| IntroductionOsteoporosis is a systemic skeletal disease, with the characteristics of reduction in bone mass, deterioration in bone microstructure, increase in brittleness, and inclination for bone fracture. Osteoporosis frequently occurs in elder people, especially postmenopausal women.Bone is a dynamic tissue that undergoes continuous remodeling/renewal to maintain skeletal appearance and structural integrity. In adults, bone destruction and formation are kept in a dynamic balance, and bone mass is maintained in a steady state, which is determined by mechanical usage and largely by homeostatic factors. Starting from the fifth decade of life or menopause for women, bone destruction begins to exceed bone formation, resulting in local or systemic bone loss and bone fragility, vertebrae collapses, fractures, and ultimately disabling the mobility of individuals having bone-related disorders such as osteoporosis.The increasing risk of osteoporotic fracture in seniors is associated with progressive loss of cancellous and cortical bone, partially caused by reduced osteoblastic bone formation. Recently, several studies have unveiled a link between accumulation of bone marrow fat and age-related bone loss, as in the cases of glucocorticoid treatment, immobilization, and ovariectomy. Marrow fat not only exerts a passive role by simply occupying the space, but also participates in lipid metabolism and functions as a localized energy reservoir, thus supporting blood cell lineage and osteoblasts in bone formation. Adipocytes may influence bone formation through the secretion of fatty acids and adipokines that regulate the differentiation of marrow precursors into osteoblasts. There is an inverse correlation between accumulation of adipocyte in bone marrow tissue and bone volume in osteoporotic patients. An increasing body of evidence supports a reciprocal relationship between MSC-derived adipocytes and MSC-derived osteoblasts. Bone marrow mesenchymal stem cells (BMSCs) have the capacity for renewal and the potential to differentiate into several cell types including osteoblasts, chondrocytes, adipocytes, astrocytes, myocytes, oligodendrocytes, and neurons. Differentiation of BMSCs into osteoblast and adipocyte lineages is particularly related to the maintenance of normal bone homeostasis.Therefore, the underlying mechanism, which regulates the differential direction of BMSCs is crucial for the study of etiology of osteoporosis. Particularly, Wnt signal transduction pathway regulates cell differentiation and growth via paracrine or autocrine. The canonical Wnt signaling pathway is activated when Wnts interact with Lrp/Fzd receptor complexes. Receptor engagement activates an unknown kinase(s)(K) that phosphorylates the cytoplasmic tail of Lrp5/6. These phosphorylated residues (P) serve as docking sites for Axin, APC, Dsh, and β-Catenin complex. The interaction results in the inhibition of GSK-3activity, causing the prevention of β-Catenin degradation and accumulation of β-Catenin in the cytoplasm. Upon reaching a certain concentration level, β-Catenin translocates to the nucleus where it associates with the Tcf/Lef family of transcription factors to regulate the expression of the target genes and bone formation.ObjectivesTo demonstrate that adipocytes have an inhibitory effect on osteoblastogenesis and elucidate the underlying molecular mechanisms during adipocyte-regulated osteoblastogenesis.Methods1. hBMSCs were cultured in two types of co-culture modes. In each mode, three groups were designated according to the quantity of adipocytes as follows:Group i, hBMSCs were treated without adipogenic induction; Group ii, hBMSCs were stimulated under an adipogenic condition using a half dose of stimulators; Group iii, hBMSCs were stimulated under a standard adipogenic condition. Mode A, co-culture with intercellular contact (direct regulation):osteoblasts and adipocytes were present in the same plate. Briefly, human hBMSCs were induced into adipocytes according to the above grouping for14days, followed by a uniform osteogenic stimulation in adipocyte maintenance medium for another14days.Mode B, co-culture without intercellular contact (indirect regulation):osteoblasts and adipocytes were plated on separate surfaces within the same well of transwell plates. Briefly, human hBMSCs were induced into adipocytes according to the above grouping for14days within the netwell inserts. At the same time, human hBMSCs were cultured on the lower surfaces of the Snapwell. These two parts were later assembled and cells were maintained in adipocyte maintenance medium using uniform osteogenic stimulation as above for another14days.2. To analyze the activity of the alkaline phosphatase (ALP) on day14, we performed ALP staining and quantitative ALP activity assay using the Alkaline Phosphatase Activity Assay Kit in Mode A and B. To analyze