Short- and long-term exposure to high glucose induces unique transcriptional changes in osteoblasts in vitro.

Bone is increasingly recognized as a target for diabetic complications. In order to evaluate the direct effects of high glucose on bone, we investigated the global transcriptional changes induced by hyperglycemia in osteoblasts in vitro. Rat bone marrow-derived mesenchymal stromal cells were differentiated into osteoblasts for 10 days, and prior to analysis, they were exposed to hyperglycemia (25 mM) for the short-term (1 or 3 days) or long-term (10 days). Genes and pathways regulated by hyperglycemia were identified using mRNA sequencing and verified with qPCR. Genes upregulated by 1-day hyperglycemia were, for example, related to extracellular matrix organization, collagen synthesis and bone formation. This stimulatory effect was attenuated by 3 days. Long-term exposure impaired osteoblast viability, and downregulated, for example, extracellular matrix organization and lysosomal pathways, and increased intracellular oxidative stress. Interestingly, transcriptional changes by different exposure times were mostly unique and only 89 common genes responding to glucose were identified. In conclusion, short-term hyperglycemia had a stimulatory effect on osteoblasts and bone formation, whereas long-term hyperglycemia had a negative effect on intracellular redox balance, osteoblast viability and function.

Evaluation of bone marrow glucose uptake and adiposity in male rats after diet and exercise interventions.

Objectives: Obesity impairs bone marrow (BM) glucose metabolism. Adult BM constitutes mostly of adipocytes that respond to changes in energy metabolism by modulating their morphology and number. Here we evaluated whether diet or exercise intervention could improve the high-fat diet (HFD) associated impairment in BM glucose uptake (BMGU) and whether this associates with the morphology of BM adipocytes (BMAds) in rats. Methods: Eight-week-old male Sprague-Dawley rats were fed ad libitum either HFD or chow diet for 24 weeks. Additionally after 12 weeks, HFD-fed rats switched either to chow diet, voluntary intermittent running exercise, or both for another 12 weeks. BMAd morphology was assessed by perilipin-1 immunofluorescence staining in formalin-fixed paraffin-embedded tibial sections. Insulin-stimulated sternal and humeral BMGU were measured using [18F]FDG-PET/CT. Tibial microarchitecture and mineral density were measured with microCT. Results: HFD rats had significantly higher whole-body fat percentage compared to the chow group (17% vs 13%, respectively; p = 0.004) and larger median size of BMAds in the proximal tibia (815 µm2 vs 592 µm2, respectively; p = 0.03) but not in the distal tibia. Switch to chow diet combined with running exercise normalized whole-body fat percentage (p < 0.001) but not the BMAd size. At 32 weeks of age, there was no significant difference in insulin-stimulated BMGU between the study groups. However, BMGU was significantly higher in sternum compared to humerus (p < 0.001) and higher in 8-week-old compared to 32-week-old rats (p < 0.001). BMAd size in proximal tibia correlated positively with whole-body fat percentage (r = 0.48, p = 0.005) and negatively with humeral BMGU (r = -0.63, p = 0.02). HFD significantly reduced trabecular number (p < 0.001) compared to the chow group. Switch to chow diet reversed this as the trabecular number was significantly higher (p = 0.008) than in the HFD group. Conclusion: In this study we showed that insulin-stimulated BMGU is age- and site-dependent. BMGU was not affected by the study interventions. HFD increased whole-body fat percentage and the size of BMAds in proximal tibia. Switching from HFD to a chow diet and running exercise improved glucose homeostasis and normalized the HFD-induced increase in body fat but not the hypertrophy of BMAds.

Perilipin-1 immunostaining improves semi-automated digital quantitation of bone marrow adipocytes in histological bone sections.

Bone marrow adipocytes (BMAds) are not just passive fillers inside the bone marrow compartment but respond to various metabolic changes. Assessment of those responses requires evaluation of the number of BMAds and their morphology for which laborious and error-prone manual histological analysis remains the most widely used method. Here, we report an alternative image analysis strategy to semi-automatically quantitate and analyse the morphology of BMAds in histological bone sections. Decalcified, formalin-fixed paraffin-embedded histological sections of long bones of Sprague-Dawley rats were stained with either haematoxylin and eosin (HE) or by immunofluorescent staining for adipocyte-specific protein perilipin-1 (PLIN1). ImageJ-based commands were constructed to detect BMAds sized 200 µm2 or larger from standardized 1 mm2 analysis regions by either classifying the background colour (HE) or the positive and circular PLIN1 fluorescent signal. Semi-automated quantitation strongly correlated with independent, single-blinded manual counts regardless of the staining method (HE-based: r=0.85, p<0.001; PLIN1 based: r=0.95, p<0.001). The detection error was higher in HE-stained sections than in PLIN1-stained sections (14% versus 5%, respectively; p<0.001), which was due to false-positive detections of unstained adipocyte-like circular structures. In our dataset, the total adiposity area from standardised ROIs in PLIN-1-stained sections correlated with that in whole-bone sections (r=0.60, p=0.02).

Glucose transporters GLUT1, GLUT3, and GLUT4 have different effects on osteoblast proliferation and metabolism.

Bone is an active tissue that undergoes constant remodeling. Bone formation requires energy and one of the energy sources of bone-forming osteoblasts is glucose, which is transported inside the cells via glucose transporters. However, the role of class I glucose transporters in the differentiation and metabolism of osteoblasts and their precursors, bone marrow mesenchymal stromal cells (BMSCs) remains inconclusive. Our aim was to characterize the expression and contribution of main class I glucose transporters, GLUT1, GLUT3, and GLUT4, during osteoblast proliferation and differentiation. To investigate the role of each GLUT, we downregulated GLUTs with siRNA technology in primary rat BMSCs. Live-cell imaging and RNA-seq analysis was used to evaluate downstream pathways in silenced osteoblasts. Glucose transporters GLUT1, GLUT3, and GLUT4 had distinct expression patterns in osteoblasts. GLUT1 was abundant in BMSCs, but rapidly and significantly downregulated during osteoblast differentiation by up to 80% (p < 0.001). Similar downregulation was observed for GLUT4 (p < 0.001). In contrast, expression levels of GLUT3 remained stable during differentiation. Osteoblasts lacked GLUT2. Silencing of GLUT4 resulted in a significant decrease in proliferation and differentiation of preosteoblasts (p < 0.001) and several pathways related to carbohydrate metabolism and cell signaling were suppressed. However, silencing of GLUT3 resulted in increased proliferation (p < 0.001), despite suppression of several pathways involved in cellular metabolism, biosynthesis and actin organization. Silencing of GLUT1 had no effect on proliferation and less changes in the transcriptome. RNA-seq dataset further revealed that osteoblasts express also class II and III glucose transporters, except for GLUT7. In conclusion, GLUT1, -3 and -4 may all contribute to glucose uptake in differentiating osteoblasts. GLUT4 expression was clearly required for osteoblast proliferation and differentiation. GLUT1 appears to be abundant in early precursors, but stable expression of GLUT3 suggest also a role for GLUT3 in osteoblasts. Presence of other GLUT members may further contribute to fine-tuning of glucose uptake. Together, glucose uptake in osteoblast lineage appears to rely on several glucose transporters to ensure sufficient energy for new bone formation.