyaluronan) before adding the monocytes (C) (reproduced with permission from FEBS).

FIGURE 10.

Western blot analyses for RUNX, PPARγ, C/EBPα, GRP94, cyclin D3, and hyaluronan synthase 2 (HAS2) in RMSC cultures. RMSCs were cultured for 120 h in low glucose, in high glucose alone, with 4-MU-xyl (xyl), or with heparin (Hep) as indicated. The Western blot analysis shows an example of one set of cultures. The bar graphs show the results from analyses of three cultures for each treatment (mean ± S.D.) normalized to the results for the low glucose-treated cultures. Unpaired Student's t test was used to compare the means of two groups (*, p < 0.01).