OBJECTIVETo study the relationship between insulin resistance and methylation of insulin receptor (INSR) gene in the endometrium of women with polycystic ovary syndrome (PCOS).

METHODSBased on the HOMA index, 35 patients with PCOS were divided into insulin resistant group (IR group, n=18) and non-resistant group (NIR group, n=18). The patients age, serum estriol, testosterone, FSH and LH, fasting insulin and fasting blood glucose were compared between the two groups. The endometrial samples were obtained from the patients to examine DNA methylation status of INSR gene in the endometrial cells using methylation-specific PCR.

RESULTSThe BMI, WHR, fasting glucose, fasting insulin, and HOMA index differed significantly between the two groups (P<0.05). PCR analysis showed partial methylation in the promoter region of INSR gene in 13 samples in IR group and 11 samples in NIR group, without detection of full methylation of the INSR gene in either group. The methylation status showed no significant difference between the two groups (P=0.328).

CONCLUSIONPartial methylation of the INSR gene occurs in the endometria of PCOS patients, but this study does not provide a strong evidence supporting the relationship between insulin resistance and INSR gene methylation in women with PCOS.

Adult ; DNA Methylation ; Endometrium ; metabolism ; Female ; Humans ; Insulin Resistance ; Polycystic Ovary Syndrome ; genetics ; metabolism ; Receptor, Insulin ; genetics ; metabolism

More and more evidence suggests that microRNA is widely involved in the regulation of cardiovascular function. Our preliminary experiment showed that miR-494-3p was increased in heart of diabetic rats, and miR-494-3p was reported to be related to metabolism such as obesity and exercise. Therefore, this study was aimed to explore the role of miR-494-3p in diabetic myocardial insulin sensitivity and the related mechanism. The diabetic rat model was induced by high fat diet (45 kcal% fat, 12 weeks) combined with streptozotocin (STZ, 30 mg/kg), and cardiac tissue RNA was extracted for qPCR. The results showed that the level of miR-494-3p was significantly up-regulated in the myocardium of diabetic rats compared with the control (P < 0.05). The level of miR-494-3p in H9c2 cells cultured in high glucose and high fat medium (HGHF) was significantly increased (P < 0.01) with the increase of sodium palmitate concentration, whereas down-regulation of miR-494-3p in HGHF treated cells led to an increase in insulin-stimulated glucose uptake (P < 0.01) and the ratio of p-Akt/Akt (P < 0.05). Over-expression of miR-494-3p in H9c2 cell line significantly inhibited insulin-stimulated glucose uptake and phosphorylation of Akt (P < 0.01). Bioinformatics combined with Western blotting experiments confirmed insulin receptor substrate 1 (IRS1) as a target molecule of miR-494-3p. These results suggest that miR-494-3p reduces insulin sensitivity in diabetic cardiomyocytes by down-regulating IRS1.
Animals ; Diabetes Mellitus, Experimental ; physiopathology ; Down-Regulation ; Insulin ; Insulin Receptor Substrate Proteins ; physiology ; Insulin Resistance ; MicroRNAs ; genetics ; Myocytes, Cardiac ; physiology ; Rats

