Correction: Runx1 is a central regulator of osteogenesis for bone homeostasis by orchestrating BMP and WNT signaling pathways.

[This corrects the article DOI: 10.1371/journal.pgen.1009233.].

Knockout and Double Knockout of Cathepsin K and Mmp9 reveals a novel function of Cathepsin K as a regulator of osteoclast gene expression and bone homeostasis.

Cathepsins play a role in regulation of cell function through their presence in the cell nucleus. However, the role of Cathepsin K (Ctsk) as an epigenetic regulator in osteoclasts remains unknown. Our data demonstrated that Ctsk-/-Mmp9-/- mice have a striking phenotype with a 5-fold increase in bone volume compared with WT. RNA-seq analysis of Ctsk-/- , Mmp9-/- and Ctsk-/-/Mmp9-/- osteoclasts revealed their distinct functions in gene expression regulation, including reduced Cebpa expression, increased Nfatc1 expression, and in signaling pathways activity regulation. Western blots and qPCR data validated these changes. ATAC-seq profiling of Ctsk-/- , Mmp9-/-, and Ctsk-/-/Mmp9-/- osteoclasts indicated the changes resulted from reduced chromatin openness in the promoter region of Cebpa and increased chromatin openness in Nfatc1 promoter in Ctsk-/-/Mmp9-/- osteoclasts compared to that in osteoclasts of WT, Ctsk/- and Mmp9-/- . We found co-localization of Ctsk with c-Fos and cleavage of H3K27me3 in wild-type osteoclasts. Remarkably, cleavage of H3K27me3 was blocked in osteoclasts of Ctsk-/- and Ctsk-/-/Mmp9-/- mice, suggesting that Ctsk may epigenetically regulate distinctive groups of genes' expression by regulating proteolysis of H3K27me3. Ctsk-/-/Mmp9-/- double knockout dramatically protects against ovariectomy induced bone loss. We found that Ctsk may function as an essential epigenetic regulator in modulating levels of H3K27me3 in osteoclast activation and maintaining bone homeostasis. Our study revealed complementary and unique functions of Ctsk as epigenetic regulators for maintaining osteoclast activation and bone homeostasis by orchestrating multiple signaling pathways and targeting both Ctsk and Mmp9 is a novel therapeutic approach for osteolytic diseases such as osteoporosis.

Spheroid co-culture of BMSCs with osteocytes yields ring-shaped bone-like tissue that enhances alveolar bone regeneration.

Oral and maxillofacial bone defects severely impair appearance and function, and bioactive materials are urgently needed for bone regeneration. Here, we spheroid co-cultured green fluorescent protein (GFP)-labeled bone marrow stromal cells (BMSCs) and osteocyte-like MLO-Y4 cells in different ratios (3:1, 2:1, 1:1, 1:2, 1:3) or as monoculture. Bone-like tissue was formed in the 3:1, 2:1, and 1:1 co-cultures and MLO-Y4 monoculture. We found a continuous dense calcium phosphate structure and spherical calcium phosphate similar to mouse femur with the 3:1, 2:1, and 1:1 co-cultures, along with GFP-positive osteocyte-like cells encircled by an osteoid-like matrix similar to cortical bone. Flake-like calcium phosphate, which is more mature than spherical calcium phosphate, was found with the 3:1 and 2:1 co-cultures. Phosphorus and calcium signals were highest with 3:1 co-culture, and this bone-like tissue was ring-shaped. In a murine tooth extraction model, implantation of the ring-shaped bone-like tissue yielded more bone mass, osteoid and mineralized bone, and collagen versus no implantation. This tissue fabricated by spheroid co-culturing BMSCs with osteocytes yields an internal structure and mineral composition similar to mouse femur and could promote bone formation and maturation, accelerating regeneration. These findings open the way to new strategies in bone tissue engineering.

GPR125 positively regulates osteoclastogenesis potentially through AKT-NF-κB and MAPK signaling pathways.

