Femoral bone mass and new bone around the implants were reduced in high-carbohydrate, high-fat diets-induced hyperlipidemia rats
We successfully constructed the rat model of hyperlipidemia, which was confirmed by elevated levels of TC and LDL-C, and reduced level of HDL-C (Fig. 1A). Compared to the control group, the biochemical markers P1NP related to bone formation decreased significantly, while CTX-1 related to bone resorption increased significantly in the hyperlipidemia group (Fig. 1B). Our results indicated that long-term high-fat diet led to cancellous bone loss at distal femur characterized by reduced cancellous BMD, BV/TV, Tb.N, and increased Tb.Sp. (Fig. 1C, D). However, the cortical parameters were observed to be not obviously changed (Fig. 1E, F); Hyperlipidemia led to the decrease of new bone around implants characterized by reduced BV/TV, Tb.Th, Tb.N and TB.Sp (Fig. 1G, H). Moreover, HE staining results showed that incomplete trabecular bone structures, increased marrow adiposity within the bone marrow cavity (Fig. 1I) and inafiltration of adipocytes in the trabecular spaces of new bone tissue around the implants were seen in the femur of hyperlipidemic rats (Fig. 1J).
Fig. 1 Femoral bone mass and new bone around the implants were reduced in high-fat diet-induced hyperlipidemia rats. (A) Serum levels of TC, TG, LDL -C, HDL-C. (B) Serum biochemical test. (C) Representative micro-CT images of distal femur. (D) Trabecular bone parameters: BMD, BV / TV, Tb. N, Tb. Sp, and Tb. Th. (E) Representative micro-CT images of the cortical bone in the middle area of the femora. (F) Cortical bone parameters: Ct.Th and Ct.Ar. (G) Representative micro-CT images of the femoral implants in rats. (H) Parameters of new bone around implants: BV/TV, Tb.Th, Tb.N and Tb.Sp .(I)Images of distal humerus stained with HE. (J) Bone microtructure around the femoral implant stained with HE. The black arrows indicate adipocytes. Data are reported as means ±SD. *P < 0.05 Vs. RD group. RD: regulated diet. HCHF: high-carbohydrate, high-fat diet.
BMMSCs derived from HFD rats displayed impaired osteogenic differentiation capacity and enhanced adipogenic differentiation capacity
BMMSCs derived from RD and HCHF rats were used to analyze the cell biological properties. As shown in Fig. 2A, the primary BMMSCs derived from RD and HCHF rats were fusiform or triangular, mixed with round, highlighted and unattached hybrid cells. The self-renewal capacity of HCHF-BMMSCs were significantly reduced than RD-BMMSCs as indicated by CFU analysis (Fig. 2B). BMMSCs of both origins were characterized by positive expression of CD90, CD29 and CD44 while negative expression of the hematopoietic markers CD45 and CD11b (Fig. 2C). HCHF-BMMSCs exhibited aberrant differentiation commitment. Significant reduction of osteogenic differentiation ability was observed in HCHF-BMMSCs. HCHF-BMMSCs formed fewer alizarin red-positive nodules, which was in line with the decreased expression of Runx2, ALP and Col1 at both the mRNA and protein levels (Fig. 2D, F). Compared with RD-BMMSCs, HCHF-BMMSCs showed increased adipogenic differentiation and this was evident from the Oil red staining (Fig. 2E). PPAR-γ is the adipocyte-specific factor essential for adipocyte differentiation. From our results it was confirmed that the expression of PPAR-γ mRNA and protein were increased in HCHF-BMMSCs (Fig. 2G).
Fig. 2 BMMSCs derived from high-fat diet rats displayed with decreased self-renewal capacity, osteogenic differentiation capacity and increased adipogenic differentiation capacity. (A) Cell morphology (scale bar = 200μm). (B) colony forming unit analysis. (C) Immunophenotypic Characterization of BMMSCs analyzed by flow cytometry. (D) Cytological staining of alizarin red to detect matrix mineralization in BMMSCs culture induced for osteogenesis for 21 d (scale bar = 200μm), and quantitative analysis performed using cetylpyridinium chloride. (E) Cytological staining of oil red O to detect fat droplets in the cytoplasm of BMMSCs induced for adipogenic differentiation for 14 d (scale bar = 50μm), and quantitative analysis performed using isopropanol. (F) q RT-PCR and Western blot were used to assess the mRNA and protein levels of Runx2, ALP, Col 1 in BMMSCs culture induced for osteogenesis for 14 d. (G) q RT-PCR and Western blot were used to assess the mRNA and protein levels of PPAR-γ in BMMSCs culture induced for adipogenic for 14 d. Data are reported as mean ± SD. *P< 0.05 Vs. control group. **P< 0.01 Vs. control group. RD: regulated diet. HCHF: high-carbohydrate, high-fat diet.
Autophagy levels declined in BMMSCs derived from high-carbohydrate, high-fat diet
Research shows that autophagy occupies important roles in bone homeostasis. In this study, a series of methods were adopted to examine autophagy levels of RD-BMMSCs and HCHF-BMMSCs. During the study, we measured the expressions of Beclin1 and microtubuler associated protein 1 light chain 3 (LC3), which is of key importance in the initiation and maturation of autophagosome, and P62, an indicator of autophagosomal degradation. Impaired autophagy was found in HCHF-BMMSCs. The gene expression levels of Beclin1 and Lc3 decreased, while P62 increased in HCHF-BMMSCs (Fig. 3A). Decreased expression of Beclin1 and reduced LC3II/LC3I ratio, as well as accumulated P62 were observed in HCHF-BMMSCs by Western blot (Fig. 3B). Further, the vacuoles labeled with mCherry-GFP-LC3B were detected by confocal laser scanning microscope (LSCM) to examine the status of autophagic flux. Colocalization of RFP-LC3 and GFP-LC3 dots (yellow) were lower in HFD-BMMSCs (Fig. 3C). Autophagosomes are spherical structures with double-layer membranes enclosing damaged or unnecessary organelles. Autophagosomes can be identified by TEM, and we found that HCHF-BMMSCs possessed fewer autophagosomes than RD-BMMSCs (Fig. 3D).
