Genome engineering and disease modeling via programmable nucleases for insulin gene therapy;promises of CRISPR/Cas9 technology

Targeted genome editing is a continually evolving technology employing programmable nucleases to specifically change,insert,or remove a genomic sequence of interest.These advanced molecular tools include meganucleases,zinc finger nucleases,transcription activator-like effector nucleases and RNA-guided engineered nucleases(RGENs),which create double-strand breaks at specific target sites in the genome,and repair DNA either by homologous recombination in the presence of donor DNA or via the error-prone non-homologous end-joining mechanism.A recently discovered group of RGENs known as CRISPR/Cas9 gene-editing systems allowed precise genome manipulation revealing a causal association between disease genotype and phenotype,without the need for the reengineering of the specific enzyme when targeting different sequences.CRISPR/Cas9 has been successfully employed as an ex vivo gene-editing tool in embryonic stem cells and patient-derived stem cells to understand pancreatic beta-cell development and function.RNA-guided nucleases also open the way for the generation of novel animal models for diabetes and allow testing the efficiency of various therapeutic approaches in diabetes,as summarized and exemplified in this manuscript.

Gene therapy for type 1 diabetes mellitus in rats by gastrointestinal administration of chitosan nanoparticles containing human insulin gene

AIM:To study the expression of human insulin gene in gastrointestinal tracts of diabetic rats. METHODS: pCMV.Ins, an expression plasmid of the human insulin gene, wrapped with chitosan nanoparticles, was transfected to the diabetic rats through lavage and coloclysis, respectively. Fasting blood glucose and plasma insulin levels were measured for 7 d. Reverse transcription polymerase chain reaction (RT-PCR) analysis and Western blot analysis were performed to confirm the expression of human insulin gene. RESULTS: Compared with the control group, the fasting blood glucose levels in the lavage and coloclysis groups were decreased significantly in 4 d (5.63 ± 0.48 mmol/L and 5.07 ± 0.37 mmol/L vs 22.12 ± 1.31 mmol/L, respectively, P < 0.01), while the plasma insulin levels were much higher (32.26 ± 1.81 μIU/mL and 32.79 ± 1.84 μIU/mL vs 14.23 ± 1.38 μIU/mL, respectively, P < 0.01). The human insulin gene mRNA and human insulin were only detected in the lavage and coloclysis groups. CONCLUSION: Human insulin gene wrapped with chitosan nanoparticles can be successfully transfected to rats through gastrointestinal tract, indicating that chitosan is a promising non-viral vector.

Glucose-responsive artificial promoter-mediated insulin gene transfer improves glucose control in diabetic mice

AIM:To investigate the effect of insulin gene therapy using a glucose-responsive synthetic promoter in type 2 diabetic obese mice.METHODS:We employed a recently developed novel insulin gene therapy strategy using a synthetic promoter that regulates insulin gene expression in the liver in response to blood glucose level changes.We intravenously administered a recombinant adenovirus expressing furin-cleavable rat insulin under the control of the synthetic promoter(rAd-SP-rINSfur) into diabetic Lepr db/db mice.A recombinant adenovirus expressing β-galactosidase under the cytomegalovirus promoter was used as a control(rAd-CMV-βgal).Blood glucose levels and body weights were monitored for 50 d.Glucose and insulin tolerance tests were performed.Immunohistochemical staining was performed to investigate islet morphology and insulin content.RESULTS:Administration of rAd-SP-rINSfur lowered blood glucose levels and normoglycemia was maintained for 50 d,whereas the rAd-CMV-βgal control virus-injected mice remained hyperglycemic.Glucose tolerance tests showed that rAd-SP-rINSfur-treated mice cleared exogenous glucose from the blood more efficiently than control virus-injected mice at 4 wk [area under the curve(AUC):21 508.80 ± 2248.18 vs 62 640.00 ± 5014.28,P < 0.01] and at 6 wk(AUC:29 956.60 ± 1757.33 vs 60 016.60 ± 3794.47,P < 0.01).In addition,insulin sensitivity was also significantly improved in mice treated with rAd-SP-rINSfur compared with rAd-CMV-βgal-treated mice(AUC:9150.17 ± 1007.78 vs 11 994.20 ± 474.40,P < 0.05).The islets from rAd-SP-rINSfur-injected mice appeared to be smaller and to contain a higher concentration of insulin than those from rAd-CMV-βgal-injected mice.CONCLUSION:Based on these results,we suggest that insulin gene therapy might be one therapeutic option for remission of type 2 diabetes.