G-protein-coupled receptors (GPCRs) signaling is critical to cell differentiation and activation. However, the function of GPCRs in osteoclast differentiation and activation remains unclear. We found that the G-protein coupled receptor 125 (GPCR 125) gene (Gpr125) gene was highly expressed in osteoclasts through RNA-sequencing technology, qRT-PCR, and Western blot analysis. We characterized the role of GPCR125 in osteoclast differentiation and activation by loss-of-function and gain-of-function methods in osteoclasts. Osteoclasts with lentivirus-mediated GPR125 silencing demonstrated a dramatic reduction in differentiation and impaired bone resorption function. In contrast, overexpression of Gpr125 in osteoclasts increased NFATC1 expression and enhanced osteoclast differentiation and enhanced osteoclast-mediated bone resorption. These results indicated that GPCR125 positively regulates osteoclast formation and function. Following receptor activator of nuclear factor kappa-Β ligand (RANKL) stimulation, the expression levels of MAPK signaling pathway proteins phosphorylated-ERK (p-ERK) and phosphorylated-p38 (p-p38) were significantly decreased in the Gpr125 knockdown (sh-GPR125) group compared to its control group. We also found that phosphorylated AKT (p-AKT) expression was downregulated, as well as nuclear factor kappa-B (NF-κB) signaling pathway protein phosphorylated-IKB alpha (p-IKBα). Our results demonstrated that GPCR125 positively regulates osteoclasts via RANKL-stimulated MAPK and AKT-NF-κB signaling pathways, and GPCR125 could potentially be utilized as a novel therapeutic target in bone related diseases including osteoporosis.

Association of Sex Hormones and Fat Distribution in Men with Different Obese and Metabolic Statuses.

PURPOSE: Although several studies have explored the association of sex hormones with glucose metabolism, the association between sex hormones and body fat distribution, which is closely related to insulin resistance, has not been fully elucidated. We have tried to explore the relationship of testosterone (T) and estradiol (E2) with visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) mass in Chinese men with different obese and metabolic statuses. PATIENTS AND METHODS: A total of 128 men from the Health Management Center of the Second Xiangya Hospital, Central South University were collected and grouped in accordance with their obese and metabolic syndrome (MS) statuses: metabolically healthy non-overweight/obese men (MHNO), metabolically healthy overweight/obese men (MHO) and metabolically unhealthy overweight/obese men (MUO). Multiple regression analyses were performed to estimate contributions of sex hormones levels to the variations of body fat distribution and the contributions of body fat distribution to the variations of sex hormone levels. RESULTS: With fat mass parameters as independent variables, SAT had a strong negative association with T in MHNO (ß = -2.772, P = 0.034), VAT was positively correlated with E2 in MHO (ß = 22.269, P = 0.009), and SAT was negatively associated with T in MUO (ß = -3.315, P = 0.010). With sex hormones as independent variables, E2 positively correlated with VAT (ß = -176.259, P = 0.048), while T negatively correlated with VAT in MHO (ß = 183.150, P = 0.029). In MUO, an inverse association of T with SAT was observed (ß = -213.689, P = 0.021). CONCLUSION: E2 and VAT had a mutual influence, thus resulting in a vicious circle, and the negative correlation between T and VAT may be related to the decrease of the MS occurrence in the MHO group. There were bi-directional relationships between sex hormones and fat distribution in men with different obese and metabolic statuses. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR-EOC-16010194. Retrospectively registered.

Hyperglycemia and blood glucose deterioration are risk factors for severe COVID-19 with diabetes: A two-center cohort study.

We aimed to assess whether blood glucose control can be used as predictors for the severity of 2019 coronavirus disease (COVID-19) and to improve the management of diabetic patients with COVID-19. A two-center cohort with a total of 241 confirmed cases of COVID-19 with definite outcomes was studied. After the diagnosis of COVID-19, the clinical data and laboratory results were collected, the fasting blood glucose levels were followed up at initial, middle stage of admission and discharge, the severity of the COVID-19 was assessed at any time from admission to discharge. Hyperglycemia patients with COVID-19 were divided into three groups: good blood glucose control, fair blood glucose control, and blood glucose deterioration. The relationship of blood glucose levels, blood glucose control status, and severe COVID-19 were analyzed by univariate and multivariable regression analysis. In our cohort, 21.16% were severe cases and 78.84% were nonsevere cases. Admission hyperglycemia (adjusted odds ratio [aOR], 1.938; 95% confidence interval [95% CI], 1.387-2.707), mid-term hyperglycemia (aOR, 1.758; 95% CI, 1.325-2.332), and blood glucose deterioration (aOR, 22.783; 95% CI, 2.661-195.071) were identified as the risk factors of severe COVID-19. Receiver operating characteristic (ROC) curve analysis, reaching an area under ROC curve of 0.806, and a sensitivity and specificity of 80.40% and 68.40%, respectively, revealed that hyperglycemia on admission and blood glucose deterioration of diabetic patients are potential predictive factors for severe COVID-19. Our results indicated that admission hyperglycemia and blood glucose deterioration were positively correlated with the risk factor for severe COVID-19, and deterioration of blood glucose may be more likely to the occurrence of severe illness in COVID-19.

