[Peroxisome proliferator activated receptor gamma Pro12Ala: old topic of conversation and new question].

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.

[Adenovirus construction of expression and its function of connective tissue growth factor].

OBJECTIVE: To construct and identify a adenovirus vector of the expression of connective tissue growth factor (CTGF) and to explore the role of CTGF in the metabolism of glucose and lipid. METHODS: The over-expressed plasmid of CTGF was cloned, and then the CTGF sequences were cloned into pAdTrack-CMW vector. The reformed E. coli BJ5183-sensitive bacteria that contain pAdEasy-1 were transformed with lined vector cut by Pme I enzyme. The recombinant adenovirus vector was cut with Pac I enzyme and obtained, then transfected 293A cells to produce virus. Through three times of amplification, the adenovirus infected the primary hepatocytes to determine the infection efficiency and CTGF expression. The mice were starved for several time periods, and then the liver RNA was extracted for real-time PCR to detect the expressions of CTGF under different nutritional conditions. RESULTS: The adenovirus of CTGF was successfully produced with an infection efficiency of 90%. The expressions of the CTGF were different under different nutritional conditions and showed a coincidence with the expression of peroxisome proliferators-activated receptor gamma coactivator 1 alpha. After the mice were starved for 24h, the expression of CTGF increased by (2.38 +/- 0.51) folds; after the mice were starved for 48 h, the expression of CTGF increased by (2.95 +/- 0.57) folds (P < 0.05). CONCLUSION: CTGF is speculated to be involved in the metabolism of glucose and lipids.

PGC-1alpha coactivates estrogen-related receptor-alpha to induce the expression of glucokinase.

Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) is a key regulator of cellular energy metabolism and regulates processes such as adaptive thermogenesis, hepatic gluconeogenesis, fatty acid oxidation, and mitochondrial biogenesis by coactivating numerous nuclear receptors and transcription factors. Here, we demonstrate the presence of the ERRalpha binding site in the regulatory sequence of the glucokinase gene and that PGC-1alpha coactivates ERRalpha to stimulate the transcription of glucokinase. Simultaneous overexpression of PGC-1alpha and ERRalpha potently induced the glucokinase gene expression and its enzymatic activity in primary hepatocytes; however, expression of either PGC-1alpha or ERRalpha alone had no significant effect. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed the interaction of ERRalpha with the glucokinase promoter. Finally, the knockdown of endogenous ERRalpha with specific siRNA (siERRalpha) or pharmacological inhibition of ERRalpha with XCT790 attenuated insulin-induced glucokinase expression. Taken together, this research identifies glucokinase as a novel target of PGC-1alpha/ERRalpha and underscores the regulatory function of ERRalpha in insulin-dependent enzyme regulation.

A second protein marker of caveolae: caveolin-2.

Caveolin-2, a protein about 20 kD, is a major component of the inner surface of caveolae, small invaginations of the plasma membrane. Similar with caveolin-1 and caveolin-3, it serves as a protein marker of caveolae. Caveolin-1 and -2 are located next to each other at 7q31.1 on human chromosome, the proteins encoded are co-localized and form a stable hetero-oligomeric complex, distributing similarly in tissue and cultured cells. Caveolin-3 is located on different chromosomes but confirmed to interact with caveolin-2. Caveolin-2 is similar to caveolin-1 in many respects but differs from the latter in functional domains, especially in G-protein binding domain and caveolin scaffolding domain. The mRNAs of both caveolin-1 and caveolin-2 are most abundantly expressed in white adipose tissue and are induced during differentiation of 3T3-L1 cells to adipocytes. Caveolin-2-deficient mice demonstrate clear pulmonary defects, with little or no change in caveolin-1 expression and caveolae formation, suggesting that caveolin-2 plays a selective role in lung functions. Caveolin-2 is also involved in lipid metabolism and human cancers.

Sulfated tyrosines 27 and 29 in the N-terminus of human CXCR3 participate in binding native IP-10.