Duplication and diversification of insulin genes in ray-finned fish

Insulin is a key hormone for the regulation of metabolism in vertebrates.Insulin is produced by pancreatic islet cells in response to elevated glucose levels and leads to the uptake of glucose by tissues such as liver and adipose tissue to store energy.Insulin also has additional functions in regulating development.Previous work has shown that the proglucagon gene,which encodes hormones counter regulating insulin,is duplicated in teleost fish,and that the peptide hormones encoded by these genes have diversified in function.I sought to determine whether similar processes have occurred to insulin genes in these species.Searches of fish genomes revealed an unexpected diversity of insulin genes.A triplication of the insulin gene occurred at the origin of teleost fish,however one of these three genes,insc,has been lost in most teleost fish lineages.The two other insulin genes,insa and insb,have been retained but show differing levels of selective constraint suggesting that they might have diversified in function.Intriguingly,a duplicate copy of the insa gene,which I named insab,is found in many fish.The coding sequenee encoded by insab genes is under weak selective constraint,with its predicted protein sequences losing their potential to be processed into a two-peptide hormone.However,these sequences have retained perfectly conserved cystine residues,suggesting that they maintain insulin's three-dimensional structure and therefore might modulate the processing and secretion of insulin produced by the other genes.

A SINGLE TETRACYCLINE-REGULATED VECTOR DEVISED FOR CONTROLLED INSULIN GENE EXPRESSION

Objective To construct a single plasmid vector mediating doxycycline-inducible recombined human insulin gene expr-ession in myotube cell line. Methods An expression cassette of rtTAnls driven by promoter of human cytomegalovirus and a furin-cuttable recom-bined human insulin expression cassette driven by a reverse poly-tetO DNA motif were cloned into a single plasmid vector (prTR-tetO-mINS). The prTR-tetO-mINS and pLNCX were co-transfected into a myotube cell line (C2C12) and pLNCX vector were used as a control. After selection with G418, the transfected cells were induced with doxycycline at concentra-tions of 0, 2, and 10 μg/mL. RT-PCR was used to determine expression levels of recombinant insulin mRNA at the 5th day. Insulin production in cell cultures medium (at different incubation time) and cell extracts (at the 7th day) were analyzed with human pro/insulin RIA kits. Results Immune reactive insulin (IRI) level in cell medium was found increased at 24 hours of doxycycline incubation, and still increased at the 5th day. After withdrawn of doxycycline, IRI decreased sharply and was at baseline three days later. IRI and human insulin mRNA levels were positively related to different levels of doxycycline. A 25-fold increase in IRI was found against background expression at the 7th day. Conclusion Human insulin expression can be successfully regulated by doxycycline and the background was very low. This single tet-on insulin expression system may provide a new approach to a controlled insulin gene therapy in skeletal muscle.

Retrovirus vector transfection of rat insulin gene into pancreas decrease blood glucose of diabetic rat