Changes in Bone Mineral Density Following Conventional Oral Phosphonate Treatment of Hypophosphatemic Osteomalacia: A Non-Randomized Controlled Study.

PURPOSE: There are limited clinical studies aimed at solving the problem of the efficiency of conventional treatment with oral phosphate and calcitriol in adults with hypophosphatemic osteomalacia (HO). In addition, there still had no good non-hazardous markers to evaluate the severity of bone loss of osteomalacia before and after treatment. Therefore, the purpose of this study was to assess the efficacy of conventional treatment with a self-blended phosphate supplementation and calcitriol on patients with HO and whether bone mineral density (BMD) can be helpful for monitoring the efficacy. PATIENTS AND METHODS: A total of 21 HO patients and 105 healthy controls were enrolled. All patients were tested for serum biomarkers and BMD of the lumbar spine (L1-L4), femoral neck, and total left hip. After three years of treatment, 11 of 21 HO patients were recalled for BMD measurement. According to the administration of drugs, HO patients with calcium and calcitriol were divided into three phosphate treatment groups: patients in group A (n = 3) received continuous phosphate supplementation, patients in group B (n = 5) received intermittent phosphate supplementation and patients in group C (n = 3) received no phosphate supplementation. RESULTS: The diagnoses of 21 HO patients were 5 cases of hereditary hypophosphatemic rickets, 4 cases of Fanconi syndrome with the features of renal tubular acidosis and vitamin D deficiency, and 12 cases of hereditary vitamin D abnormality. The average initial serum phosphorus level of the patient group was approximately 50% lower than that of the control group. Lower BMD was significantly observed in the HO group than the control group at the lumbar spine and total hip. Continuous treatment with the phosphate supplement could increase BMD in the lumbar spine and total hip by 33.4-52.3% and in the femoral neck increased by 43.2-79.3% compared with baseline, and the effect appears to be continued once treatment is discontinued. CONCLUSION: These findings suggest that conventional therapy can improve bone mineral defects in patients with HO, especially in the femoral neck. Detection of BMD in HO patients is a good tool to assess the extent of bone defects and the therapeutic effect. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR-OOC-16010095. Registered 7 December 2016. Retrospectively registered.

Long Non-coding RNA 332443 Inhibits Preadipocyte Differentiation by Targeting Runx1 and p38-MAPK and ERK1/2-MAPK Signaling Pathways.

Long non-coding RNAs (lncRNAs) have emerged as integral regulators of pathophysiological processes, but their specific roles and mechanisms in adipose tissue development remain largely unknown. Here, through microarray analysis, co-expression, and tissue specific analysis of adipocyte tissues after fasting for 72 h, we found that Lnc-FR332443 expression was dramatically decreased, as well as the expression of Runx1. The UCSC database and Ensembl database indicated that Lnc-FR332443 is the antisense lncRNA of Runx1. Lnc-FR332443 and Runx1 are highly enriched in adipose tissue and downregulated during adipogenic differentiation. Adipose tissue-specific knockdown of Lnc-FR332443 increased fat mass in vivo, and specific knockdown of Lnc-FR332443 in 3T3-L1 preadipocytes promoted adipogenic differentiation. In this process, Runx1 expression was decreased when Lnc-FR332443 was downregulated in adipocytes or 3T3-L1 preadipocytes, and vice versa, when Lnc-FR332443 was upregulated, the expression of Runx1 was increased. However, overexpression of Runx1 decreased the expression of the adipocyte cell marker genes PPARγ, C/EBPα and FABP4 significantly, while not affected the expression of Lnc-FR332443. Mechanistically, Lnc-FR332443 positively regulates Runx1 expression in mouse adipocytes and suppresses adipocyte differentiation by attenuating the phosphorylation of MAPK-p38 and MAPK-ERK1/2 expression. Thus, this study indicated that Lnc-FR332443 inhibits adipogenesis and which might be a drug target for the prevention and treatment of obesity.