Fig. 3 Autophagy level declined in BMMSCs from high-fat diet rats. Beclin1, Lc3 and P62 mRNA were examined by qPCR. (A) Beclin1, Lc3 and P62 mRNA were examined by qPCR.; (B) The protein level of Beclin1, LC3 and P62 in RD- and HFD-BMMSCs were examined by Western blot. (C) LSCM Images of BMMSCs transfected with AdPlus-mCherry-GFP-LC3B adenovirus. The fusion dot number (yellow) indicated the autophagy level (scale bar =20μm). (D) Autophagosomes of RD- and HFD-BMMSCs detected by TEM. Top row: scale bar = 5μm. Bottom row: scale bar = 1μm. Arrows indicated the typical autophagosomes. Data are reported as mean ± SD. *P < 0.05 Vs. RD group. RD: regulated diet. HCHF: high-carbohydrate, high-fat diet.
Autophagy was essential in maintaining the differentiation balance of BMMSCs derived from high-fat diet rats
Previous studies suggest that the autophagy is involved in regulation of cellular differentiation. HCHF-BMMSCs manifested degraded osteogenic potential and impaired autophagy activity. Therefore, we concentrated on the autophagy to explore the probable cause of the anomalous differentiation of HCHF-BMMSCs. The rapamycin promoted osteogenic differentiation, but inhibited the adipogenic differentiation of HCHF-BMMSCs through activation of autophagy. The increased expression of RUNX2 and decreased PPAR-γ were further confirmed by our results (Fig. 3A, B). The 3-MA, an autophagy inhibitor, inhibited the osteogenic differentiation, but increased the adipogenic differentiation of RD-BMMSCs, as shown by alizarin red and oil red staining. The decreased expression of RUNX2 but elevated expression of PPAR-γ was further confirmed by the results shown in Fig. 3C, D. These results indicated that autophagy has an essential role in differentiation of BMMSCs, and activating autophagy with rapamycin partially restored the anomalous differentiation of HFD-BMMSCs.
Fig. 4 Autophagy was essential in maintaining the differentiation balance of BMMSCs derived from high-fat diet rats. (A) Western blot was performed to examine expressions of LC3, P62 and Runx2 protein level and alizarin red staining was performed to detect mineralized nodules formed in HCHF-BMMSCs cultured in osteo-differentiation medium in the presence of rapamycin. (B) Western blot was performed to examine expressions of LC3, P62 and PPAR-γ protein level and Oil Red O staining was performed to detect lipid droplet formed in HCHF-BMMSCs cultured in adipo-differentiation medium in the presence of rapamycin. (C) Western blot was performed to examine expressions of LC3, P62 and Runx2 protein level and alizarin red staining was performed to detect mineralized nodules formed in RD-BMMSCs cultured in osteo-differentiation medium in the presence of 3-MA. (D) Western blot was performed to examine expressions of LC3, P62 and PPAR-γ protein level and Oil Red O staining was performed to detect lipid droplet formed in RD-BMMSCs cultured in adipo-differentiation medium in the presence of 3-MA. Oil Red O staining (Scale bar =50μm). RD: regulated diet. HCHF: high-carbohydrate, high-fat diet.
AMPK/mTOR signaling pathway was involved in the regulation of autophagy and osteogenesis of BMMSCs derived from high-carbohydrate, high-fat diet
As shown in Fig. 5A, the BMMSCs in the HCHF group were found to have increased p-mTOR/mTOR, insignificantly changed p-Akt/Akt, and decreased p-AMPK/AMPK compared with the rats in the control group. These results suggested that AMPK/mTOR signaling pathway was presumably associated with the impaired autophagy of BMMSCs after long-time exposure to the high-carbohydrate, high-fat diet. As the first found AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) is widely used in AMPK and related experimental research. The BMMSCs in the HCHF group after treatment with AICAR demonstrated increased p-AMPK/AMPK, decreased p-mTOR/mTOR, enhanced autophagy activity which was evident from the increased LC3II/LC3I ratio and reduced P62 expression by Western blot (Fig. 5B). We also observed enhanced osteogenic ability which was evident from increased expression of Runx2 and more alizarin red-positive nodules (Fig. 5C). These results indicated that AMPK/mTOR signaling pathway was related to the impairment of autophagy and osteogenesis of BMMSCs derived from high-carbohydrate, high-fat diet.
Fig. 5. AMPK/mTOR signaling pathway was related to the impairment of autophagy and osteogenesis of BMMSCs derived from high-fat diet rats. (A) Western blot was performed to examine expressions of p-mTOR, mTOR, p-Akt, Akt, p-AMPK, AMPK, LC3 and P62 protein in RD- and HFD-BMMSCs. (B) Western blot was performed to examine expressions of p-mTOR, mTOR, p-AMPK, AMPK, LC3 and P62 protein in HCHF-BMMSCs in the presence of AICAR. (C) Western blot was performed to examine expression of Runx2 protein and alizarin red staining was performed to detect mineralized nodules formed in HCHF-BMMSCs cultured in osteo-differentiation medium in the presence of AICAR. Data are reported as mean ± SD. *P < 0.05 Vs. RD group. RD: regulated diet. HCHF: high-carbohydrate, high-fat diet.