Comparison of gene expression in cynomolgus monkeys with preclinical type II diabetes induced by different high energy diets.

BACKGROUND: Cynomolgus disease models that are similar to the preclinical stage of human type 2 diabetes mellitus (T2DM) were established by feeding middle-aged cynomolgus monkeys different high energy diets to study the differential expression of diabetes-related genes. METHODS: A total of 36 male monkeys were randomly divided into four groups and fed human diets with high sugar, high fat, double high sugar and fat, and a normal diet. The preclinical diabetes phase was determined by monitoring the metabolic characteristic indices and the results of oral glucose tolerance tests (OGTT). The mRNA expression of 45 diabetes-related genes in peripheral blood leukocytes was analyzed using real-time PCR. RESULTS: A total of 22, 25, and 21 genes were significantly up-regulated (P < 0.05) and 5, 7, and 5 genes were significantly down-regulated (P < 0.05) in the above three induced groups, respectively, compared with the control group. Of the 45 tested genes, the expression profiles of 21 genes were consistent. Most of the expression levels in the double high sugar-and-fat individuals were slightly lower than those in the high glucose and high fat groups, although the expression patterns of the three groups were essentially similar. CONCLUSION: The different high energy diets all induced diabetes and shared some phenotypic properties with human T2DM. Most of the expression patterns of the related genes were identical. The gene expression profiles could be used as references for the study of early diagnostic indicators and T2DM pathogenesis.

[Establishment and application of cell models with stable expression of hOCTN1/2].

Human carnitine/organic cation transporter 1 and 2（hOCTN1 and hOCTN2） mediate transport of endogenous and exogenous compounds. The present study aimed to establish cell models with stable expression of hOCTN1 or hOCTN2 to study interactions with compounds and transporters. MDCK cells were transfected with pcDNA3.1 (+) plasmid vector containing hOCTN1 or hOCTN2（pcDNA3.1(+)-hOCTN1/2), several stable transfected clones were obtained after G418 screening. hOCTN1 and hOCTN2 clones were screened with ergothioneine and mildronate respectively as substrates to identify the best candidates. We explored interactions of endogenous substances, alkaloids, flavonoids and ACEIs with hOCTN1/2. As a result, the cellular accumulation of ergothioneine in MDCK-hOCTN1 or mildronate in MDCK-hOCTN2 was 122 and 108 folds of the control cells, respectively. The kinetic parameters, K(m) and V(max) of ergothioneine, mediated by MDCK-hOCTN1, were 8.19 ± 0.61 µmol·L-1 and 1 427 ± 49 pmol·mg(-1)(protein)·min(-1); while K(m) and V(max) of mildronate by MDCK- hOCTN2 were 52.3 ± 4.3 µmol·L(-1) and 2 454 ± 64 pmol·mg(-1)(protein)·min(-1). Dopamine, glutamine, piperine, berberine, nuciferine, lisinopril and fosinopril could inhibit ergothioneine or mildronate uptake by MDCK- hOCTN1/2. In conclusion, cell models with good stable hOCTN1 and hOCTN2 functions have been established successfully, which can be applied to the study of interactions between compounds and transporters of hOCTN1 and hOCTN2.

[Comparison of gene expression between naturally occurring and diet-induced T2DM in cynomolgus monkeys].

To explore pathological alteration of T2DM in cynomolgus monkeys, gene expression profiles of peripheral blood leukocytes from spontaneous and diet-induced T2DM models was analyzed using quantitative real-time PCR. Among 36 T2DM associated genes tested, 19 genes (including G6PC, CCR2B, CTLA4) displayed a similar expression pattern in both spontaneous and diet-induced T2DM models and were significantly up-regulated or down-regulated compared to controls. Interestingly, expression abundance of all up-regulated genes in the diet-induced T2DM was stronger, although not significantly, than spontaneous models, indicating diet-induced T2DM in monkeys should be a reliable research model for changes in gene expression. The characteristic gene expression pattern obtained here may be useful for the clinical diagnosis of T2DM.

[Correlation between cardiovascular risk factors and the expression of cardiovascular disease related genes in cynomolgus monkeys].

The correlation between cardiovascular risk factors and cardiovascular disease related genes plays an important role in early-warning risk and early diagnosis. Thirty middle-aged male crab-eating macaques were fed a moderately atherogenic diet (0.053 mg cholesterol/kJ and 40% of calories as saturated fat) for twelve months. According to cardiovascular risk factors, we selected low-risk and high-risk crab-eating macaques, then analyzed the expression of 113 cardiovascular related genes by real-time PCR. A total of 65 genes were detected in peripheral blood leukocytes by real-time PCR. Sixteen up-regulated genes and nineteen down-regulated genes were detected in low-risk and high-risk crab-eating macaques compared to normal crab-eating macaques (P<0.05), in addition to fifteen genes that showed unique expression patterns (P<0.05). We also detected 42 genes in human peripheral blood leukocytes. The expression patterns of 22 genes were consistent between human and crab-eating macaques. These results narrowed the scope of genes for further research.

[Screening of spontaneous diabetes mellitus in middle-and old-aged cynomolgus monkey].

To screen spontaneous diabetic mellitus and explore methods for its rapid identification, the basal and inferred levels of blood glucose of 440 overweight, middle- and old-aged cynomolgus monkeys were analyzed. Diagnostic diabetes was further validated by the oral glucose tolerance test (OGTT) and urine glucose. The average level of blood glucose of these cynomolgus monkeys was (3.88±0.98) mmol/L, which was lower than the level for suspected diabetes (5.0 mmol/L). Of them, 56 (12.72%) monkeys were identified with levels of blood glucose greater than 5.0 mmol/L and diagnosed as the diabetic subjects. This population showed impaired glucose tolerance using the OGTT and 39 of the 56 (69.23%) had glucose positive urine. The methods for screening diabetic mellitus used in this study were simple, quick, and limited the harm to animals. However, the incidence of diabetes was higher in these tested monkeys than in the regular human population in China (9.7%), suggesting that these methods are useful for screening diabetic disease in a large population but not suitable for all cynomolgus monkeys.

[Differential expression of six obesity-related genes with different disease phases of T2DM in cynomolgus monkey].

Type 2 diabetes mellitus (T2DM) is a metabolic disease with a strong genetic component that is very prevalent in the world. The aim of this study is to investigate the association of a set of six obesity-related genes with the different disease phases of T2DM in a model using middle or aged cynomogus monkeys. A total of 25 male monkeys were used and fed with high-fat diet (15% lard). The disease development and progression of T2DM were monitored through the levels of plasma glucose and lipid. The mRNA expression of 6 genes was evaluated using real-time PCR on monocyte isolated from monkey peripheral blood. The 2-hour plasma glucose levels followed oral glucose tolerance test (OGTT) were (11.06+/-6.05) mmol/L and (13.12+/-2.89) mmol/L respectively (P<0.01), and the fasting plasma glucose level was (7.58+/-1.56) mmol/L (vs controls, P<0.01), indicating that we developed successful the models of pre-diabetic and diabetic disease in the cynomolgus monkey. Of the six tested genes, CDKN2B, IGF2BP2, and FTO genes were significantly up-regulated with disease progression in T2DM. We found that the expression of IGF2BP2 and FTO increased 65.92 and 4.30 folds in the developed T2DM. We conclude that the genes of CDKN2B, IGF2BP2, and FTO can be used as early diagnostic and prognostic biomarkers in type 2 diabetes.