Quantifying the electron-donating and -accepting capacities of wastewater for evaluating and optimizing biological wastewater treatment processes.

Biological processes have been widely used for the treatment of both domestic and industrial wastewaters. In such biological processes, pollutants are converted into pollution-free substances by microorganisms through oxidation-reduction reactions. Thus, how to quantify the internal oxidation-reduction properties wastewaters and seek out targeted countermeasures is essential to understand, operate, and optimize biological wastewater treatment systems. So far, no such approach is available yet. In this work, a novel concept of electron neutralization-based evaluation is proposed to describe the internal oxidation-reduction properties of wastewater. Pollutants in wastewater are defined as electron donor substances (EDSs) or electron acceptor substances (EASs), which could give or accept electrons, respectively. With such an electron neutralization concept, several parameters, i.e., electron residual concentration (R), economy-related index (E and Er), and economical evaluation index (Y and Yr), are defined. Then, these parameters are used to evaluate the performance and economic aspects of currently applied wastewater treatment processes and even optimize systems. Three case studies demonstrate that the proposed concept could be effectively used to reduce wastewater treatment costs, assess energy recovery, and evaluate process performance. Therefore, a new, simple, and reliable methodology is established to describe the oxidation-reduction properties of wastewater and assess the biological wastewater treatment processes.

Runx1 is a central regulator of osteogenesis for bone homeostasis by orchestrating BMP and WNT signaling pathways.

Runx1 is highly expressed in osteoblasts, however, its function in osteogenesis is unclear. We generated mesenchymal progenitor-specific (Runx1f/fTwist2-Cre) and osteoblast-specific (Runx1f/fCol1α1-Cre) conditional knockout (Runx1 CKO) mice. The mutant CKO mice with normal skeletal development displayed a severe osteoporosis phenotype at postnatal and adult stages. Runx1 CKO resulted in decreased osteogenesis and increased adipogenesis. RNA-sequencing analysis, Western blot, and qPCR validation of Runx1 CKO samples showed that Runx1 regulates BMP signaling pathway and Wnt/ß-catenin signaling pathway. ChIP assay revealed direct binding of Runx1 to the promoter regions of Bmp7, Alk3, and Atf4, and promoter mapping demonstrated that Runx1 upregulates their promoter activity through the binding regions. Bmp7 overexpression rescued Alk3, Runx2, and Atf4 expression in Runx1-deficient BMSCs. Runx2 expression was decreased while Runx1 was not changed in Alk3 deficient osteoblasts. Atf4 overexpression in Runx1-deficient BMSCs did not rescue expression of Runx1, Bmp7, and Alk3. Smad1/5/8 activity was vitally reduced in Runx1 CKO cells, indicating Runx1 positively regulates the Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 signaling pathway. Notably, Runx1 overexpression in Runx2-/- osteoblasts rescued expression of Atf4, OCN, and ALP to compensate Runx2 function. Runx1 CKO mice at various osteoblast differentiation stages reduced Wnt signaling and caused high expression of C/ebpα and Pparγ and largely increased adipogenesis. Co-culture of Runx1-deficient and wild-type cells demonstrated that Runx1 regulates osteoblast-adipocyte lineage commitment both cell-autonomously and non-autonomously. Notably, Runx1 overexpression rescued bone loss in OVX-induced osteoporosis. This study focused on the role of Runx1 in different cell populations with regards to BMP and Wnt signaling pathways and in the interacting network underlying bone homeostasis as well as adipogenesis, and has provided new insight and advancement of knowledge in skeletal development. Collectively, Runx1 maintains adult bone homeostasis from bone loss though up-regulating Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 and WNT/ß-Catenin signaling pathways, and targeting Runx1 potentially leads to novel therapeutics for osteoporosis.