mineralization on day28, Alizarin Red S staining was performed in Mode A. To examine adipogenesis on day14, we performed oil red staining in Mode A. Proteins were separately extracted from the insert wells and snap wells in Mode B to determine the expression levels of osteoblastogenesis marker-Runx2and adipogenesis marker-Ppary in Mode B.3. To understand the influence of MSC-derived adipocytes on osteoblast differentiation, mRNA expression profile in osteoblasts from Snapwells in Groups i, ii, and iii in Mode B were analyzed using mRNA microarray. The expression level of S100A6mRNA was validated by RT-PCR.4. To study the mechanism underlying the influence of adipocytes on osteoblastogenesis during MSC differentiation, we studied changes in protein expression during adipocyte-regulated osteoblastogenesis. Total proteins from cultured cells in Mode B were extracted. The proteins were separated using immobilized pH gradients2-DE and visualized by Sypro-Ruby staining. The differential protein spots were cut from the gels and subjected to in-gel digestion with trypsin. The peptides were desalted by using C18ziptips, and then subjected to identification by MALDI-TOF/TOF MS. The expression levels of Runx2, β-Catenin, S100A6and Calreticulin were confirmed using Western blot analysis.5. To further understand the mechanism underlying inhibition on osteoblastogenesis by adipocytes, medium proteins from adipocyte maintenance medium from Mode B on day4after osteogenic stimulation were analyzed using a human adipokine array.6. Using the ELISA Development kit, we determined the amount of active TGF-β1in the co-culture media for MSCs from Group i to iii in Mode B after4,7,10, and14days.7. Anti-human TGF-P neutralizing antibodies were added to both the upper and lower chambers in group iii, at5μg/ml (treatment2, Trt2) and10μg/ml (treatment3, Trt3) in Mode B at different time points,0day,4days,7days and10days of the culture. Equal volume of PBS was applied in negative control (treatment1, Trt1). After14days of co-culture, cells were collected for ALP staining, analysis of ALP activity and Western blot.Results1. In mode A, the quantity of adipocytes, as shown in oil red staining, increased significantly from Group i to Group iii, while the number of osteoblasts reduced progressively from Group i to Group iii as shown in ALP-positive area and calcified area. The inverse correlation of MSC-derived adipocytes and osteoblasts was also shown in the indirect co-culture Mode B. The results of Western blot analysis showed that Runx2was significantly down-regulated in Groups ii and iii compared to Group i. Conversely, the levels of Ppary were low in Group i but significantly higher in Groups ii and iii.2. The results from mRNA microarray revealed that247genes were significantly down-regulated, and163genes were substantially up-regulated when comparing Group i to Groups ii and iii. The genes related to osteoblastogenesis, COL1A1, Notchl and S100A6, were over-expressed in Group i, and significantly down-regulated in the other groups; yet the genes related to adipogenesis, Calreticulin, Ppary and SMAD6were low in Group i but up-regulated in Groups ii and iii. The expression of S100A6was validated by RT-PCR. Among the differentially expressed proteins, proteins related to osteoblast functions, such as Annexin A2and S100A6, were down-regulated significantly in Groups ii and iii compared to Group i. In contrast, the level of Calreticulin was low in Group i, but up-regulated remarkably in Groups ii and iii. The differentially expressed proteins were further verified by Western blot analysis. S100A6and Calreticulin appeared in both mRNA and protein expression profiles.3. Among the differential expressions of secreted adipokines, there were23adipokines, such as TGF-β, which were significantly up-regulated in Groups ii and iii as compared to Group i; on the other hand, there were25adipokines, such as MCP-3, which were remarkably down-regulated in Group ii and iii as compared to Group i. ELISA assay shows that the levels of active TGF-P were much higher in the media of Group ii and iii. The difference was detected starting from day4and lasted for14days. To illustrate TGF-P regulation of osteoblastogenesis, we neutralized the TGF-β produced by adipocytes in Group iii of the co-culture. The percentage of osteoblasts and the ALP activity after TGF-P neutralizing were increased in Trt2and Trt3compared to Trt1. The expression levels of bone formation markers such as Runx2, S100A6and β-Catenin were significantly up-regulated in Trt2and Trt3compared to Trt1. Conversely, the level of Calreticulin was high in Trt1but significantly reduced in Trts2and3by Western blot analysis.ConclusionIn summary, the present study demonstrates that adipocytes regulate osteoblast differentiation indirectly through secreted factors. The increase in TGF-P in the