Human and animal diabetes mellitus were controlled by a dietary treatment supplemented with either a sulfonylurea drug or insulin injection. Insulin injections were inconvenient and the hypoglycemia induced by insulin-overdose could be fatal. Sulfonylurea drugs were administered orally, however, do not typically provide satisfactory control of blood glucose as a starting treatment in 25% - 30% patients. Therefore, it was imperative to develop a method for the control of human and animal diabetes mellitus. Recently, insulin gene transferred and expressed in non-pancreatic cells as a means for the treatment of diabetes was developed rapidly in the expanding gene therapy. Retrovirus, lentivirus, adenovirus, adenoassociated virus and herpes simplex had been used as viral vectors, and the constructed viral-insulin gene was successfully transferred into diabetic rat cells. A gene, containing promoter, enhancer and rat type I insulin gene (a-chain, b-chain and signal peptide), was constructed into a retrovirus vector in the study. The constructed viral-insulin gene was transferred into mouse fibroblast cell. The insulin concentration in 3-day cultured mouse fibroblast cells was 4806.35 ± 53.72 pg/ml. The insulin concentration for the viral vector containing enhancer and promoter of rat insulin gene was higher than the vector containing only insulin gene by a 61% increase in the cultured mouse fibroblast cells. The enhancer and promoter activity of rat insulin gene would be an important determinant for the expression of insulin gene. The secreted amount of insulin by retrovirus vector contained enhancer/promoter gene in this study could achieve as high concentrations (4806.35 ± 53.72 pg/ml) as the insulin injection therapy. Blood glouse decreased sig- nificantly for at last 10 days demonstrated that transfection, direction injection of viral-insulin gene into pancreas of diabetic rat, was successful. These studies suggest that the retrovirus vector might be used to transfer the insulin gene in vitro and in vivo.

Effects of Human Insulin Gene Transfection on the Adipogenic Differentiation of Human Umbilical Cord Mesenchymal Stem Cells in Silk Fibroin Scaffolds <i>in Vitro</i>

The resorption of the transplanted fat over time limited the use of autologous fat for the reconstruction of soft tissue defect. Tissue engineering (TE) adipose with silk fibroin scaffold could be a promising substitute for soft tissue filling. In this study, we try to develop a tissue engineering adipose in vitro by seeding silk fibroin scaffold with human umbilical cord mesenchymal stem cells (hUCMSCs) after transfected with recombinant human insulin gene lentivirus. Our aim was to observe the effects of the insulin gene transfection on the adipogenesis of hUCMSCs when cultured with silk fibroin scaffolds. The hUCMSCs infected with recombinant lentiviral pLenti6.3-insulin-IRES-EGFP were seeded on silk fibroin scaffolds and cultured in adipogenic differentiation medium for 5 - 7 days. The expression of adipogenic gene PPARγ-2 was tested by RT-PCR after 7 days culture of adipogenic induction. The accumulation of cytoplasmic droplets of neutral lipids was assessed by Oil Red O staining. The RNA and protein expression of transfected insulin gene in hUCMSCs were detected by QPCR and western blot. The effect of recombinant lentivirus transfection on the growth and proliferation of hUCMSCs was observed by MTT test. We observed that the 2-ΔΔCt value of insulin gene expression of hUCMSCs in the transfected group was 300.25 times higher than that in the untransfected group. The western blot showed that a positive band was discerned at the site of a relative molecular mass of 8 × 103 Dalton in transfected group. After adipogenic culture for 7 days, under the fluorescent inverted phase-contrast microscope, after Oil Red O staining, a lot of adipocytes appeared in silk fibroin scaffold;round adipose droplets showed intracellularly;the size of the adipocytes was not homogenous, and the density of adipocytes in transfected group was significantly higher than that in untransfected group (P = 0.007, P < 0.01). RT-PCR results showed that the expression of adipogenic gene PPARγ-2 in transfected group was much stronger than that in untransfected group. MTT test showed that there was no significant difference in optical density (A) at each time point between transfected group and nontransfected group (P = 0.056, P > 0.05). And there was also no significant difference in optical density (A) between cell group and cell-scalffold group (P = 0.066, P > 0.05). We concluded that insulin gene could obviously promote the adipogenic differentiation of hUCMSCs, and a tissue engineering adipose could be constructed by the silk fibroin scaffolds seeded with human insulin gene-modified hUCMSCs effectively in vitro.

