BACKGROUNDSteroid-induced osteonecrosis of the femoral head (ONFH) is a common clinical disease, with a high disability rate. At present, efficient prevention and treatment of steroid-induced ONFH is still lacking. The peroxisome proliferator-activated receptor-γ (PPARγ) is recognized as an important pathogenic gene for the development of steroid-induced ONFH. RNA interference (RNAi) is a tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target proteins. Therefore, down-regulation of PPARγ expression by RNAi may prevent the incidence of steroid-induced ONFH.

METHODSAccording to the principles of siRNA design, three duplex siRNA sequences (971 - 989, 1253 - 1271 and 1367 - 1385) derived from the PPARγ gene (NM_001082148) were synthesized. These duplexes were annealed, purified and ligated into 1.0-cytomegalovirus (CMV) shuttle vector. The shuttle vector was transfected into HEK293 cells. The HEK293 generated recombinant adenovirus vector carrying PPARγ siRNA sequences was purified and the titer of recombinant adenovirus was determined.

RESULTSAfter the annealing of single-strand DNA oligo encoding short hairpin RNA (shRNA) sequences, products were identified by gel electrophoresis. These products were ligated into the 1.0-CMV shuttle vector and the recombinant shuttle vectors 1.0-CMV-971, 1.0-CMV-1253 and 1.0-CMV-1367 were constructed. These sequences of these recombinant vectors were confirmed. We then successfully constructed the recombinant adenovirus vector carrying siRNA targeting PPARγ. After purification, the virus titer was higher than 10(10) plaque forming unit (PFU)/ml.

CONCLUSIONIn this study, three recombinant adenovirus shuttle vectors carrying siRNA targeting PPARγ, including shuttle vectors 1.0-CMV-971, 1.0-CMV-1253 and 1.0-CMV-1367, were successfully constructed and high titers of recombinant adenovirus were obtained.

Adenoviridae ; genetics ; Genetic Vectors ; genetics ; PPAR gamma ; genetics ; RNA, Small Interfering ; genetics

This study aimed to explore the effect of miR-23b-3p on the differentiation of goat intramuscular preadipocytes, and to confirm whether miR-23b-3p plays its roles via targeting the PDE4B gene. Based on the pre-transcriptome sequencing data obtained previously, the miR-23b-3p, which was differentially expressed in goat intramuscular adipocytes before and after differentiation, was used as an entry point. real-time quantitative-polymerase chain reaction (qPCR) was used to detect the expression pattern of miR-23b-3p during the differentiation of goat intramuscular preadipocytes. The effects of miR-23b-3p on adipose differentiation and adipose differentiation marker genes were determined at the morphological and molecular levels. The downstream target genes of miR-23b-3p were determined using bioinformatics prediction as well as dual luciferase reporter assay to clarify the targeting relationship between miR-23b-3p and the predicted target genes. The results indicated that overexpression of miR-23b-3p reduced lipid droplet accumulation in goat intramuscular adipocytes, significantly down-regulated the expression levels of adipogenic marker genes AP2, C/EBPα, FASN, and LPL (P < 0.01). In addition, the expressions of C/EBPβ, DGAT2, GLUT4 and PPARγ were significantly downregulated (P < 0.05). After interfering with the expression of miR-23b-3p, lipid droplet accumulation was increased in goat intramuscular adipocytes. The expression levels of ACC, ATGL, AP2, DGAT2, GLUT4, FASN and SREBP1 were extremely significantly up-regulated (P < 0.01), and the expression levels of C/EBPβ, LPL and PPARγ were significantly up-regulated (P < 0.05). It was predicted that PDE4B might be a target gene of miR-23b-3p. The mRNA expression level of PDE4B was significantly decreased after overexpression of miR-23b-3p (P < 0.01), and the interference with miR-23b-3p significantly increased the mRNA level of PDE4B (P < 0.05). The dual luciferase reporter assay indicated that miR-23b-3p had a targeting relationship with PDE4B gene. MiR-23b-3p regulates the differentiation of goat intramuscular preadipocytes by targeting the PDE4B gene.
Animals ; MicroRNAs/metabolism* ; Goats/genetics* ; PPAR gamma/metabolism* ; Adipogenesis/genetics* ; Cell Differentiation/genetics* ; Luciferases ; RNA, Messenger

Fatty Liver ; genetics ; Female ; Humans ; Male ; PPAR gamma ; genetics ; Polymorphism, Restriction Fragment Length

OBJECTIVETo breed neual stem cell-specific peroxisome proliferator-activated receptor &gamma; (PPAR&gamma;) knockout mice.

METHODSTwo transgenic mouse models, namely B6.PPAR&gamma;loxp/loxp and B6.Nestin-Cre were interbred, and the first- generation offsprings were backcrossed with B6.PPAR&gamma;loxp/loxp to obtain the second-generation mice. Genomic DNA was extracted from the second-generation mice for PCR to amplify the loxp and Cre gene fragments followed by agarose gel electrophoresis to verify their sizes. The mice with the PPAR&gamma;loxp/loxp.Nestin-Cre (KO) genotype were selected as the neural stem cell-specific knockout PPAR&gamma; mice, with B6.PPAR&gamma;loxp/loxp (loxp) mice as the control. Tissue samples were collected from specific regions of the mouse brain and peripheral tissue for detecting the expression of PPAR&gamma; mRNA using RT-PCR and real-time quantitative PCR.