medium, in accord with the increase in adipocyte numbers, could affect the expressions of calreticulin and S100A6to suppress canonical Wnt signaling activity and osteoblast differentiation. Our study elucidates the mechanism by which adipocytes regulate osteoblast differentiation, thus providing clues for how TGF-P inhibits osteoblast differentiation in later phases. These results revealed partial reasons for reduction in bone mass and bone formation in osteoporotic patients, offering new clues for further study on the inhibitory mechanism of adipocytes on bone formation in the future. Meanwhile, our study discovered new target proteins for the prevention and treatment of osteoporosis. IntroductionType2diabetes is one of the most common endocrine diseases in developed countries, which is caused by absolute or relative deficiencies in insulin secretion or insulin action. Chronic hyperglycemia is the major biochemical alteration in type2diabetes. Moreover, individuals with type2diabetes usually display a marked disruption of lipid metabolism, with an abnormal accumulation of fat in various tissues including the liver. Hyperglycemia and hyperlipidemia not only impair β-cell function and increase insulin resistance in peripheral tissues, such as the muscle, liver and adipose tissue, but also induce oxidative stress reactions, which cause initiation and progression of diabetes-related diseases such as diabetic cardiovascular disease, diabetic nephropathy and diabetic retinopathy. Consequently, regulation of dyslipidemia and reduction in oxidative stress have been regarded as important treatment methods for alleviating diabetes and its complications.Phlorizin(glucose,1-[2-(p.D-glucopyranosyloxy)-4,6-dihydroxyphenyl]-3-(4-hyd roxyphenyl)-1-propanone) is a member of the chalcone class of organic compounds and is mainly distributed in the plants of the genus Malus. Phlorizin has multiple pharmacological activities, such as anti-oxidative, estrogenic and anti-estrogenic activities, memory improvement and cardioprotective activities. Reports show that phlorizin inhibits intestinal glucose uptake and renal glucose reabsorption by inhibiting the sodium D-glucose cotransporter. Phlorizin has been reported to normalize the effects of insulin on glucose metabolism in the liver and other peripheral tissues. However, little is known regarding the effect of phlorizin on hepatic damage associated with type2diabetes. With the completion of human genome sequencing project, life science has entered post-genomic era. Proteomics is regarded as the most important part in post-genomic studies. The rapidly emerging field of quantitative proteomics provides a powerful technique-isobaric tag for relative and absolute quantitation (iTRAQ) labeling combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), for the identification and characterization of protein profiles. This technique can lessen variation, enhance throughput and enable quantitative analysis.ObjectiveTo investigate the protective mechanism of phlorizin on hepatic damage in diabetic db/db mice through profiling the global protein expression changes.Methods1. Male C57BLKS/J db/db and age-matched db/m mice (n=24,7weeks of age) were kept under observation for one week prior to the start of the experiments. C57BLKS/J db/m mice were selected as the control group (n=8). The db/db mice were randomly divided into two groups:the vehicle-treated diabetic group (DM, n=8) administered normal saline solution and the other diabetic group treated with phlorizin at a dosage of20mg/kg (DMT, n=8). Phlorizin was administered in normal saline solution by intragastric administration for10weeks. Each group of mice was observed without any administration of other hypoglycemic therapy throughout the experiment. At the end of the intervention, all mice were fasted overnight and then sacrificed. Fasting blood was collected, and the liver tissue was dissected. The tissues and sera were kept at-80℃until further analysis.2. Animals were weighed every week. The levels of fasting blood glucose (FBG), blood triglycerides (TG) and blood total cholesterol (TC) were determined using DVI-1650Automatic Biochemistry and Analysis Instrument (Bayer, Germany) at the end of the treatment.3. In order to ascertain whether phlorizin has any beneficial effect on hepatocellular damage in db/db mice, the excised parts of the livers were immediately fixed in10%paraformaldehyde and embedded in paraffin. After solidification,5-μm sections were cut from the blocks. After hematoxylin and eosin (H&E) staining, the sections were examined using light microscopy.4. To investigate the protective mechanism of phlorizin on hepatic damage in diabetic db/db mice, we studied the global protein expression changes in the liver using iTRAQ approach. Total proteins were extracted from the pooled liver tissue from each of four mice from