Insulin gene enhancer protein 1 mediates glycolysis and tumorigenesis of gastric cancer through regulating glucose transporter 4

Background:Insulin gene enhancer protein 1,(ISL1),a LIM-homeodomain transcription factor,is involved in multiple tumors and is associated with insulin secretion and metabolic phenotypes.However,the role of ISL1 in stimulating glycolysis to promote tumorigenesis in gastric cancer(GC)is unclear.In this study,we aimed to characterize the expression pattern of ISL1 in GC patients and explore its molecular biological mechanism in glycolysis and tumorigenesis.Methods:We analyzed the expression and clinical significance of ISL1 in GC using immunohistochemistry and real-time polymerase chain reaction(PCR).Flow cytometry and IncuCyte assays were used to measure cell proliferation after ISL1 knockdown.RNA-sequencing was performed to identify differentially expressed genes,followed by Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis and Gene Set Enrichment Analysis(GSEA)to reveal key signaling pathways likely regulated by ISL1 in GC.Alteration of the glycolytic ability of GC cells with ISL1 knockdown was validated by measuring the extracellular acidification rate(ECAR)and oxygen consumption rate(OCR)and by detecting glucose consumption and lactate production.The expression of glucose transporter 4(GLUT4)and ISL1 was assessed by Western blotting,immunohistochemistry,and immunofluorescent microscopy.The luciferase reporter activity and chromatin immunoprecipitation assays were performed to determine the transcriptional regulation of ISL1 on GLUT4.Results:High levels of ISL1 and GLUT4 expression was associated with short survival of GC patients.ISL1 knockdown inhibited cell proliferation both in vitro and in vivo.KEGG analysis and GSEA for RNA-sequencing data indicated impairment of the glycolysis pathway in GC cells with ISL1 knockdown,which was validated by reduced glucose uptake and lactate production,decreased ECAR,and increased OCR.Mechanistic investigation indicated that ISL1 transcriptionally regulated GLUT4 through binding to its promoter.Conclusion:ISL1 facilitates glycolysis and tumorigenesis in GC via the transcriptional regulation of GLUT4.

Study of Mutation in Tyrosine Protein Kinase of Insulin Receptor Gene in Patients with Polycystic Ovarian Syndrome

Objective To explore the molecular mechanism of insulin resistance in the patients with polycystic ovarian syndrome (PCOS)Methods Polymerase chain reaction, silver staining-single strand conformation poly-morphism(PCR-SSCP) and DNA direct sequencing were used to detect the mutation of insulin receptor (INSR) gene in exon 17-21 with the abdominal wall adipose tissue from 31 patients with PCOS (PCOS Group) and 30 patients with pure hysteromyoma in reproductive lift (Control Group).Results Tiventy-two variant SSCP patterns in exon 17 of INSR gene were detected. Direct sequence analysis of exon 17 showed that homozygous nonsense mutation was two alleles single nucleotide polymorphism (SNP) at the codon 1058 (CAC→CAT). Exons 18-21were not detected with any significantly mutation. The INSR gene His1058C→ T substitution collecting rate and insulin resistance were significantly higher in the PCOS group than in the control group (P = 0. 0293, P<0. 05, P<0. 01). Conclusion It is suggested that the SNP in codon 1058 of the INSR gene might be related with the insulin resistance in PCOS patients, which has hereditary tendency. And the missense mutation,nonsense mutation and frameshift mutation at exons 18-21 in tyrosine protein kinase region of INSR gene for PCOS patients were not frequently observed.

The Relationship of Insulin-receptor Gene Polymorphism to Ischemic Stroke?