Serum miR-503 is a Candidate Biomarker for Differentiating Metabolic Healthy Obesity from Metabolic Unhealthy Obesity.

PURPOSE: Overweight and obesity are associated with metabolic diseases. However, a subgroup of the overweight/obese population does not present metabolic abnormalities. Hence, there is an urgent need to identify biomarkers that can distinguish different obesity phenotypes and metabolic status. PATIENTS AND METHODS: A total of 98 individuals were divided into three groups: metabolically healthy normal weight (MHNW), metabolically healthy obese (MHO), and metabolically unhealthy obese (MUO). Participants were evaluated for anthropometric and biochemical parameters and serum BMPR1A concentration and miR-503 level. Receiver operating characteristic (ROC) curve analysis and logistic regression analysis were performed. RESULTS: The level of miR-503 was significantly higher in the MHO group compared with that in the MUO group, but no difference was observed between the MHNW and MHO groups. Meanwhile, no significant differences in serum BMPR1A concentration were observed between the three groups. ROC curve analysis showed that miR-503 could be used as a marker to distinguish the MUO from the MHO. Logistic regression analysis suggested that miR-503 was an important related factor associated with an unhealthy metabolic state in overweight/obese subjects. CONCLUSION: miR-503 can be considered as a suitable biomarker to distinguish between the MUO and MHO, which may be a related factor for the incidence of metabolic disorders in overweight/obese subjects.

Insulin receptor substrate-1 inhibits high-fat diet-induced obesity by browning of white adipose tissue through miR-503.

Genetic variation of insulin receptor substrate 1 (IRS-1) was found to modulate the insulin resistance of adipose tissues, but the underlying mechanism was not clear. To investigate how the IRS-1 was involved in the browning of white adipose tissue through miRNA, we identified a mutated Irs-1 (Irs-1-/- ) mice model and found that this mice had a reduced subcutaneous WAT (sWAT) and increased brown adipose tissue (BAT) in the interscapular region. So we isolated the bone marrow stromal cells and analyzed differentially expressed miRNAs and adipogenesis-related genes with miRNA arrays and PCR arrays. Irs-1-/- mice showed decreased miR-503 expression, but increased expression of its target, bone morphogenetic protein receptor type 1a (BMPR1a). Overexpression of miR-503 in preadipocytes downregulated BMPR1a and impaired adipogenic activity through the phosphotidylinositol 3-kinase (PI3K/Akt) pathway, while the inhibitor had the opposite effect. In both Irs-1-/- and cold-induced models, sWAT exhibited BAT features, and showed tissue-specific increased BMPR1a expression, PI3K expression, and Akt phosphorylation. Thus, our results showed that IRS-1 regulated brown preadipocyte differentiation and induced browning in sWAT through the miR-503-BMPR1a pathway, which played important roles in high-fat diet-induced obesity.

Runx1 up-regulates chondrocyte to osteoblast lineage commitment and promotes bone formation by enhancing both chondrogenesis and osteogenesis.

One of the fundamental questions in bone biology is where osteoblasts originate and how osteoblast differentiation is regulated. The mechanism underlying which factors regulate chondrocyte to osteoblast lineage commitment remains unknown. Our data showed that Runt-related transcription factor 1 (Runx1) is expressed at different stages of both chondrocyte and osteoblast differentiation. Runx1 chondrocyte-specific knockout (Runx1f/fCol2α1-cre) mice exhibited impaired cartilage formation, decreased bone density, and an osteoporotic phenotype. The expressions of chondrocyte differentiation regulation genes, including Sox9, Ihh, CyclinD1, PTH1R, and hypertrophic chondrocyte marker genes including Col2α1, Runx2, MMP13, Col10α1 in the growth plate were significantly decreased in Runx1f/fCol2α1-cre mice chondrocytes. Importantly, the expression of osteoblast differentiation regulation genes including Osx, Runx2, ATF4, and osteoblast marker genes including osteocalcin (OCN) and osteopontin (OPN) were significantly decreased in the osteoblasts of Runx1f/fCol2α1-cre mice. Notably, our data showed that osteoblast differentiation regulation genes and marker genes are also expressed in chondrocytes and the expressions of these marker genes were significantly decreased in the chondrocytes of Runx1f/fCol2α1-cre mice. Our data showed that chromatin immunoprecipitation (ChIP) and promoter mapping analysis revealed that Runx1 directly binds to the Indian hedgehog homolog (Ihh) promoter to regulate its expression, indicating that Runx1 directly regulates the transcriptional expression of chondrocyte genes. Collectively, we revealed that Runx1 signals chondrocyte to osteoblast lineage commitment and promotes endochondral bone formation through enhancing both chondrogenesis and osteogenesis genes expressions, indicating Runx1 may be a therapeutic target to enhance endochondral bone formation and prevent osteoporosis fractures.