OBJECTIVE: To investigate the effect CD36 deficiency on muscle insulin signaling in mice fed a normal-fat diet and explore the possible mechanism. METHODS: Wild-type (WT) mice and systemic CD36 knockout (CD36^-/-) mice with normal feeding for 14 weeks (n=12) were subjected to insulin tolerance test (ITT) after intraperitoneal injection with insulin (1 U/kg). Real-time PCR was used to detect the mRNA expressions of insulin receptor (IR), insulin receptor substrate 1/2 (IRS1/2) and protein tyrosine phosphatase 1B (PTP1B), and Western blotting was performed to detect the protein expressions of AKT, IR, IRS1/2 and PTP1B in the muscle tissues of the mice. Tyrosine phosphorylation of IR and IRS1 and histone acetylation of PTP1B promoter in muscle tissues were detected using co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP), respectively. RESULTS: CD36^-/- mice showed significantly lowered insulin sensitivity with obviously decreased area under the insulin tolerance curve in comparison with the WT mice (P < 0.05). CD36^-/- mice also had significantly higher serum insulin concentration and HOMA-IR than WT mice (P < 0.05). Western blotting showed that the p-AKT/AKT ratio in the muscle tissues was significantly decreased in CD36^-/- mice as compared with the WT mice (P < 0.01). No significant differences were found in mRNA and protein levels of IR, IRS1 and IRS2 in the muscle tissues between WT and CD36^-/- mice (P>0.05). In the muscle tissue of CD36^-/- mice, tyrosine phosphorylation levels of IR and IRS1 were significantly decreased (P < 0.05), and the mRNA and protein levels of PTP1B (P < 0.05) and histone acetylation level of PTP1B promoters (P < 0.01) were significantly increased as compared with those in the WT mice. Intraperitoneal injection of claramine, a PTP1B inhibitor, effectively improved the impairment of insulin sensitivity in CD36^-/- mice. CONCLUSION CD36 is essential for maintaining muscle insulin sensitivity under physiological conditions, and CD36 gene deletion in mice causes impaired insulin sensitivity by up-regulating muscle PTP1B expression, which results in detyrosine phosphorylation of IR and IRS1.
Animals ; Gene Deletion ; Histones/genetics* ; Insulin ; Insulin Receptor Substrate Proteins/metabolism* ; Insulin Resistance/genetics* ; Membrane Cofactor Protein/genetics* ; Mice ; Mice, Knockout ; Muscles/metabolism* ; Phosphoric Monoester Hydrolases/metabolism* ; Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; RNA, Messenger/metabolism* ; Receptor, Insulin/metabolism* ; Tyrosine/genetics* ; Up-Regulation

OBJECTIVE: To study the expression of tumor associated vascular insulin receptor (TVIR) in colorectal cancer with or without metabolic syndrome (MS) and its relationship with the pathological features of colorectal cancer. METHODS: The expression of TVIR in 220 colorectal cancer specimens was detected by tissue microarray and immunohistochemistry. The relationships between the expression of TVIR and the pathological features (pathological subtypes, histological grade, invasion depth, lymph node metastasis and TNM stage) of colorectal cancer with/without MS were analyzed. RESULTS: The insulin receptor expression was observed in colorectal cancer tissue or border area between cancer and normal tissue, but not in normal intestinal tissue. The high-expression rates of TVIR in MS group was remarkably lower than that of non-MS group (21.6%vs. 41.0%, CONCLUSIONS s: High-expression of TVIR is associated with aggressive pathological features such as invasion, lymph node metastasis and high TNM stage of colorectal cancer, especially for those patients without MS. TVIR could be a useful biological marker for prognosis of colorectal cancer.
Biomarkers, Tumor/genetics* ; Colorectal Neoplasms/physiopathology* ; Gene Expression Regulation, Neoplastic ; Humans ; Neoplasm Staging ; Prognosis ; Receptor, Insulin/genetics*

Acanthosis Nigricans ; diagnosis ; genetics ; pathology ; Adolescent ; Antigens, CD ; genetics ; Biomarkers ; blood ; DNA Mutational Analysis ; Donohue Syndrome ; diagnosis ; genetics ; pathology ; Humans ; Insulin Resistance ; genetics ; Male ; Mutation ; genetics ; Polymerase Chain Reaction ; Receptor, Insulin ; genetics ; Sequence Analysis, DNA

Obesity has become a severe public health problem across the world, and seriously affects the health and life quality of human beings. Here we generated lepr and mc4r mutant zebrafish via the CRISPR/Cas9 technique, and performed morphological and functional characterizations of those mutants. We observed that there was no significant phenotypic difference between homozygous mutants and wild-type controls before 2.5 months post-fertilization (mpf). However, the adult leprand mc4rindividuals displayed increased food intake, heavier weight, and higher body fat percentage, the characteristics of obesity phenotypes. Blood glucose test showed that overfeeding induced significantly impaired glucose tolerance in adult leprand mc4rzebrafish. Furthermore, we analyzed 76 energy metabolism-related transcripts in leprand mc4rzebrafish livers by using real-time RT-PCR, and compared the results with the published microarray data of Lepmouse livers, and found that the changes in the expression of insulin/IGF signaling (IIS) pathway genes in leprzebrafish and Lepmouse were positively correlated, suggesting that the IIS pathway maintains functional conservation between zebrafish and mammals during the evolution of the obesity-regulating molecule network.
Animals ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Insulin ; metabolism ; Leptin ; Mutation ; Obesity ; genetics ; Receptor, Melanocortin, Type 4 ; genetics ; Receptors, Leptin ; genetics ; Signal Transduction ; Zebrafish ; Zebrafish Proteins ; genetics