RESULTS AND CONCLUSIONGenotyping results showed PPAR&gamma;loxp and Cre bands in the knockout mice, which showed obviously decreased mRNA expression of PPAR&gamma;, suggesting successful establishment of neural stem cell-specific PPAR&gamma; knockout mice. The two transgenic mice we used were fertile, and their breeding pattern followed the laws of Mendelian inheritance.

Animals ; Genotype ; Mice ; Mice, Knockout ; Neural Stem Cells ; PPAR gamma ; genetics ; RNA, Messenger

Type 2 diabetes is a common multifactorial genetic syndrome, which is determined by several different genes and environmental factors. With the accomplishment of Human Genome Project and the development of the screening technology for single-nucleotide polymorphisms (SNP), many SNP researches have been carried out to determine the genetic factors involved in type 2 diabetes. This article introduces the strategies of SNP studies and reviews the SNP studies of major candidate genes for type 2 diabetes.
Adiponectin ; genetics ; Diabetes Mellitus, Type 2 ; genetics ; Genetic Predisposition to Disease ; genetics ; Humans ; PPAR gamma ; genetics ; Polymorphism, Single Nucleotide

Comments concerning Meta analysis for relationship between peroxisome proliferator activated receptor gamma Pro12Ala polymorphism and type 2 diabetes susceptibility in different cohorts in this mini review were given. The comments pointed out existent problems and presented suggestions for genetic analysis of diseases in Chinese populations.
Asian Continental Ancestry Group ; genetics ; Diabetes Mellitus, Type 2 ; genetics ; Genetic Predisposition to Disease ; Humans ; PPAR gamma ; genetics ; Polymorphism, Genetic

Congenital lipodystrophic diabetes (CLD) is a rare genetic disease characterized by generalized or topical subcutaneous fat loss combined with various metabolic disorders such as insulin resistance, dyslipidemia, and impaired glucose tolerance. Recent studies have discovered genes underlying the disease. Mutations of such genes are associated with adipogenic anomaly, especially regulational function of peroxisome proliferators-activated receptor γ (γPPAR) for lipid. This paper has provided a review for the main clinical symptoms, classification, pathogenic genes, molecular mechanism and the relationship between PPARγ and fat loss.
Cell Differentiation ; Diabetes Mellitus ; genetics ; Humans ; Insulin Resistance ; Lipodystrophy, Congenital Generalized ; genetics ; PPAR gamma ; genetics ; Transcription Factors

This study was aimed to investigate the effect of stress induced by high-intensity exercises on the cardiovascular system. In the epidemiological investigation, 200 subjects (test group) engaged in special high-intensity exercises, and 97 who lived and worked in the same environment and conditions as those in the test group, but did not participate in the exercises served as controls. In the second part of the study, 50 mice were randomly divided into control group, exhaustive swimming group, white noise group, exhaustive swimming plus white noise group, and pioglitazone intervention group. The results showed that the plasma concentrations of the myocardial injury markers heart fatty acid-binding protein (H-FABP), C-reactive protein (CRP), β-endorphin (β-EP) and levels of psychological stress were significantly increased in test group as compared with control group; special high-intensity exercises resulted in a significant elevation of the incidence of cardiac arrhythmias. Animal experiments showed that the plasma levels of corticosterone (CORT) and troponin I (TnI) were raised while the level of SOD was reduced in exhaustive swimming group, white noise group, and exhaustive swimming plus white noise group. The expression levels of PPAR&gamma; mRNA and protein were decreased in myocardial tissues in these groups as well. HE staining showed no remarkable change in myocardial tissues in all the groups. Treatment with pioglitazone significantly decreased the plasma levels of TnI and CORT, while increased the level of SOD and the expression levels of PPAR&gamma; mRNA and protein. It was concluded that the high-intensity exercises may induce a heavy physical and psychological stress and predispose the subjects to accumulated fatigue and sleep deprivation; high-intensity exercises also increases the incidence of arrhythmias and myocardial injury. PPAR&gamma; may be involved in the physical and psychological changes induced by high-intensity exercises.
Animals ; Heart Injuries ; physiopathology ; Male ; Mice ; PPAR gamma ; genetics ; physiology ; RNA, Messenger ; genetics ; Real-Time Polymerase Chain Reaction ; Stress, Physiological

OBJECTIVETo investigate the effects of rapamycin on cholesterol homeostasis and secretory function of 3T3-L1 cells.