Associations of Salivary BPIFA1 Protein in Chronic Periodontitis Patients with Type 2 Diabetes Mellitus.

AIMS: To explore the differences in salivary BPI fold containing family A, member 1 (BPIFA1) concentration among type 2 diabetes mellitus (T2DM) subjects with various severities of chronic periodontitis and to determine whether BPIFA1 in saliva can be used as a potential biomarker of T2DM. METHODS: Unstimulated saliva samples were collected from 44 subjects with T2DM and 44 without T2DM (NDM). Additionally, demographic data and general health parameters, including fasting blood glucose (FBG) and body mass index (BMI), were collected. We also detected full-mouth clinical periodontal parameters including probing pocket depth (PPD), clinical attachment level (CAL), bleeding index (BI), and plaque index (PLI). Salivary BPIFA1, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) concentrations were also detected. RESULTS: BPIFA1 in saliva was detected at relatively high levels. T2DM subjects had decreased salivary BPIFA1 concentrations (P = 0.031). In T2DM subjects with nonperiodontitis or severe periodontitis, the level of BPIFA1 was significantly lower compared with that of NDM. Salivary TNF-α concentration displayed a similar trend to BPIFA1 in the NDM group. CONCLUSIONS: BPIFA1 protein is rich in saliva and might be used as a potential predictive biomarker of T2DM, especially in patients with severe periodontitis and nonperiodontitis. This trial is registered with ChiCTR-ROC-17010310.

IRS-2 Partially Compensates for the Insulin Signal Defects in IRS-1-/- Mice Mediated by miR-33.

Insulin signaling is coordinated by insulin receptor substrates (IRSs). Many insulin responses, especially for blood glucose metabolism, are mediated primarily through Irs-1 and Irs-2. Irs-1 knockout mice show growth retardation and insulin signaling defects, which can be compensated by other IRSs in vivo; however, the underlying mechanism is not clear. Here, we presented an Irs-1 truncated mutated mouse (Irs-1-/-) with growth retardation and subcutaneous adipocyte atrophy. Irs-1-/- mice exhibited mild insulin resistance, as demonstrated by the insulin tolerance test. Phosphatidylinositol 3-kinase (PI3K) activity and phosphorylated Protein Kinase B (PKB/AKT) expression were elevated in liver, skeletal muscle, and subcutaneous adipocytes in Irs-1 deficiency. In addition, the expression of IRS-2 and its phosphorylated version were clearly elevated in liver and skeletal muscle. With miRNA microarray analysis, we found miR-33 was down-regulated in bone marrow stromal cells (BMSCs) of Irs-1-/- mice, while its target gene Irs-2 was up-regulated in vitro studies. In addition, miR-33 was down-regulated in the presence of Irs-1 and which was up-regulated in fasting status. What's more, miR-33 restored its expression in re-feeding status. Meanwhile, miR-33 levels decreased and Irs-2 levels increased in liver, skeletal muscle, and subcutaneous adipocytes of Irs-1-/- mice. In primary cultured liver cells transfected with an miR-33 inhibitor, the expression of IRS-2, PI3K, and phosphorylated-AKT (p-AKT) increased while the opposite results were observed in the presence of an miR-33 mimic. Therefore, decreased miR-33 levels can up-regulate IRS-2 expression, which appears to compensate for the defects of the insulin signaling pathway in Irs-1 deficient mice.

Insulin-like growth factor-1 promotes osteogenic differentiation and collagen I alpha 2 synthesis via induction of mRNA-binding protein LARP6 expression.