AIM: Human CXCR3, a seven-transmembrane segment (7TMS), is predominantly expressed in Th1-mediated responses. Interferon-gamma-inducible protein 10 (IP-10) is an important ligand for CXCR3. Their interaction is pivotal for leukocyte migration and activation. Tyrosine sulfation in 7TMS is a posttranslational modification that contributes substantially to ligand binding. We aimed to study the role of tyrosine sulfation of CXCR3 in the protein's binding to IP-10. METHODS: Plasmids encoding CXCR3 and its mutants were prepared by PCR and site-directed mutagenesis. HEK 293T cells were transfected with plasmids encoding CXCR3 or its variants using calcium phosphate. Transfected cells were labeled with [(35)S]-cysteine and methionine or [(35)S]-Na(2)SO(3) and then analyzed by immunoprecipitation to measure sulfation. Experiments with (125)I-labeled IP-10 were carried out to evaluate the affinity of CXCR3 for its ligand. Calcium influx assays were used to measure intercellular signal transduction. RESULTS: Our data show that sulfate moieties are added to tyrosines 27 and 29 of CXCR3. Mutation of these two tyrosines to phenylalanines substantially decreases binding of CXCR3 to IP-10 and appears to eliminate the associated signal transduction. Tyrosine sulfation of CXCR3 is enhanced by tyrosyl protein sulfotransferases (TPSTs), and it is weakened by shRNA constructs. The binding ability of CXCR3 to IP-10 is increased by TPSTs and decreased by shRNAs. CONCLUSIONS: This study identifies two sulfated tyrosines in the N-terminus of CXCR3 as part of the binding site for IP-10, and it underscores the fact that tyrosine sulfation in the N-termini of 7TMS receptors is functionally important for ligand interactions. Our study suggests a molecular target for inhibiting this ligand-receptor interaction.

Function of SIRT1 in physiology.

Sirtuins were originally defined as a family of oxidized nicotinamide adenine nucleotide (NAD+)-dependent enzymes that deacetylate lysine residues on various proteins. The sirtuins are remarkably conserved throughout evolution from archae to eukaryotes. They were named after their homology to the Saccharomyces cerevisiae gene silent information regulator 2 (Sir2). The mammalian sirtuins, SIRT1-7, are implicated in a variety of cellular functions ranging from gene silencing, control of the cell cycle and apoptosis, and energy homeostasis. As SIRT1 is a nuclear protein and is the mammalian homolog most highly related to Sir2, it has been the focus of a large number of recent studies. Here we review some of the current data related to SIRT1 and discuss its mode of action and biological role in cellular and organismal models.

Protein kinase Czeta mediates insulin-induced glucose transport through actin remodeling in L6 muscle cells.

Protein kinase C (PKC) zeta has been implicated in insulin-induced glucose uptake in skeletal muscle cell, although the underlying mechanism remains unknown. In this study, we investigated the effect of PKCzeta on actin remodeling and glucose transport in differentiated rat L6 muscle cells expressing myc-tagged glucose transporter 4 (GLUT4). On insulin stimulation, PKCzeta translocated from low-density microsomes to plasma membrane accompanied by increase in GLUT4 translocation and glucose uptake. Z-scan confocal microscopy revealed a spatial colocalization of relocated PKCzeta with the small GTPase Rac-1, actin, and GLUT4 after insulin stimulation. The insulin-mediated colocalization, PKCzeta distribution, GLUT4 translocation, and glucose uptake were inhibited by wortmannin and cell-permeable PKCzeta pseudosubstrate peptide. In stable transfected cells, overexpression of PKCzeta caused an insulin-like effect on actin remodeling accompanied by a 2.1-fold increase in GLUT4 translocation and 1.7-fold increase in glucose uptake in the absence of insulin. The effects of PKCzeta overexpression were abolished by cell-permeable PKCzeta pseudosubstrate peptide, but not wortmannin. Transient transfection of constitutively active Rac-1 recruited PKCzeta to new structures resembling actin remodeling, whereas dominant negative Rac-1 prevented the insulin-mediated PKCzeta translocation. Together, these results suggest that PKCzeta mediates insulin effect on glucose transport through actin remodeling in muscle cells.

Gene expression profile of human skeletal muscle and adipose tissue of Chinese Han patients with type 2 diabetes mellitus.

OBJECTIVE: To study the differential patterns of gene expression in skeletal muscle and adipose tissue between type 2 diabetes mellitus (T2DM) patients and healthy subjects using DNA microarray analysis. METHODS: T2DM patiens were divided into female group, young male group and old male group. DNA microarray analysis and quantitative real-time PCR were carried out to analyze the relation between gene expressions and T2DM. RESULTS: The mRNA expression of 298, 578, and 350 genes was changed in the skeletal muscle of diabetes mellitus patients compared with control subjects. The 1320, 1143, and 2847 genes were modified in adipose tissue of the three groups. Among the genes surveyed, the change of 25 and 39 gene transcripts in skeletal muscle and adipose tissue was > or = 2 folds. These differentially expressed genes were classified into 15 categories according to their functions. CONCLUSION: New genes are found and T2DM can be prevented or cured.

[Genetic analysis of complex diseases: status quo and prospects].

This review comments the status quo, especially the major problems, of the genetic analysis of the complex diseases, provides some possible solutions, and explores the further development trends in this field.

[Coactivators in energy metabolism: peroxisome proliferator-activated receptor-gamma coactivator 1 family].