each group. After the proteins were digested using filter aided proteome preparation (FASP), the peptides of each group was labeled with iTRAQ reagents (114for the peptides of the control group,116for the peptides of the DMT group, and117for the peptides of the DM group). The labeled samples were separated by strong cation exchange (SCX) chromatography. Mass spectrometric analysis was performed using a micro-liquid chromatography system and a LTQ-Velos ion trap mass spectrometer. For protein identification and statistical validation, the acquired MS/MS spectra were automatically searched against the non-redundant International Protein Index (IPI) mouse protein database (version3.72) using the Turbo SEQUEST program in the Bio WorksTM3.1software suite. Data filtering parameters were chosen to generate false-positive protein identification rates of<1%.5. The differentially expressed genes were analyzed through the use of Ingenuity Pathway Analysis.6. The expression levels of CAT,ACACA,HMGCS2and NSDHL were confirmed using Western blot analysis.Results1. The initial, final, and gain in body weight in the DM group were significantly higher than those values in the control group. However, the body weight gain was significantly inhibited from the second week after phlorizin administration in the DMT group compared to the DM group.The levels of FBG, TG and TC in the DM group were markedly elevated compared with the levels in the control group. Moreover, these three parameters were significantly decreased by10weeks of phlorizin administration in the DMT group when compared with the DM group.2. The degree of hepatocellular damage was higher in the livers of the DM group than that of the control group. Yet, phlorizin treatment significantly ameliorated the hepatocellular damage in DMT group.3. Protein profiling was analyzed using iTRAQ approach. A total of1821proteins were identified. A strict cutoff value of a1.5-fold change was used for identification of differential proteins. Two hundred and fifteen proteins were elevated in the DM group compared with the control group and were then inhibited by phlorizin treatment. Forty-six proteins were inhibited in the DM group compared with control group and then restored by phlorizin treatment. The combination of enhancement in glycolysis and suppression in pyruvate oxidation leads to accumulation of mitochondrial acetyl-CoA, which can be transported into the cytosol and utilized for de novo biosynthesis of fatty acids and cholesterol. The key-limiting enzymes in glycolysis and oxidation of pyruvate were down-regulated in the DMT group compared to the DM group. Meanwhile, the key enzymes involved in the TCA cycle were up-regulated in the DMT group, indicating the reduction in the amount of acetyl-CoA. The rate-limiting enzymes for fatty acid biosynthesis and cholesterol biosynthesis were down-regulated in the DMT group compared to the DM group. However, in the DM group, several enzymes involved in fatty acid β-oxidation were down-regulated. Part of the inhibition was reversed by phlorizin administration. Meanwhile, the key anti-oxidative enzymes appeared to be down-regulated in the DM group but were stimulated again by phlorizin treatment. The enzymes involved in generation of superoxide and hydrogen peroxide was decreased in the DMT group.4. Ingenuity Pathway Analysis with all261differentially expressed proteins showed that most of the proteins were from the cytoplasm and the top-ranked biological functions included lipid metabolism and free radical scavenging. The top two protein networks generated by the pathway analysis consisted of proteins involved in lipid metabolism, energy production and small molecule biochemistry, which was consistent with the biological function of phlorizin.5. Four candidate proteins were validated using western blot analysis. ACACA, HMGCS2and NSDHL were found to be inhibited whereas CAT was enhanced in the DMT group compared to the DM group. This result verified the reliability of the iTRAQ results.ConclusionFor the first time, we established quantitative iTRAQ profiles of global liver proteins in a db/db diabetic mouse model treated with or without phlorizin. Phlorizin ameliorated hyperlipidemia and oxidative stress by affecting the expression of a set of proteins which were involved in carbohydrate metabolism, fatty acid synthesis and (3-oxidation, cholesterol biosynthesis, and free radical scavenging in the mouse liver. At the same time, phlorizin successfully decreased body weight, blood glucose, blood TG, and blood TC. Our study provides novel information in regards to the mechanisms of phlorizin on type2diabetic hepatic damage. Phlorizin, as a natural product, may become an effective therapeutic agent in the treatment of diabetes mellitus, particularly for complications in the liver.
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