Hyperglycemia induced down-regulation of renal P-glycoprotein expression.

The purpose of this study is to investigate the regulation of P-glycoprotein expression in the kidney under diabetic condition. Renal P-glycoprotein expression was examined in inbred mice with type 1 or type 2 diabetes by Western blotting. The underlying mechanisms of P-glycoprotein regulation were examined in Madin-Darby canine kidney type II (MDCK-II) cells by Western blotting or qRT-PCR. (3)H-digoxin uptake was measured for P-glycoprotein activity in cells under various treatments. The results showed that P-glycoprotein expression was lower in kidneys of diabetic mice than in controls. In MDCK-II cells, treatments with insulin or IL-6 did not cause any change in P-glycoprotein expression, whereas TNF-α tended to increase P-glycoprotein expression at a concentration of 1 ng/ml. On the other hand, P-glycoprotein expression was reduced under high glucose conditions (450 mg/dl), while superoxide production was increased, and the reduction in P-glycoprotein expression was abolished by N-acetylcysteine (an antioxidant) and staurosporine (a nonselective PKC inhibitor). Treatment with oxidizing agents (H(2)O(2), BSO) or PMA (a PKC activator) reduced P-glycoprotein expression. Antioxidant (N-acetylcysteine or glutathione) co-treatment abolished the H(2)O(2)-induced and BSO-induced reduction in P-glycoprotein expression, whereas it did not prevent the effect of PMA. The PMA-induced P-glycoprotein down-regulation was prevented by co-treatment of LY333531 (a PKC-ß inhibitor). (3)H-digoxin levels were higher in MDCK-II cells with high glucose, PMA or H(2)O(2) treatments. In conclusion, P-glycoprotein expression is lower in kidneys of diabetic mice and in MDCK-II cells under high glucose conditions. Hyperglycemia induced reactive oxygen species and activated PKC in MDCK-II cells, leading to the decrease in P-glycoprotein expression.

Change in P-glycoprotein and caveolin protein expression in brain striatum capillaries in New Zealand obese mice with type 2 diabetes.

AIMS: To investigate the expression of P-gp and caveolins in brain striatum capillaries in inbred mice with type 2 diabetes. MAIN METHODS: Inbred mice with type 2 diabetes (male New Zealand obese; NZO) were compared with related mice without diabetes (female NZO and New Zealand White). Protein expression of P-gp and caveolins in capillaries of the brain striatum was examined by immunohistochemical analysis. P-gp efflux pump activity in the blood-brain barrier (BBB) was measured by in vivo brain microdialysis. Regulation of P-gp and caveolin expression was examined in cultured adult rat brain endothelial cells (ARBEC). KEY FINDINGS: In capillaries in the brain striatum, expression of P-gp and caveolins was higher and lower, respectively, in mice with type 2 diabetes compared with non-diabetic mice. Brain extracellular concentrations of intravenously injected rhodamine 123 were more than 50-60% lower in type 2 diabetic mice. Insulin and PMA treatments significantly increased P-gp expression, whereas the same treatments decreased caveolin expression in ARBEC. SIGNIFICANCE: Protein expression of P-gp and caveolins can be regulated in animals with type 2 diabetes. These changes may be important in modulating P-gp activity in the BBB in type 2 diabetes.

Mutations in the SLC2A10 gene cause arterial abnormalities in mice.

AIMS: Glucose transporter 10 (GLUT10), encoded by the SLC2A10 gene, is a member of the class III facilitative glucose transporter family. Mutations in the SLC2A10 gene cause arterial tortuosity syndrome (ATS) in humans. To further study the pathogenesis of the disease, we generated mice carrying GLUT10 mutations. METHODS AND RESULTS: Using a gene-driven N-ethyl-N-nitrosourea (ENU)-mutagenesis approach, we generated mice carrying GLUT10 mutations c.383G>A and c.449C>T, which resulted in missense mutations of glycine to glutamic acid (p.G128E) and serine to phenylalanine (p.S150F), respectively. Both mutant strains appeared normal at birth, gained weight appropriately and survived to adulthood (>18 months). Blood and urine glucose were normal. Echocardiogram and electrocardiogram were also normal and brain magnetic resonance angiography revealed normal cerebral arteries without tortuosity, stenosis/dilatation, or aneurysm. Histopathology revealed thickening and irregular vessel wall shape of large and medium size arteries characterized by markedly increased elastic fibres, both in number and size. There was also intima endothelial hypertrophy and deranged elastic fibres that resulted in disruption of internal elastic lamina in the aorta of older mice. CONCLUSION: Abnormal elastogenesis with early elastic fibre proliferation provides a clue to the pathogenesis of arterial tortuosity in human ATS. Availability of this mouse model will allow testing of the relationship between diabetes and its vascular complications, including diabetic retinopathy, nephropathy and peripheral vascular disease.