Objective\ To investigate the role of mutation of insulin receptor (INSR) gene in the development of ischemic stroke. Methods\ The base variations at exon 17 and 20 of INSR gene, by means of PCR SSCP were determined in 68 cases of atherothrombotic cerebral infarction (ACI), 81 cases of lacunar infarction (LI) and 62 healthy controls (HC). Results\ There were 2 alleles of T and C at exon 17 of INSR gene. The prevalence of mutant of T allele in ACI patients was more common than that in the controls. The blood pressure and the parameters of lipid metabolism in the patients with mutant were higher than those in the controls with wild type gene. However, the correlative analysis showed that the polymorphism of INSR gene was not related statistically to the blood pressure. No base variation at exon 20 was found in the study. Conclusion\ The mutation at exon 17 of INSR gene, by promoting the development of atherosclerosis, may participate in the occurrence of ischemic stroke.\;

Insulin resistance,adipokine profile and hepatic expression of SOCS-3 gene in chronic hepatitis C

AIM:to analyze adipokine concentrations,insulin resistance and hepatic expression of suppressor of cytokine signaling 3(SOCS-3)in patients with chronic hepatitis C genotype 1 with normal body weight,glucose and lipid profile.METHODS:The study group consisted of 31 patients with chronic hepatitis C and 9 healthy subjects.Total levels of adiponectin,leptin,resistin,visfatin,omentin,osteopontin and insulin were measured using an ELISA kit.The hepatic expression of SOCS-3 was determined by the use of the reverse transcription polymerase chain reaction method.RESULTS:Homeostasis model assessment for insulin resistance(HOMA-IR)values were significantly higher in hepatitis C virus(HCV)infected patients without metabolic disorders compared to healthy controls(2.24 vs 0.59,P=0.0003).Hepatic steatosis was observed in 32.2%of patients with HCV infection and was found in patients with increased HOMA-IR index(2.81 vs1.99,P=0.05)and reduced adiponectin level(5.96vs 8.37,P=0.04).Inflammatory activity(G≥2)was related to increased osteopontin concentration(34.04vs 23.35,P=0.03).Advanced liver fibrosis(S≥2)was associated with increased levels of omentin and osteopontin(436.94 vs 360.09,P=0.03 and 32.84 vs20.29,P=0.03)and reduced resistin concentration(1.40 vs 1.74,P=0.047).No correlations were reported between adipokine profile,HOMA-IR values and hepatic expression of the SOCS-3 gene.CONCLUSION:We speculated that no relationship between adipokines and HOMA-IR values may indicate that HCV can induce insulin resistance itself.Some adipokines appear to be biochemical markers of steatosis,inflammation and fibrosis in patients with chronic HCV infection.

Improvement in erectile dysfunction after insulin-like growth factor-1 gene therapy in diabetic rats

Aim： To determine whether adenoviral gene transfer of insulin like growth factor-1 （IGF-1） to the penis of streptozotocin （STZ）-induced diabetic rats could improve erectile capacity. Methods： The STZ diabetic rats were transfected with AdCMV-βgal or AdCMV-IGF-1. These rats underwent cavernous nerve stimulation to assess erectile function and their responses were compared with those of age-matched control rats 1 to 2 days after transfection. In control and transfected STZ diabetic rats, IGF-1 expression were examined by reverse transcription polymerase chain reaction （RT-PCR）, Western blot and histology. The penis β-galactosidase activity and localization of the STZ diabetic rats were also determined. Results： One to two days after transfection, the β-galactosidase was found in the smooth muscle cells of the diabetic rat penis transfected with AdCMV-βgal. One to 2 days after administration of AdCMV- IGF-1, the cavernosal pressure, as determined by the ratio of maximal intracavernous pressure-to-mean arterial pressure （ICP/MAP） and total intracavernous pressure （ICP）, was increased in response to cavernous nerve stimulation. Transgene expression was confirmed by RT-PCR, Western blot and histology. Conclusion： Gene transfer of IGF-1 significantly increased erectile function in the STZ diabetic rats. These results suggest that in vivo gene transfer of IGF- 1 might be a new therapeutic intervention for the treatment of erectile dysfunction （ED） in the STZ diabetic rats.