This study explored the mechanism underlying the stimulation of collagen synthesis and osteoblastic differentiation by insulin-like growth factor 1 (IGF1) in primary mouse osteoblasts. Primary mouse calvarial osteoblasts were cultured and treated with various doses of IGF1 before transfection with siRNA targeting the collagen type I alpha 2 (Col1a2) or La ribonucleoprotein domain family member 6 (Larp6) genes. Alkaline phosphatase (ALP) activity, osteocalcin staining, alizarin red quantification and the expression level of runt-related transcription factor 2 (RUNX2) were performed to assess the differentiation of pre-osteoblasts. Based on Western blot analysis, IGF1 up-regulated COL1A2 protein expression in the primary osteoblasts in a dose- and time-dependent manner. In addition, Col1a2 interference inhibited the differentiation and mineralization of osteoblasts. IGF1 also stimulated the differentiation of mouse primary osteoblasts and increased LARP6 expression during osteogenic differentiation. RNA-Immunoprecipitation (IP) indicated that LARP6 could bind to Col1a2 mRNA after IGF1 stimulation. However, transfection of Larp6-specific siRNA significantly reduced collagen and ALP secretion, mineralization and inhibited the expression of osteocalcin and RUNX2, indicating that Larp6 interference inhibited the differentiation ability of primary mouse calvarial osteoblasts, and these effects could not be reversed by IGF1. Thus, IGF1 could promote COL1A2 expression and osteoblast differentiation in primary mouse calvarial pre-osteoblasts by increasing LARP6 expression via a posttranscriptional mechanism.

Genetic diagnosis and treatment of a Chinese ketosis-prone MODY 3 family with depression.

BACKGROUND: To analyze the gene mutation and mental disorder of a Chinese ketosis-prone diabetes (KPD) family, and to make a precise diagnosis and give a treatment for them. METHODS: We studied a Chinese family with a clinical diagnosis of maturity-onset diabetes of the young (MODY). The clinical data and the blood samples were collected. The promotor and coding regions inclusive intron exon boundaries of the HNF1A, HNF4A were detected by polymerase chain reaction (PCR) and direct sequencing. The missense mutation was also analyzed by bioinformatics. Genetic counseling was performed twice a month to relieve the mental disorder of the persons. RESULTS: The missense mutation c.779 C>T (p.T260M) in exon4 of HNF1A gene was detected, and the symptom heterogenicity among persons in this family were found. All the members were retreated with Gliclazide and stopped to use other medicine, the blood glucose of them were well controlled. We also performed an active genetic counseling to them and the mental disorder of the proband's sister was relieved. CONCLUSIONS: A missense mutation of HNF1A gene was first found in Chinese ketosis-prone MODY family with manifestations heterogenicity among the persons. Sulphonylureas medicine and genetic counseling are efficiency ways to treat MODY 3 and its' mental disorder respectively.

Plasticity of adipose tissue in response to fasting and refeeding in male mice.

BACKGROUND: Fasting is the most widely prescribed and self-imposed strategy for treating excessive weight gain and obesity, and has been shown to exert a number of beneficial effects. The aim of the present study was to determine the exact role of fasting and subsequent refeeding on fat distribution in mice. METHODS: C57/BL6 mice fasted for 24 to 72 h and were then subjected to refeeding for 72 h. At 24, 48 and 72 h of fasting, and 12, 24, 48 and 72 h of refeeding, the mice were sacrificed, and serum and various adipose tissues were collected. Serum biochemical parameters, adipose tissue masses and histomorphological analysis of different depots were detected. MRNA was isolated from various adipose tissues, and the expressions of thermogenesis, visceral signature and lipid metabolism-related genes were examined. The phenotypes of adipose tissues between juvenile and adult mice subjected to fasting and refeeding were also compared. RESULTS: Fasting preferentially consumed mesenteric fat mass and decreased the cell size of mesenteric depots; however, refeeding recovered the mass and morphology of inguinal adipose tissues preferentially compared with visceral depots. Thermogenesis-related gene expression in the inguinal WAT and interscapular BAT were suppressed. Mitochondrial biogenesis was affected by fasting in a depot-specific manner. Furthermore, a short period of fasting led to an increase in visceral signature genes (Wt1, Tcf21) in subcutaneous adipose tissue, while the expression of these genes decreased sharply as the fasting time increased. Additionally, lipogenesis-related markers were enhanced to a greater extent greater in subcutaneous depots compared with those in visceral adipose tissues by refeeding. Although similar phenotypic changes in adipose tissue were observed between juvenile mice and adult mice subjected to fasting and refeeding, the alterations appeared earlier and more sensitively in juvenile mice. CONCLUSIONS: Fasting preferentially consumes lipids in visceral adipose tissues, whereas refeeding recovers lipids predominantly in subcutaneous adipose tissues, which indicated the significance of plasticity of adipose organs for fat distribution when subject to food deprivation or refeeding.