OBJECTIVE: To determine the effects of long-term estrogen replacement treatment on blood pressure and expressions of insulin receptor (IR) and insulin receptor substrate-1 ( IRS-1) in myocardium. METHODS: Fifty female SD rats were randomly divided into 3 groups. And then sham ( n = 16), ovariectomy (OVX, n = 17), and estrogen replacement treatment group (OVX + E2, n = 17) were established. Systolic blood pressure of tail artery was determined by tail-cuff technique before the operation and on week 12 after the operation. The expressions of IR and IRS-1 were measured by RT-PCR in myocardium of SD rats. RESULTS: Blood pressure [ (118.75+/-2.77) mmHg] in OVX was significantly higher than that in the sham [ ( 103.86+/-1.84) mmHg, P < 0.05 ] and OVX + E2 [( 107.83+/-3.24) mmHg, P < 0.05 ] rats. Expression of IRS-1 in OVX group was significantly lower ( 1.2588+/-0.1045)than that in the sham(2.2089+/-0.0988, P <0.05) and OVX + E2 groups ( 1.9100+/-0.1230, P <0.05 ). However, there was no difference on blood pressure and expression of IRS-1 between sham and OVX + E2 groups (P > 0.05 ). The difference of IR expression has no statistical significance among the 3 groups. CONCLUSION Long-term estrogen replacement treatment might protect cardiovascular system through decreasing the blood pressure and inducing the expression of IRS-1 in myocardium. However, plasma estrogen level doesn't significantly influence the IR expression.
Animals ; Blood Pressure ; drug effects ; Estradiol ; pharmacology ; Estrogen Replacement Therapy ; Female ; Insulin Receptor Substrate Proteins ; Myocardium ; metabolism ; Ovariectomy ; Phosphoproteins ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Receptor, Insulin ; biosynthesis ; genetics ; Time Factors

OBJECTIVETo investigate the molecular mechanism of the effect of alcohol on insulin sensitivity.

METHODSFour groups of Wistar rats were used, i.e. control (C) group, and low (L), moderate (M) and high (H) alcohol group. Alcohol doses of each group were 0, 0.6, 1.8 and 3.0 ml.(kg.bw)(-1).day(-1). Each group was comprised of 10 male and 10 female rats. Alcohol was given to rats by gastric intubation. Thirteen weeks later, serum was collected for testing of fasting plasma glucose and insulin. HOMA-IR index of each group were calculated. Total muscle RNA was extracted using Trizol Reagent (Promega). The expression level of IRS-1 mRNA in muscle was detected by RT-PCR.

RESULTSIn female rats, the fasting plasma glucose of group (8.36 +/- 0.57) mmol/L was higher and the fasting plasma insulin (15.25 +/- 3.32) was lower than those of group C (7.56 +/- 0.85, 20.80 +/- 3.25). The HOMA-IR of group L (1.775 3 +/- 0.138 1) was lower than that of group C (1.982 6 +/- 0.124 6) (P < 0.05), while IRS-1 mRNA (0.766 1 +/- 0.076 9) was up-regulated (P < 0.05); HOMA-IR of group M (2.202 2 +/- 0.271 0) was higher than that of group C (P < 0.01), while IRS-1 mRNA (0.501 8 +/- 0.049 2) was suppressed (P < 0.01); HOMA-IR of group H (1.850 1 +/- 0.162 8) was not significantly changed as compared with that of group C (1.982 6 +/- 0.124 6) (P > 0.05), while IRS-1 mRNA (0.418 1 +/- 0.049 1) was significantly suppressed (P < 0.01). In male rats, the fasting plasma glucose and insulin had the similar change as those of female rats. The HOMA-IR of group M (1.878 5 +/- 0.250 2) was lower than that of C group (2.147 3 +/- 0.330 8) (P < 0.05), IRS-1 mRNA was up-regulated (0.824 9 +/- 0.064 7) (P < 0.05).

