Hou-De Zhou

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.

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‐1s role in the bone metabolism, we generated Irs‐1‐deficient Irs‐1smla/smla mice. These nullmice developed a spontaneousmutation that led to an increase in trabecular thickness (Tb. Th) in12‐mo‐old, butnot in 2‐mo‐oldmice. Analyses of the bonemarrowstromal cells (BMSCs) fromthese 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 Ia2 (COL1A2), was reduced in BMSCs derived fromIrs‐1‐nullmice. COL1A2expressionwas thenshowntobe agedependent in osteoblasts and BMSCsderivedfrom 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 BMSCdifferentiation into osteocyte‐like cells, both inwild‐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. FASEB J. 30, 4214–4226 (2016). www.fasebj.org

Effect of nicotinamide mononucleotide on insulin secretion and gene expressions of PDX-1 and FoxO1 in RIN-m5f cells.

OBJECTIVE To investigate the effect of nicotinamide mononucleotide (NMN) on insulin secretion and gene expressions of pancreatic and duodenal homeobox 1(PDX-1) and forkhead box-containing protein O-1 (FoxO1), which were important transcription factors for insulin secretion. METHODS Insulin secretion level in RIN-m5f cells was detected by rat insulin ELISA detection kit. The mRNA expression levels of PDX-1 and FoxO1 in RIN-m5f cells were analyzed by real-time PCR. The protein expression of PDX-1 was measured by Western blot. RESULTS Insulin secretion levels in RIN-m5f cells treated with repaglinide (10 nmol/L) plus NMN (100 μmol/L) was significantly higher than those in the blank control, the DMSO control group, and the NMN (50 μmol/L) treated group (P<0.05). The mRNA expression levels of PDX-1 in RIN-m5f cells treated with NMN (10, 50 and 100 μmol/L) for 36 h were significantly higher than those in the control group (P<0.05, P<0.01, and P<0.001, respectively). There was marked differences in the mRNA expression levels of PDX-1 among different concentrations of NMN (P<0.001), but no significant differences in the mRNA expression level of FoxO1 (P>0.05). No significant difference was found in the protein expression levels of PDX-1 in RIN-m5f cells treated by NMN (50, 100, and 200 μmol/L) for 36 or 48 h compared with the control group (P>0.05). CONCLUSION NMN can stimulate insulin secretion and upregulate the mRNA expression of PDX-1 in RIN-m5f cells.

Effect of docetaxel on expression of eIF3a in human lung cancer A549 cell line.

OBJECTIVE To explore the dose-dependent and time-dependent effect of docetaxel on the expression of mammalian eukaryotic initiation factor 3 subunit A (eIF3a) in lung cancer cell line. METHODS The human lung cancer cell line A549 was treated with gradient concentrations of docetaxel for different time. Real-time PCR and Western blot were used to detect mRNA and protein expression levels of eIF3a and alpha-tubulin, respectively. RESULTS Docetaxel did not affect alpha-tubulin expression at either mRNA level or protein level. When A549 cells were treated with high concentration of docetaxel (30 μg/L), the expression level of eIF3a mRNA tended to increase in a time-dependent manner. Protein expression level of alpha-tubulin was not associated with eIF3a expression significantly in cells treated by docetaxel. CONCLUSION Docetaxel could slightly increase the expression of eIF3a mRNA, and eIF3a does not regulate the expression of alpha-tubulin in A549 cells treated by docetaxel.

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.

Effects of Hybrid Coat on shear bond strength of five cements: an in vitro study

PURPOSE To evaluate the sealing performance of Hybrid Coat and its influence on the shear bond strength of five dentin surface cements. MATERIALS AND METHODS Six premolars were pretreated to expose the dentin surface prior to the application of Hybrid Coat. The microscopic characteristics of the dentinal surfaces were examined with scanning electron microscopy (SEM). Then, 40 premolars were sectioned longitudinally, and 80 semi-sections were divided into a control group (untreated) and a study group (treated by Hybrid Coat). Alloy restoration was bonded to the teeth specimen using five different cements. Shear bond strength was measured by the universal testing machine. The fracture patterns and the adhesive interface were observed using astereomicroscope. RESULTS SEM revealed that the lumens of dentinal tubules were completely occluded by Hybrid Coat. The Hybrid Coat significantly improved the shear bond strength of resin-modified glass ionomer cement (RMGIC) and resin cement (RC) but weakened the performance of zinc phosphate cement (ZPC), zinc polycarboxylate cement (ZPCC) and glass ionomer cement (GIC). CONCLUSION Hybrid Coat is an effective dentinal tubule sealant, and therefore its combined use with resin or resin-modified glass ionomer cements can be applied for the prostheses attachment purpose.

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

BackgroundTo 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.MethodsWe 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.ResultsThe 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.ConclusionsA 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.

Novel frameshift mutation in the insulin (INS) gene in a family with maturity onset diabetes of the young (MODY)

Highlights This study reports a novel frameshift mutation c.212dupG (p.Gly73fs) in the insulin (INS) gene causing maturity onset diabetes of the young (MODY) 10, one of the rare types of MODY, identified in seven family members. Screening for mutations in identified MODY genes is warranted in patients who require insulin, are negative for autoantibodies but have a family history of diabetes.

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.

Dental caries and risk indicators for patients with leprosy in China

Background In leprosy, oral health is often neglected and poorly understood. This study aimed to evaluate the prevalence and risk indicators of dental caries in patients with leprosy in China. Methods This cross‐sectional, multicentre study included 613 patients with leprosy and 602 control subjects. Based on the established standards of the World Health Organization, we investigated dental caries in cluster samplings from six so‐called ‘leprosy villages’ in three Chinese provinces. Clinical oral examinations were performed and data were reported as decayed (D), missing (M) and filled (F) teeth (DMFT scores). Results The average DMFT scores were 10.39 in patients with leprosy (D = 4.43; M = 5.94; and F = 0.02) and 4.39 in control individuals (D = 2.29; M = 2.02; F = 0.08). The DMFT scores were statistically significantly different in patients with different ages, educational backgrounds and daily brushing frequency (P < 0.05). High DMFT scores were related to age, low educational levels and poor toothbrushing habits. Conclusions The results indicate that patients with leprosy have a high prevalence of severe dental caries. Effective therapy and oral health education should be enhanced for this group of patients.