A gain-of-function screen to identify genes that reduce lifespan in the adult of Drosophila melanogaster.

BACKGROUND: Several lines of evidence associate misregulated genetic expression with risk factors for diabetes, Alzheimer's, and other diseases that sporadically develop in healthy adults with no background of hereditary disorders. Thus, we are interested in genes that may be expressed normally through parts of an individual's life, but can cause physiological defects and disease when misexpressed in adulthood. RESULTS: We attempted to identify these genes in a model organism by arbitrarily misexpressing specific genes in adult Drosophila melanogaster, using 14,133 Gene Search lines. We identified 39 "reduced-lifespan genes" that, when misexpressed in adulthood, shortened the flies' lifespan to less than 30% of that of control flies. About half of these genes have human orthologs that are known to be involved in human diseases. For about one-fourth of the reduced-lifespan genes, suppressing apoptosis restored the lifespan shortened by their misexpression. We determined the organs responsible for reduced lifespan when these genes were misexpressed specifically in adulthood, and found that while some genes induced reduced lifespan only when misexpressed in specific adult organs, others could induce reduced lifespan when misexpressed in various organs. This finding suggests that tissue-specific dysfunction may be involved in reduced lifespan related to gene misexpression. Gene ontology analysis showed that reduced-lifespan genes are biased toward genes related to development. CONCLUSIONS: We identified 39 genes that, when misexpressed in adulthood, shortened the lifespan of adult flies. Suppressing apoptosis rescued this shortened lifespan for only a subset of the reduced-lifespan genes. The adult tissues in which gene misexpression caused early death differed among the reduced-lifespan genes. These results suggest that the cause of reduced lifespan upon misexpression differed among the genes.

A novel mutation, Ser159Pro in the NeuroD1/BETA2 gene contributes to the development of diabetes in a Chinese potential MODY family.

OBJECTIVES: During examining the prevalence of mutations in NeuroD1/BETA2 gene in Chinese early-onset type 2 diabetic probands, a novel missense mutation, Ser159Pro in a potential MODY family was identified. To investigate the role of the mutation in early-onset diabetes, we studied its transcriptional activity on human insulin gene and clinical characteristics of the family with the mutation. METHODS: Bi-directional sequencing of NeuroD1/BETA2 was performed in 85 early-onset type 2 diabetic probands without mutations in HNF4alpha, glucokinase, HNF1alpha, IPF-1 and HNF1beta genes, 95 late-onset type 2 diabetics with strong diabetic history and 87 non-diabetic control subjects. The function of the Ser159Pro to the transcription of a human insulin promotor-linked luciferase reporter gene in rat INS-1 cells was tested using Dual-Luciferase Reporter Assay System. Clinical phenotypes of the family with the Ser159Pro mutation were examined and analyzed. RESULTS: A novel mutation, Ser159Pro were found in a 27-years-old proband with both parents had diabetes. The mutation was transmitted in the heterozygous state and co-segregated with diabetes in four out of five carriers from the paternal side. Expect for the proband, all of other members with this mutation in the family, however, were diagnosed with diabetes after 50-years-old. The functional study showed that the mutant protein exhibited a 25% reduction in transcriptional activity of insulin gene when compared with the wild type. CONCLUSIONS: These results suggest that the novel Ser159Pro mutation in the NeuroD1/BETA2 gene contributes to the development of diabetes in a Chinese potential MODY family.

K(ATP) channel knockout mice crossbred with transgenic mice expressing a dominant-negative form of human insulin receptor have glucose intolerance but not diabetes.

Impaired insulin secretion and insulin resistance are thought to be two major causes of type 2 diabetes mellitus. There are two kinds of diabetic model mice: one is a K(ATP) channel knockout (Kir6.2KO) mouse which is defective in glucose-induced insulin secretion, and the other is a transgenic mouse expressing the tyrosine kinase-deficient (dominant-negative form of) human insulin receptor (hIR(KM)TG), and which has insulin resistance in muscle and fat. However, all of these mice have no evidence of overt diabetes. To determine if the double mutant Kir6.2KO/hIR(KM)TG mice would have diabetes, we generated mutant mice by crossbreeding, which would show both impaired glucose-induced insulin secretion and insulin resistance in muscle and fat. We report here that: 1) blood glucose levels of randomly fed and 6 h fasted double mutant (Kir6.2KO/hIR(KM)TG) mice were comparable with those of wild type mice; 2) in intraperitoneal glucose tolerance test (ipGTT), Kir6.2KO/hIR(KM)TG mice had an impaired glucose tolerance; and 3) during ipGTT, insulin secretion was not induced in either Kir6.2KO/hIR(KM)TG or Kir6.2KO mice, while the hIR(KM)TG mice showed a more prolonged insulin secretion than did wild type mice; 4) hyperinsulinemic euglycemic clamp test revealed that Kir6.2KO, Kir6.2KO/hIR(KM)TG and hIR(KM)TG mice, showed decreased whole-body glucose disposal compared with wild type mice; 5) Kir6.2KO, but not Kir6.2KO/hIR(KM)TG mice had some obesity and hyperleptinemia compared with wild type mice. Thus, the defects in glucose-induced insulin secretion (Kir6.2KO) and an insulin resistance in muscle and fat (hIR(KM)TG) were not sufficient to lead to overt diabetes.

Increased insulin sensitivity and hypoinsulinemia in APS knockout mice.