Insulin receptor substrate gene polymorphisms are associated with metabolic syndrome but not with its components

Aim: Metabolic syndrome (MetS) is a major risk factor for both diabetes mellitus and cardiovascular disease (CVD). The aims of the study were 1) to investigate the insulin receptor substrate-1 (IRS-1) and insulin receptor substrate-2 (IRS-2) gene polymorphisms in patients with MetS and 2) to examine the relationships between gene polymorphisms and components of MetS. Patients & Methods: The study population included 100 patients with MetS and 30 patients without MetS as control group. Metabolic syndrome (MS) was defined as in ATP III. Entire coding exons of IRS-1 and IRS-2 genes were amplified by polymerase chain reaction (PCR). Insulin resistance (IR) was estimated using the homeostasis model assessment (HOMA). Results: In patients with MetS, 34 (34%), had G972R (rs1801278) gene polymorphism and 66 (66%) had no nucleotide substitutions at the IRS-1 gene (p circumference, blood pressure, triglyceride, HDL-Cholesterol, LDL-Cholesterol and HOMA-IR levels. Conclusion: Insulin receptor substrate-1 and 2 gene polymorphisms were associated with metabolic syndrome but not its components.

Variation in Expression of Insulin-like Growth Factors( IGFs) System Genes in Ovarian and Uterine Tissues of Aging Rats

Variations in expression of insulin-like growth factors （IGFs） system genes in rat ovarian and uterine tissues were investigated by real-time quantitative PCR amplification using RT2 ProfilerTM PCR Array Rat Insulin Signaling Pathway （PARN-030A） chips. According to results of gene detection, a total of 15 differeutially expressed genes were screened in ovarian tissues, including 3 upregulated genes and 10 downregulated genes; a total of 22 differentially expressed genes were screened in uterine tissues, including 5 upregulated genes and 17 downregulated genes. These differentially expressed genes might be related to the insulin resistance.

1,25-Dihydroxyvitamin D_(3) effects on the regulation of the insulin receptor gene in the hind limb muscle and heart of streptozotocin-induced diabetic rats

In the present study, we examine the effects of the treatment with 1,25-dihydroxyvitamin D3 [150 IU/Kg (3.75 μg/Kg) once a day, for 15 days] to non-diabetic and streptozotocin-induced diabetic rats. The results indicate that treatment with 1,25-dihydroxyvitamin D3 had minor effects in non-diabetic rats. The same treatment in streptozotocin-induced diabetic rats, although it did not correct the hyperglycemia and hypoinsulinemia induced by the diabetes, caused other actions that could mean beneficial effects on the amelioration of diabetes e.g., it avoided body weight loss, increased calcium and phosphorus plasma levels, and corrected the over-expression of the insulin receptor mRNA species of 9.5 and 7.5 Kb present in the hind limb muscle and heart of these animals. These genomic 1,25-dihydroxyvitamin D3 effects could involve transcriptional mechanisms of repression mediated by vitamin D response elements in the rat insulin receptor gene promoter. Using computer analysis of this promoter, we propose the -249/-235 bp VDRE (5’GGGTGACCCGGGGTT3’) with a pyrimidine (T) in the (+7) position of the3’half-site as the best candidate for negative control by 1,25-dihydroxy-vitamin D3. In addition, posttranscriptional mechanisms of regulation could also be implicated. Thus, computer inspection of the5’untranslated region of the rat insulin receptor pre-mRNA indicated the presence of a virtual internal ribosome entry segment whereas the computer inspection of the3’untranslated region localized various destabilizing sequences, including various AU-rich elements. We propose that through these virtual cis-regulatory sequences, 1,25-dihydroxyvitamin D3 could control the translation and stability of insulin receptor mRNA species in the hind limb muscle and heart of diabetic rats.