METHODSThe in vitro cultured 3T3-L1 cells (preadipocytes) were divided into control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group. Intracellular cholesterol level was measured by oil red O staining and high performance liquid chromatography. The secretion levels of leptin and adiponectin were assayed by enzyme-linked immunosorbent assay. The mRNA and protein expressions of peroxisome proliferator-activated receptor (PPARgamma) were assayed by quantitative real-time polymerase chain reaction and Western blot.

RESULTSOil red O staining showed rapamycin down-regulated 3T3-L1 cells differentiation and lipid accumulation. Quantitative measurement of cholesterol with high performance liquid chromatography showed that the concentrations of free cholesterol in rapamycin treatment groups had a significant reduction. The concentrations of free cholesterol in the control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were (12.89 +/- 0.16), (9.84 +/- 0.45), (9.39 +/- 0.46), and (8.61 +/- 0.34) mg/ml, respectively (P < 0.05), and the concentrations of total cholesterol were (12.91 +/- 0.50), (9.94 +/- 0.96), (10.45 +/- 2.51), and (9.53 +/- 0.63) mg/ml, respectively. The leptin concentrations in the control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were (19.02 +/- 0.52), (16.98 +/- 0.11), (15.62 +/- 0.01), and (13.84 +/- 0.66) ng/ml, respectively. The mRNA expressions of PPARgamma in the rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were significantly lower than that in control group (P < 0.05). The protein expressions of PPARgamma in the rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were 80%, 74%, and 61% of that in control group (P < 0.05). After the cells were treated with rapamycin 100 nmol/L, PPARgamma blocking agent GW9662 10 micromol/L, and PPARgamma agonist troglitazone 10 micromol/L, respectively, for 96 hours, the mRNA expression of PPARgamma was (0.60 +/- 0.14), (0.67 +/- 0.03), and (1.30 +/- 0.14) of that in control group (P < 0.05). The protein expression showed a similar trend with mRNA expression (P < 0.05). After the cells were treated with rapamycin 100 nmol/L, PPARgamma blocking agent GW9662 10 micromol/L, and PPARgamma agonist troglitazone 10 micromol/L, respectively, for 96 hours, the expression of leptin in the control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group was (19.02 +/- 0.52), (15.62 +/- 0.10), and (14.45 +/- 1.01) and (18.07 +/- 0.66) ng/ml, respectively (P < 0.05 compared with the control group).

CONCLUSIONSBy downregulating the expression of PPARgamma, rapamycin can decrease cholesterol accumulation in 3T3-L1 cells and inhibit its leptin-secreting capability. This finding may provide a possible explanation for rapamycin-induced hyperlipidemia in clinical practice.

3T3-L1 Cells ; Adipocytes ; drug effects ; metabolism ; Animals ; Cholesterol ; metabolism ; Leptin ; metabolism ; Mice ; PPAR gamma ; genetics ; metabolism ; Sirolimus ; pharmacology

OBJECTIVETo study the effect of beta3 adrenergic receptor (beta3AR) Trp64Arg and peroxisome proliferator activated receptor gamma 2 (PPARgamma2) Prol2Ala polymorphisms on insulin resistance.

METHODSOne hundred and eight dizygotic twin pairs were enrolled in this study. Microsatellite polymorphism was used to diagnose zygosity of twins. Insulin sensitivity was estimated with logarithm transformed homeostasis model assessment (HOMA). PCR-RFLP analysis was performed to detect the variants. As a supplement to the sib-pair method, identity by state (IBS) was used to analyze the association of polymorphisms with insulin sensitivity.

RESULTSThe genotype frequencies of Trp64Trg, Trp64Arg, and Arg64Arg were 72.3%, 23.8%, and 3.9%, respectively, while the genotype frequencies of Prol2Pro, Prol2Ala, and Alal2Ala were 89.9%, 9.6%, and 0.5%, respectively. For beta3AR Trp64Arg the interclass co-twin correlations of Waist-to-hip ratio (WHR), blood glucose (GLU), and insulin (INS), homeostasis model assessment insulin resistance index (HOMA-IR) of the twin pairs sharing 2 alleles of IBS were greater than those sharing 0-1 allele of IBS, and HOMA-IR had statistic significance. For PPARgamma2 Pro12Ala most traits of twin pairs sharing 2 alleles of IBS had greater correlations and statistic significance in body mass index (BMI), WHR, percent of body fat (PBF) and GLU, but there were low correlations of either insulin or HOMA-IR of twin pairs sharing 1 or 2 alleles of IBS. The combined effects of the two variations showed less squared significant twin-pair differences of INS and HOMA-IR among twins sharing 4 alleles of IBS.

CONCLUSIONSBeta3AR Trp64Arg and PPARgamma2 Pro12Ala polymorphisms might be associated with insulin resistance and obesity, and there might be slight synergistic effects between this two gene loci, and further studies are necessary to confirm this finding.

Adolescent ; Child ; Child, Preschool ; Genotype ; Humans ; Insulin Resistance ; genetics ; Obesity ; genetics ; PPAR gamma ; genetics ; Polymorphism, Genetic ; Receptors, Adrenergic, beta-3 ; genetics ; Twins, Dizygotic ; genetics ; metabolism