Peroxisome proliferator-activated receptor gamma coactivator 1 (PGC1) family is highly expressed in tissues with high energy metabolism. They coactivate transcription factors in regulating genes engaged in processes such as gluconeogenesis, adipose beta-oxydation, lipoprotein synthesis and secretion, mitochondrial biogenesis, and oxidative metabolism. Protein conformation studies demonstrated that they lack DNA binding domains and act as coactivators through physical interaction with transcription factors. PGC1 activity is regulated at transcription level or by multiple covalent chemical modifications such as phosphorylation, methylation and acetylation/deacetylation. Abnormal expression of PGC1 coactivators usually is closely correlated with diseases such as diabetes, obesity, hyperglycemia, hyperlipemia, and arterial and brain neuron necrosis diseases.

[Research advances in Sirt1 gene].

As the most homologic homologue of silent information regulator 2 of yeast, Sirt1 gene is extensively expressed in mature tissues, and is rich in early embryo and reproductive cells. It is involved in the regulation of gene transcription, energy metabolism and cell aging. It promotes fat mobilization in adipocytes and glucose production in liver and regulates insulin secretion in islet beta cell. Furthermore, Sirt1 gene is an essential endogenous apoptosis inhibitor. In future, it may be used as new drug targets or applied in other disease management modalities.

[Current situation researching of methylation in tumor].

The disorders of DNA and histone methylation have a close relationship with the development and progression of tumors. Epigenetic regulation is critical in maintaining the stability and integrity of the expression profiles of different cell types by modifying DNA methylation and histone methylation. However, the abnormal changes of methylation often result in the development and progression of tumors. This review summarized the theory of tumor genomic and histone methylation, detection methods of methylation and their applications, and the clinical application of methylation as biological markers and drug targets.

Protein kinase C-zeta regulation of GLUT4 translocation through actin remodeling in CHO cells.

Actin remodeling plays a crucial role in insulin-induced translocation of glucose transporter 4 (GLUT4) from the cytoplasm to the plasma membrane and subsequent glucose transport. Protein kinase C (PKC) zeta has been implicated in this translocation process, although the exact mechanism remains unknown. In this study, we investigated the effect of PKCzeta on actin cytoskeleton and translocation of GLUT4 in CHO-K1 cells expressing myc-tagged GLUT4. Insulin stimulated the phosphorylation of PKCzeta at Thr410 with no apparent effect on its protein expression. Moreover, insulin promoted colocalization of PKCzeta and actin that could be abolished by Latrunculin B. The overexpression of PKCzeta mimicked the insulin-induced change in actin cytoskeleton and translocation of GLUT4. These effects were also completely abrogated by Latrunculin B treatment. Using cell-permeable pseudosubstrate (PS) inhibitor of PKCzeta, the response to insulin could be alleviated. Our results strongly suggest that PKCzeta mediates the stimulatory effect of insulin on GLUT4 translocation through its interaction with actin cytoskeleton.

Atypical protein kinase C in glucose metabolism.

Type 2 diabetes mellitus is a multigenic disease with evident genetic predisposition, and complex pathogenesis in which environmental and genetic factors interact. The disorder of body utilization glucose is a crucial reason for causing diabetes. Atypical PKCs, belonging to Ser/Thr protein kinase, have many important biological functions in vivo, and may be involved in the pathogenesis of diabetes mellitus. APKCs participate in glucose metabolism by regulating glucose transport and absorption, glycogen synthesis, and insulin secretion. The exact mechanism by which aPKCs participate in glucose metabolism remains unclear. So far, the clarification of which will be helpful for the prevention and cure of type 2 diabetes.

[Analysis of genetic mutation in patients with nonsyndromic hearing loss received cochlear implant].

OBJECTIVE: To investigate the prevalence of mutations of the gap junction protein (GJB) 2 and mitochondria 12SrRNA in patients with nonsyndromic hearing loss who received cochlear implant. METHODS: Genomic DNA was extracted from the peripheral blood samples obtained from 100 Chinese patients who had received cochlear implantation, 96 with prelingual hearing loss and 4 with postlingual hearing loss, all very severe. Sixteen of the 100 patients had the history of application of aminoglycosides, among which 12 were with prelingual hearing loss and 4 with postlingual hearing loss. PCR was performed and the products were sequenced by automated DNA sequencer. RESULTS: GJB2 mutations were detected in 34 of the 100 cochlear implant recipients (34%), all with prelingual hearing loss, among which 27 (27%) had 235delC mutation. Among the 16 patients who had used aminoglycosides, two had the mutation A1555G, and one carried the mitochondrial genetic mutation delT961Cn. CONCLUSION: Mutation of GJB2 gene is the major cause of deafness in cochlear implant recipients, with a high frequency of 235delC mutation. Mitochondria genetic mutation A1555G is the common form of mutation in postlingual deafness with a history of aminoglycoside injection.

[Screening of SLC26A4 (PDS) gene mutation in cochlear implant recipients with inner ear malformation].