CONCLUSIONSThe present study showed that low-to-moderate dose of alcohol could increase insulin sensitivity; while alcohol abuse could decrease insulin sensitivity. Sex difference in this effect was found. Changes of IRS-1 mRNA expression may be involved in the molecular mechanism of the effects of alcohol on insulin sensitivity.

Animals ; Dose-Response Relationship, Drug ; Ethanol ; pharmacology ; Female ; Insulin ; blood ; Insulin Receptor Substrate Proteins ; Insulin Resistance ; Male ; Muscle, Skeletal ; metabolism ; Phosphoproteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Up-Regulation

OBJECTIVETo construct the expression vector of siRNA targeting parathyroid hormone 1 receptor (PTH1R) gene and evaluate its effect on the cell cycle of INS-1 cells.

METHODSThe sequences of PTH1R gene was retrieved from Genbank, and 4 pairs of oligonucleotides were synthesized and inserted into pSUPERretro RNAi, which was identified by RT-PCR and sequence analysis. The vectors were then transfected into INS-1 cells, in which the expression of PTH1R was observed by Western blotting to evaluate the transfection efficiency. The cell cycle of INS-1 cells in high glucose medium was detected by flow cytometry.

RESULTSRT-PCR and sequence analysis confirmed the correct construction of the siRNA recombinant expression vector targeting PTH1R gene. The vectors were successfully transfected into INS-1 cells, and the most effective vector was selected by Western blotting. Transfection with the siRNA for PTH1R gene silencing resulted in the inhibition of INS-1 form entering the S phase.

CONCLUSIONThe successful construction of the recombinant PTH1R-siRNA vectors establishes a basis for further study of protective role of the PTH1R gene in INS-1 cells in high glucose medium.

Cell Cycle ; drug effects ; Genetic Vectors ; genetics ; Glucose ; pharmacology ; Humans ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; RNA, Small Interfering ; genetics ; Receptor, Parathyroid Hormone, Type 1 ; genetics ; metabolism

OBJECTIVETo understand the role of insulin-receptor gene in the development of insulin resistance on a population-based study in China.

METHODSPolymerase chain reaction (PCR) was used to amplify the EXON2 of the insulin-receptor gene and all amplified products were analyzed by direct sequencing.

RESULTSThree genotypes of single nucleotide at the site of 2257 in EXON2 of the insulin-receptor gene were identified. In 237 of the 345 cases (68.7%), homozygote genotype of CC phenotype was found with a gene frequency of 0.825; 13 cases (3.77%) showed homozygote genotype of TT phenotype with a gene frequency of 0.175 and the rest 95 cases (27.54%) showed heterozygote genotype of CT phenotype. Data were in agreement with the test of Hardy-Weinberg balance (chi(2) = 0.2898, upsilon = 3 - 2 = 1, 0.5 < P < 0.75). The serum level of triglyceride and the HOMA-IR index, the status of insulin resistance assessed by homeostasis model assessment (HOMA), were lower in the TT or CT genotype group than that in the CC genotype group (P < 0.05). Comparing with CC genotype, the proportion of CT genotype (18.4%) in the insulin resistance group (large than 75th percentile of HOMA-IR) was significantly lower than that in the control group (30.6%) (P = 0.022, OR = 0.493). The proportion of TT genotype (2.3%) in insulin resistance group was lower than that in the control group (4.3%, P = 0.297). logistic analysis revealed that after adjusting possible confounding factors such as taking anti-hypertensive and anti-diabetic medications, serum triglyceride and high density lipoprotein cholesterol (HDL-C), hypertension, BMI, smoking history, the OR value of people in the insulin resistance group with CT genotype was 0.448 (95% CI: 0.214 to 0.940) compared to the group with CC genotype.

CONCLUSIONThe hybridization CT genotype at the site of 2257 in EXON2 of insulin-receptor gene might have a candidate gene to serve as a protective factor for insulin resistance.

Adult ; China ; Exons ; genetics ; Female ; Gene Frequency ; Genotype ; Humans ; Insulin Resistance ; genetics ; Logistic Models ; Male ; Middle Aged ; Multivariate Analysis ; Polymerase Chain Reaction ; Polymorphism, Genetic ; genetics ; Receptor, Insulin ; genetics ; Risk Factors ; Sequence Analysis, DNA ; Triglycerides ; blood