A polymorphism in New Zealand inbred mouse strains that inactivates phosphatidylcholine transfer protein.

New Zealand obese (NZO/HlLt) male mice develop polygenic diabetes and altered phosphatidylcholine metabolism. The gene encoding phosphatidylcholine transfer protein (PC-TP) is sited within the support interval for Nidd3, a recessive NZO-derived locus on Chromosome 11 identified by prior segregation analysis between NZO/HlLt and NON/Lt. Sequence analysis revealed that the NZO-derived PC-TP contained a non-synonymous point mutation that resulted in an Arg120His substitution, which was shared by the related NZB/BlNJ and NZW/LacJ mouse strains. Consistent with the structure-based predictions, functional studies demonstrated that Arg120His PC-TP was inactive, suggesting that this mutation contributes to the deficiencies in phosphatidylcholine metabolism observed in NZO mice.

Adverse hepatic and cardiac responses to rosiglitazone in a new mouse model of type 2 diabetes: relation to dysregulated phosphatidylcholine metabolism.

Given the heterogeneous nature of metabolic dysfunctions associated with insulin resistance and type 2 diabetes (T2D), a single pharmaceutical cannot be expected to provide complication-free therapy in all patients. Thiazolidinediones (TZD) increase insulin sensitivity, reduce blood glucose and improve cardiovascular parameters. However, in addition to increasing fat mass, TZD have the potential in certain individuals to exacerbate underlying hepatosteatosis and diabetic cardiomyopathy. Pharmacogenetics should allow patient selection to maximize therapy and minimize risk. To this end, we have combined two genetically diverse inbred strains, NON/Lt and NZO/Lt, to produce a "negative heterosis" increasing the frequency of T2D in F1 males. As in humans with T2D, treatment of diabetic and hyperlipemic F1 males with rosiglitazone (Rosi), an agonist of peroxisome proliferator-activated gamma receptor (PPARgamma), reverses these disease phenotypes. However, the hybrid genome perturbed both major pathways for phosphatidylcholine (PC) biosynthesis in the liver, and effected remarkable alterations in the composition of cardiolipin in heart mitochondria. These metabolic defects severely exacerbated an underlying hepatosteatosis and increased levels of the adipokine, plasminogen activator inhibitor-1 (PAI-1), a risk factor for cardiovascular events. This model system demonstrates how the power of mouse genetics can be used to identify the metabolic signatures of individuals who may be prone to drug side effects.

Differential endocrine responses to rosiglitazone therapy in new mouse models of type 2 diabetes.

Polygenic mouse models for obesity-induced type 2 diabetes (T2D) more accurately reflect the most common manifestations of the human disease. Two inbred mouse strains (NON/Lt and NZO/HlLt) separately contributed T2D susceptibility- conferring quantitative trait loci to F1 males. Although chronic administration of rosiglitazone (Rosi) in diet (50 mg/kg) effectively suppressed F1 diabetes, hepatosteatosis was an undesired side effect. Three recombinant congenic strains (designated RCS1, -2, and -10) developed on the NON/Lt background carry variable numbers of these quantitative trait loci that elicit differential weight gain and male glucose intolerance syndromes of variable severity. We previously showed that RCS1 and -2 mice responded to chronic Rosi therapy without severe steatosis, whereas RCS10 males were moderately sensitive. In contrast, another recombinant congenic strain, RCS8, responded to Rosi therapy with the extreme hepatosteatosis observed in the F1. Longitudinal changes in multiple plasma analytes, including insulin, the adipokines leptin, resistin, and adiponectin, and plasminogen activator inhibitor-1 (PAI-1) allowed profiling of the differential Rosi responses in steatosis-exacerbated F1 and RCS8 males vs. the resistant RCS1 and RCS2 or moderately sensitive RCS10. Of these biomarkers, PAI-1 most effectively predicted adverse drug responses. Unexpectedly, mean resistin concentrations were higher in Rosi-treated RCS8 and RCS10. In summary, longitudinal profiling of multiple plasma analytes identified PAI-1 as a useful biomarker to monitor for differential pharmacogenetic responses to Rosi in these new mouse models of T2D.