OBJECTIVE: To determine the prevalence of SLC26A4 (PDS) gene mutations in cochlear implant recipients with inner ear malformation, and the correlation between SLC26A4 (PDS) gene mutation and inner ear malformation and intra-operative testing of the electrically evoked auditory nerve compound action potentials (ECAP). METHODS: Peripheral blood samples were collected from 48 cochlear implant recipients with temporal bone malformation and 50 healthy controls. Genomic DNA was extracted from the blood; PCR and direct sequencing were used to detect the mutations of SLC26A4 (PDS) gene. During the implantation of artificial cochlea the 48 recipients underwent intraoperative neural response telemetry (NRT) to measure the electrically evoked auditory nerve compound action potentials (ECAP). RESULTS: SLC26A4 (PDS) mutations were detected in 70.3% (26/37) of the patients with enlarged vestibular aqueduct (EVA), and 18.2% (2/11) of the patients with other malformations of inner ear. Fifteen different mutations were identified, 8 of which had never been previously reported. The IVS7-2A>G mutation was the most prevalent mutation of SLC26A4 (PDS) gene, accounting for 45.9% (17/37) in the EVA patients. No association was detected between SLC26A4 mutation and ECAP. CONCLUSION: Mutations in the SLC26A4 (PDS) gene is a major cause of EVA, with IVS7-2A>G as the most common mutation form.

[Liver X receptor: crucial mediator in lipid and carbohydrate metabolism].

Liver X receptors (LXRs) are members of the nuclear receptor superfamily and are activated by oxysterols and intermediates in the cholesterol synthetic pathway. The pivotal role of LXRs in the metabolic conversion of cholesterol to bile acids has been well established. Furthermore, insulin induces LXRa in hepatocytes, resulting in the suppression of key enzymes in gluconeogenesis, including phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and fructose-1, 6-bisphosphatase (FBPase). LXRs also play an important role in fatty acid metabolism by activating the sterol regulatory element-bing protein 1c gene (SREBP1c). This articles reviews the molecular mechanisms by which LXRs act to influence the lipid and carbohydrate metabolism.

[Regulation of tyrosylprotein sulfotransferases activity by sulfotyrosine].

OBJECTIVE: To investigate the role of sulfated tyrosine in regulating the activity of tyrosylprotein sulfotransferases (TPST) 1 and TPST2. METHODS: Constructs of TPST 1 and TPST2 were amplified by polymerase chain reaction (PCR), then fused into immunoglobulin G1 Fc region. All the variants in which sulfated tyrosines were mutated to phenylalanine were made by the PCR-based Quick Change method and confirmed by sequencing the entire reading frame. Small hairpin RNA (shRNA) constructs-targeting nucleotides 259-275 of TPST1 and nucleotides 73-94 of TPST2 were generated and subcloned into pBluescript. Human embryonic kidney (HEK) 293T cells were transfected with these plasmids. One day later, cells were split: one part was labeled with 35S-cysteine and methionine or 35S-Na2SO3 overnight, the second part was used for 125I labeled binding experiment, and the third part was retained for binding and flow cytometry. RESULTS: Tyrosines at position 326 of TPST1 and position 325 of TPST2 were sulfated posttranslationally. Tyrosine sulfation of TPSTs was effectively inhibited by sulfation inhibitors, including specific shRNAs and non-specific NaCIO3. shRNAs reduced the sulfation of C3a receptor and C5a receptor, and partially blocked the binding of these two receptors to their respective ligands. CONCLUSIONS: The activities of TPSTs were regulated by tyrosine sulfation. Inhibition of sulfotyrosine decreases the binding ability of C3a receptor and C5a receptor to their respective ligands.

[High prevalence of connexin-26 (GJB2) mutation in cochlear implant recipients].

OBJECTIVE: To determine the prevalence of GJB2 gene mutations in patients undergoing cochlear implantation. METHODS: We enrolled 115 cochlear implant recipients for mutation screening. Genomic DNA was extracted from the blood of all patients, amplified in PCR and analyzed for single strand conformation polymorphism (SSCP) or direct sequencing to detect mutations of GJB2 gene. RESULTS: The result shows that the GJB2 mutations are detected in 36.5% (42/115) of the cochlear implant recipients, and especially 41% (41/100) of the non-syndromic deafness patients, only 1 inner ear malformation patient was detected with GJB2 mutations. This study found 11 different variations in the GJB2 gene. The 235delC mutation was the most prevalent mutation accounting for 18.3% (41/230) in all cochlear implant recipients and 21% (42/200) in non-syndromic deafness group. 187G > T and 230G > A mutations were novel mutation of GJB2 gene in the Chinese population. CONCLUSION: Mutations in the GJB2 gene were a major cause of deafness in cochlear implant recipients, the carrier frequency of 235delC mutation was highest.