Insulin Effect on Gene Expression of Dental Pulp Cell during Osteodifferentiation

Insulin, an old but still actual molecule, has many roles in various cellular processes including bone formation and also angiogenesis. Insulin effects on different cell types were investigated, and we intended to check its effect on dental pulp cells (DPC) during osteoblast differentiation. First, bone differentiation ability of isolated dental pulp cells was assessed by alizarin red (AR) staining. Insulin increased dental pulp cell proliferation after 25 hours of culture. It increased mRNA expression of osteogenic markers such as Col1, RunX2, ALP, Osc, Mef2C and expression of genes involved in TGF b pathway such as Smad3, TSP1, VEGF at different time points.

cDNA Microarray Analysis of Insulin Resistance-associated Genes in Fructose-fed Rats

This study was conducted to investigate the gene expression in fructose-fed rat skeletal muscle by cDNA chip which could provide support to elucidate the molecular mechanisms underlying insulin resistance. The rats were divided into two groups, one of which was normal control and the other was fed with fructose-rich diet. The mRNA was isolated and purified from the skeletal muscle of two groups. The mRNA from two kinds of tissue was reverse transcribed to cDNA with Cy3-dUTP and Cy5-dUTP separately to prepare hybridization probes. The mixed probes were hybridized to cDNA microarray. The microarray was scanned, analyzed and repeated for two times. Among the total 4 096 tested genes, 140 genes were differently expressed, 62 up-regulated,78 down-regulated, the expression of Ptprd and Gilz and multiple genes of oxidative metabolism is associated in insulin resistance. The differential expression of gene may be related to the pathogenesis of insulin resistance.

Differentiation of embryonic stem cells into insulin-producing cells promoted by Nkx2.2 gene transfer

AIM: To investigate the ability of a genetically altered embryonic stem (ES) cell line to generate insulin-producing cells in vitro following transfer of the Nkx2.2 gene.METHODS: Hamster Nkx2.2 genes were transferred into mouse ES cells. Parental and Nkx2.2-transfected ES cells were initiated toward differentiation in embryoid body (EB)culture for 5 d and the resulting EBs were transferred to an attached culture system. Dithizone (DTZ), a zincchelating agent known to selectively stain pancreatic beta cells, was used to detect insulin-producing cells.The outgrowths were incubated in DTZ solution (final concentration, 100 μg/mL) for 15 min before being examined microscopically. Gene expression of the endocrine pancreatic markers was also analyzed by RT-PCR. In addition, insulin production was determined immunohistochemically and its secretion was examined using an ELISA.RESULTS: DTZ-stained cellular clusters appeared after approximately 14 d in the culture of Nkx2.2-transfected ES cells (Nkx-ES cells), which was as much as 2 wk earlier, than those in the culture of parental ES cells (wt-ES). The frequency of DTZ-positive cells among total cultured cells on day 28 accounted for approximately 1.0% and 0.1% of the Nkx-ES- and wt-ES-derived EB outgrowths, respectively. The DTZ-positive cellular clusters were found to be immunoreactive to insulin, while the gene expressions of pancreatic-duodenal homeobox 1(PDX1), proinsulin 1 and proinsulin 2 were observed in the cultures that contained DTZ-positive cellular clusters.Insulin secretion was also confirmed by ELISA, whereas glucose-dependent secretion was not demonstrated.CONCLUSION: Nkx2.2-transfected ES cells showed an ability to differentiate into insulin-producing cells.

Relationship between β3-AR Gene and Obesity, Type 2 Diabetes, Insulin Resistance in Chinese Han Population

Objective: To explore the relationship between the β3-adrenergic receptor(β3-AR)gene and obesity, T2DM. insulin resistance in Chinese Han population. Methods: Fifty-three healthy subjects, 105 subjects with simple obesity, 63 type 2 diabetic patients without obesity, and 114 type 2 diabetic patients with obesity were studied with the technique of PCR-RFLP in codon 64 of the exon region of β3-AR gene representing the variation Trp/Arg. Results:Compared with the subjects of Trp homozygous group, the individuals with Arg allele were more elevated in WHR,MBP,SBP,DBP,FBS,PBS, FINS,PINS, FCP,PCP and lower in ISI. Frequency of Arg allele was higher in HINS sub-group without T2DM. Cnclusion: The results indicate that the Trp/Arg variation might lead to insulin resistance, obesity and T2DM.β3-AR gene is supposed to be the candidate gene of insulin resistance, obesity and T2DM in ChineseHan population.