A tyrosine kinase adaptor protein containing pleckstrin homology and SH2 domains (APS) is rapidly and strongly tyrosine phosphorylated by insulin receptor kinase upon insulin stimulation. The function of APS in insulin signaling has heretofore remained unknown. APS-deficient (APS(-/-)) mice were used to investigate its function in vivo. The blood glucose-lowering effect of insulin, as assessed by the intraperitoneal insulin tolerance test, was increased in APS(-/-) mice. Plasma insulin levels during fasting and in the intraperitoneal glucose tolerance test were lower in APS(-/-) mice. APS(-/-) mice showed an increase in the whole-body glucose infusion rate as assessed by the hyperinsulinemic-euglycemic clamp test. These findings indicated that APS(-/-) mice exhibited increased sensitivity to insulin. However, overexpression of wild-type or dominant-negative APS in 3T3L1 adipocytes did not affect insulin receptor numbers, phosphorylations of insulin receptor, insulin receptor substrate-1, or Akt and mitogen-activated protein kinase. The glucose uptake and GLUT4 translocation were not affected by insulin stimulation in these cells. Nevertheless, the insulin-stimulated glucose transport in isolated adipocytes of APS(-/-) mice was increased over that of APS(+/+) mice. APS(-/-) mice also showed increased serum levels of leptin and adiponectin, which might explain the increased insulin sensitivity of adipocytes.

Combined effect of ACE inhibitor and exercise training on insulin resistance in type 2 diabetic rats.

The aim of this study was to investigate whether a combined treatment of ACE inhibitor and exercise training is more effective than either treatment alone in alleviating the insulin resistant states in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, a model of type 2 diabetes. OLETF rats (25 weeks old) were randomly divided into 5 groups; sedentary control, exercise-trained, temocapril (ACE inhibitor; 2 mg/kg/day)-treated, with and without exercise, and losartan (AT1 receptor antagonist; 1 mg/kg/day)-treated. Long-Evans Tokushima Otsuka rats were used as a non-diabetic control. Body weight, the amount of abdominal fat and blood pressure were higher for OLETF rats than for control rats. However, glucose infusion rate (GIR), an index of insulin resistance, was decreased greatly in OLETF rats. The fasting levels of blood glucose, insulin and lipids were also increased in the diabetic strain. In OLETF rats, both temocapril and losartan reversed hypertensive states significantly, whereas GIR and hyperlipidemia were improved when rats were treated with ACE inhibitors, but not with the AT1 receptor antagonist. Exercise training decreased body weight and the amount of abdominal fat, and also increased GIR in parallel with improved dislipidemia. The combination of the ACE inhibitor with exercise training also improved obesity, hyperinsulinemia, dislipidemia and fasting level of blood glucose, and this combination resulted in the greatest improvement of insulin resistance. These results suggest that the combination of ACE inhibitor and exercise training may be a beneficial treatment for mixed diabetic and hypertensive conditions.

Exercise training improves acetylcholine-induced endothelium-dependent hyperpolarization in type 2 diabetic rats, Otsuka Long-Evans Tokushima fatty rats.

We investigated whether endothelium-derived relaxing (EDRF) and hyperpolarizing factor (EDHF) is impaired in type 2 diabetic rats (Otsuka Long-Evans Tokushima Fatty (OLETF) rat) and whether the exercise training improves impaired EDRF and EDHF. Diabetic rats were divided into the sedentary and exercise-trained groups at the age of 16 weeks. Long-Evans Tokushima Otsuka (LETO) rats were used as age-matched non-diabetic controls. EDRF as well as EDHF induced by acetylcholine in the presence of indomethacine and L-nitro N-arginine was significantly attenuated in the diabetic rats, and was further impaired with age. Exercise training significantly improved it. Both insulin resistance and abdominal fat accumulation were significantly greater in the diabetic rats, compared with the non-diabetic rats, but were decreased in exercise-trained rats. Urinary NO(2) secretion was decrease in the diabetic rats at each age, and it was improved by exercise training. The results of the study indicated that exercise training prevented impairment of EDHF, as well as EDRF in type 2 diabetic rats, presumably due to improvement of hyperglycemia and insulin resistance and increase in the production of nitric oxide by exercise training.

Effect of eicosapentaenoic acid ethyl ester v. oleic acid-rich safflower oil on insulin resistance in type 2 diabetic model rats with hypertriacylglycerolaemia.

The purpose of the present study was to test whether hyperlipidaemia and insulin resistance in type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats can be improved by dietary supplementation with purified eicosapentaenoic acid (EPA) or oleic acid (OA). Male OLETF rats were fed powdered chow (510 g fat/kg) alone (n 8) or chow supplemented with 10 g EPA- (n 8) or OA- (n 8) rich oil/kg per d from 5 weeks until 30 weeks of age. An oral glucose tolerance test and hyperinsulinaemic euglycaemic clamp was performed at 25 and 30 weeks of age. EPA supplementation resulted in significantly (P<0.05) reduced plasma lipids, hepatic triacylglycerols, and abdominal fat deposits, and more efficient in vivo glucose disposal compared with OA supplementation and no supplementation. OA supplementation was associated with significantly increased insulin response to oral glucose compared with EPA supplementation and no supplementation. Inverse correlation was noted between glucose uptake and plasma triacylglycerol levels (r -086, P<0.001) and abdominal fat volume (r -0.80, P<0.001). The result of oral glucose tolerance test study showed that the rats fed EPA tended to improve glucose intolerance, although this was not statistically significant. Levels of plasma insulin at 60 min after glucose was significantly increased in rats fed OA compared with the other two groups. The results indicate that long-term feeding of EPA might be effective in preventing insulin resistance in diabetes-prone rats, at least in part, due to improving hypertriacylglycerolaemia.