Pharmacogenetic analysis of rosiglitazone-induced hepatosteatosis in new mouse models of type 2 diabetes.

Although thiazolidinediones suppress hyperglycemia in diabetic (NON x NZO)F1 males, these mice exhibit unusual sensitivity to drug-induced exacerbation of an underlying hepatosteatosis only rarely experienced in human patients. To establish the pharmacogenetic basis for this sensitivity, a panel of recombinant congenic strains (RCSs) with varying degrees of obesity and diabetes was generated by fixing selected NZO HlLt alleles on the diabetes- and hepatosteatosis-resistant NON/Lt background. Four new strains in this panel were exposed to chronic rosiglitazone treatment. Only one, NONcNZO8 (designated RCS8), exhibited an F1-like hepatosteatotic response. In both the F1 and RCS8 males, this adverse effect correlated with rosiglitazone suppression of already impaired hepatic phosphatidylcholine biosynthetic enzymes in both arms of the biosynthetic pathway, the phosphatidylethanolamine methyl- transferase pathway, and the CDP-choline pathway, including choline kinase and CTP-cholinephosphate cytidylyltransferase. This adverse response was not reproduced by CL316,243, a beta3-adrenergic receptor agonist with potent antihyperlipemic effects. Genome comparison showed that RCS8 differed from the other strains in carrying NZO-derived genome on virtually all of chromosome 16 and in smaller segments on chromosomes 6, 14, and 17. Thus, these RCSs present a panel of new mouse models exhibiting differential levels of obesity and diabetes as well as different drug responses. This panel can be used to screen for treatments for type 2 diabetes and its complications.

Production of polyclonal antibodies against territrem B and detection of territrem B in the conidia of Aspergillus terreus 23-1 by immunoelectron microscopy.

Territrem B, a fungal metabolite isolated from Aspergillums terreus 23-1, is a tremorgenic mycotoxin. Immunoelectron microscopy using anti-territrem B polyclonal antibody was used to detect territrem B in the fungal body of A. terreus 23-1 at different times of culture without shaking on potato dextrose (PD) agar medium. The anti-territrem B serum was produced by immunization of rabbits with 4beta-hydroxymethyl-4beta-demethylterritrem B-sccinate bound by a linker to keyhole limpet hemocyanin. This antiserum recognized territrems and immunoelectron microscopy using this antiserum, and colloidal gold-conjugated anti-rabbit IgG antibodies showed that territrem B was localized to the fungal body of A. terreus 23-1. Territrem B was first seen in the cytoplasm of the conidia after 4 days' culture on PD agar medium. Maximal territrem B production in the conidia was seen on the 14th day of culture; however, territrem B was not formed in the hyphae at any stage of culture. These results are consistent with the previous finding that the formation of territrems is related to fungal sporulation.

Contributions of dysregulated energy metabolism to type 2 diabetes development in NZO/H1Lt mice with polygenic obesity.