Insulin receptor substrate-1 time-dependently regulates bone formation by controlling collagen Iα2 expression via miR-342.

Insulin promotes bone formation via a well-studied canonical signaling pathway. An adapter in this pathway, insulin-receptor substrate (IRS)-1, has been implicated in the diabetic osteopathy provoked by impaired insulin signaling. To further investigate IRS-1's role in the bone metabolism, we generated Irs-1-deficient Irs-1smla/smla mice. These null mice developed a spontaneous mutation that led to an increase in trabecular thickness (Tb.Th) in 12-mo-old, but not in 2-mo-old mice. Analyses of the bone marrow stromal cells (BMSCs) from these mice revealed their differential expression of osteogenesis-related genes and miRNAs. The expression of miR-342, predicted and then proven to target the gene encoding collagen type Iα2 (COL1A2), was reduced in BMSCs derived from Irs-1-null mice. COL1A2 expression was then shown to be age dependent in osteoblasts and BMSCs derived from Irs-1smla/smla mice. After the induction of osteogenesis in BMSCs, miR-342 expression correlated inversely with that of Col1a2 Further, Col1a2-specific small interfering RNA (siRNA) reduced alkaline phosphatase (ALP) activity and inhibited BMSC differentiation into osteocyte-like cells, both in wild-type (WT) and Irs-1smla/smla mice. Conversely, in Irs-1smla/smla osteocytes overexpressing COL1A2, ALP-positive staining was stronger than in WT osteocytes. In summary, we uncovered a temporal regulation of BMSC differentiation/bone formation, controlled via Irs-1/miR-342 mediated regulation of Col1a2 expression.-Guo, Y., Tang, C.-Y., Man, X.-F., Tang, H.-N., Tang, J., Wang, F., Zhou, C.-L., Tan, S.-W., Feng, Y.-Z., Zhou, H.-D. Insulin receptor substrate-1 time-dependently regulates bone formation by controlling collagen Iα2 expression via miR-342.

MiR-503 inhibits adipogenesis by targeting bone morphogenetic protein receptor 1a.

Adipogenesis plays a key role in the regulation of whole-body energy homeostasis and is critically related to obesity. To overcome obesity and its associated disorders, it is necessary to elucidate the molecular mechanisms involved in adipogenesis. An adipogenesis-related miRNA array analysis demonstrated that miR-503 was differentially expressed before and after adipocyte differentiation; however, the exact role of miR-503 in adipocyte differentiation is unclear. Thus, the objective of this study was to further examine miR-503 in adipocyte differentiation. We found significantly decreased expression of miR-503 during adipocyte differentiation process. Using bioinformatic analysis, miR-503 was identified as a potential regulator of Bone Morphogenetic Protein Receptor 1a (BMPR1a). We then validated BMPR1a as the target of miR-503 using a dual luciferase assay, and found decreased miR-503 and increased BMPR1a expression during adipogenesis. Overexpression of miR-503 in preadipocytes repressed expression of BMPR1a and adipogenic-related factors such as CCAAT/enhancer binding protein a (C/EBPα), proliferator-activated receptor-gamma (PPARγ), and adipocyte protein 2 (AP2). In addition, miR-503 overexpression impaired the phosphoinositol-3 kinase (PI3K)/Akt pathway. Inhibition of miR-503 had the opposite effect. Additionally, BMPR1a interference by siRNA attenuated adipocyte differentiation and the accumulation of lipid droplets via downregulating the PI3K/Akt signaling pathway. Our study provides the first evidence of the role miR-503 plays in adipocyte differentiation by regulating BMPR1a via the PI3K/Akt pathway, which may become a novel target for obesity therapy.