New Zealand Obese (NZO) male mice develop a polygenic juvenile-onset obesity and maturity-onset hyperinsulinemia and hyperglycemia (diabesity). Here we report on metabolic and molecular changes associated with the antidiabesity action of CL316,243 (CL), a beta(3)-adrenergic receptor agonist. Dietary CL treatment initiated at weaning reduced the peripubertal rise in body weight and adiposity while promoting growth without suppressing hyperphagia. The changes in adiposity, in turn, suppressed development of hyperinsulinemia, hyperleptinemia, hyperlipidemia, and hyperglycemia. These CL-induced alterations were reflected by decreased adipose tissue mass, increased expression of transcripts for uncoupling protein-1 (UCP-1), peroxisome proliferator-activated receptor alpha (PPARalpha), peroxisome proliferater-activated receptor coactivator-1 (PGC-1), and robust development of brown adipocyte function in white fat. Increased drug-mediated energy dissipation elicited a 1.5 degrees C increase in whole body temperature under conditions of increased food intake but with no change in physical activity. Indirect calorimetry of mice treated with CL showed both increased energy expenditure and a restoration of a prominent diurnal pattern in the respiratory exchange ratio suggesting improved nutrient sensing. Our data suggest that CL promotes increased energy dissipation in white and brown fat depots by augmenting thermogenesis and by metabolic re-partitioning of energy in a diabesity-protective fashion. This is the first report demonstrating the effects of dietary beta(3)-agonist in preventing the onset of diabesity in a polygenic rodent model of type 2 diabetes.

Increasing prevalence of methicillin-resistant Staphylococcus aureus causing nosocomial infections at a university hospital in Taiwan from 1986 to 2001.

A rapid emergence of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) infection (from 26.3% in 1986 to 77% in 2001) was found. The susceptibility of 200 nonduplicate blood isolates of MRSA and 100 MRSA isolates causing refractory bacteremia to 22 antimicrobial agents disclosed that glycopeptides, quinupristin-dalfopristin, and linezolid remained the most active agents.

Evidence on the presence of secretin cells in the gastric antral and oxyntic mucosa.

Secretin is released from upper small intestinal mucosa to drive pancreatic secretion of fluid and bicarbonate and inhibit gastric acid secretion. Recently, we found that, in isolated, vascularly perfused rat stomach model, the inhibition of acid secretion by pituitary adenylate cyclase activating polypeptide (PACAP) was mediated in part via local release of secretin. However, the presence of secretin-producing cells and mRNA in gastric mucosa, particularly in oxyntic mucosa, has not been established. The present study was carried out to establish the presence of secretin cells by immunohistochemical and mRNA by biochemical methods in gastric mucosa. Secretin cells were identified in antral mucosa (27.8 +/- 2.0 cells/mm(2)) and corpus (4.7 +/- 0.5 cells/mm(2)). They were distinguishable, through double immunostaining, from gastrin and somatostatin cells in the antrum and from somatostatin cells in the corpus. The results of reverse transcription (RT)-PCR and Southern blot indicated that a secretin gene transcript of 454 bp was present in the mRNA extracts of both antral and corpus mucosae. The results indicated that secretin mRNA is present in gastric mucosa. In conclusion, secretin-producing cells and mRNA are present in gastric mucosa and the locally released secretin may exert a paracrine effect to inhibit acid secretion.

Lipid metabolome-wide effects of the PPARgamma agonist rosiglitazone.

Successful therapy for chronic diseases must normalize a targeted aspect of metabolism without disrupting the regulation of other metabolic pathways essential for maintaining health. Use of a limited number of single molecule surrogates for disease, or biomarkers, to monitor the efficacy of a therapy may fail to predict undesirable side effects. In this study, a comprehensive metabolomic assessment of lipid metabolites was employed to determine the specific effects of the peroxisome proliferator-activated receptor gamma (PPARgamma) agonist rosiglitazone on structural lipid metabolism in a new mouse model of Type 2 diabetes. Dietary supplementation with rosiglitazone (200 mg/kg diet) suppressed Type 2 diabetes in obese (NZO x NON)F1 male mice, but chronic treatment markedly exacerbated hepatic steatosis. The metabolomic data revealed that rosiglitazone i) induced hypolipidemia (by dysregulating liver-plasma lipid exchange), ii) induced de novo fatty acid synthesis, iii) decreased the biosynthesis of lipids within the peroxisome, iv) substantially altered free fatty acid and cardiolipin metabolism in heart, and v) elicited an unusual accumulation of polyunsaturated fatty acids within adipose tissue. These observations suggest that the phenotypes induced by rosiglitazone are mediated by multiple tissue-specific metabolic variables. Because many of the effects of rosiglitazone on tissue metabolism were reflected in the plasma lipid metabolome, metabolomics has excellent potential for developing clinical assessments of metabolic response to drug therapy.