Loss of coupling between calcium influx, energy consumption and insulin secretion associated with development of hyperglycaemia in the UCD-T2DM rat model of type 2 diabetes.

AIMS/HYPOTHESIS: Previous studies on isolated islets have demonstrated tight coupling between calcium (Ca(2+)) influx and oxygen consumption rate (OCR) that is correlated with insulin secretion rate (ISR). To explain these observations, we have proposed a mechanism whereby the activation of a highly energetic process (Ca(2+)/metabolic coupling process [CMCP]) by Ca(2+) mediates the stimulation of ISR. The aim of the study was to test whether impairment of the CMCP could play a role in the development of type 2 diabetes. METHODS: Glucose- and Ca(2+)-mediated changes in OCR and ISR in isolated islets were compared with the time course of changes of plasma insulin concentrations observed during the progression to hyperglycaemia in a rat model of type-2 diabetes (the University of California at Davis type 2 diabetes mellitus [UCD-T2DM] rat). Islets were isolated from UCD-T2DM rats before, 1 week, and 3 weeks after the onset of hyperglycaemia. RESULTS: Glucose stimulation of cytosolic Ca(2+) and OCR was similar for islets harvested before and 1 week after the onset of hyperglycaemia. In contrast, a loss of decrement in islet OCR and ISR in response to Ca(2+) channel blockade coincided with decreased fasting plasma insulin concentrations observed in rats 3 weeks after the onset of hyperglycaemia. CONCLUSIONS/INTERPRETATION: These results suggest that phenotypic impairment of diabetic islets in the UCD-T2DM rat is downstream of Ca(2+) influx and involves unregulated stimulation of the CMCP. The continuously elevated levels of CMCP induced by chronic hyperglycaemia in these islets may mediate the loss of islet function.

Effects of rifampin and mefenamic acid on the pharmacokinetics and pharmacodynamics of dapagliflozin.

AIMS: Dapagliflozin is a selective sodium glucose cotransporter 2 (SGLT2) inhibitor that decreases serum glucose by reducing renal glucose reabsorption, thereby promoting urinary glucose excretion. Dapagliflozin is primarily metabolized via the uridine diphosphate-glucuronosyltransferase (UGT)1A9 pathway to its major inactive metabolite, dapagliflozin 3-O-glucuronide. The aim of this study was to evaluate the potential for drug-drug interaction between dapagliflozin and two potential UGT1A9 modulators. METHODS: The results of two open-label, non-randomized, single-sequence studies are reported in which the effects of rifampin (a pleiotropic drug-metabolizing enzyme inducer; study 1) and mefenamic acid (a strong UGT1A9 inhibitor; study 2) were evaluated on the pharmacokinetics and pharmacodynamics (assessed by urinary glucose excretion [UGE]) of dapagliflozin in healthy subjects. In study 1, 14 subjects received single doses of dapagliflozin 10 mg alone and in the presence of rifampin 600 mg QD (6 days). In study 2, 16 subjects received single doses of dapagliflozin 10 mg alone and in the presence of mefenamic acid 250 mg q6h (5 days). RESULTS: Rifampin reduced total exposure (area under the concentration-time curve from time 0 to infinity [AUC0-inf]) to dapagliflozin by 22% and mefenamic acid increased dapagliflozin AUC0-inf by 51%. No clinically meaningful effect of rifampin or mefenamic acid on the pharmacokinetics of dapagliflozin or on dapagliflozin-mediated urinary glucose excretion was observed. CONCLUSION: Modest changes in dapagliflozin exposure were seen with rifampin and mefenamic acid with minor changes in UGE, none of which were considered clinically meaningful.

Consumption of fructose-sweetened beverages for 10 weeks reduces net fat oxidation and energy expenditure in overweight/obese men and women.

BACKGROUND/OBJECTIVES: The results of short-term studies in humans suggest that, compared with glucose, acute consumption of fructose leads to increased postprandial energy expenditure and carbohydrate oxidation and decreased postprandial fat oxidation. The objective of this study was to determine the potential effects of increased fructose consumption compared with isocaloric glucose consumption on substrate utilization and energy expenditure following sustained consumption and under energy-balanced conditions. SUBJECTS/METHODS: As part of a parallel arm study, overweight/obese male and female subjects, 40-72 years, consumed glucose- or fructose-sweetened beverages providing 25% of energy requirements for 10 weeks. Energy expenditure and substrate utilization were assessed using indirect calorimetry at baseline and during the 10th week of intervention. RESULTS: Consumption of fructose, but not glucose, led to significant decreases of net postprandial fat oxidation and significant increases of net postprandial carbohydrate oxidation (P<0.0001 for both). Resting energy expenditure (REE) decreased significantly from baseline values in subjects consuming fructose (P=0.031) but not in those consuming glucose. CONCLUSIONS: Increased consumption of fructose for 10 weeks leads to marked changes of postprandial substrate utilization including a significant reduction of net fat oxidation. In addition, we report that REE is reduced compared with baseline values in subjects consuming fructose-sweetened beverages for 10 weeks.

Lack of pharmacokinetic interaction between dapagliflozin, a novel sodium-glucose transporter 2 inhibitor, and metformin, pioglitazone, glimepiride or sitagliptin in healthy subjects.

AIMS: Dapagliflozin increases urinary glucose excretion by selectively inhibiting renal sodium-glucose transporter 2, an insulin-independent mechanism of action that may be complementary to that of other oral antidiabetes drugs. The current studies assessed the potential for pharmacokinetic (PK) interaction between dapagliflozin and pioglitazone, metformin, glimepiride or sitagliptin in healthy subjects following single-dose administration. METHODS: In open-label, randomized, three-period, three-treatment crossover studies, 24 subjects received 50 mg dapagliflozin, 45 mg pioglitazone or the combination, while 18 subjects received 20 mg dapagliflozin, 1000 mg metformin or the combination. In an open-label, randomized, five-period, five-treatment, unbalanced crossover study, 18 subjects first received 20 mg dapagliflozin, 4 mg glimepiride or the combination, and afterward 100 mg sitagliptin or sitagliptin plus 20 mg dapagliflozin. Blood samples were taken over 72 h of each treatment period. Lack of PK interaction was defined as the ratio of geometric means and 90% confidence interval (CI) for combination:monotherapy being within the range of 0.80-1.25. RESULTS: Co-administration of dapagliflozin with pioglitazone, metformin, glimepiride or sitagliptin had no effect on dapagliflozin maximum plasma concentration (C(max) ) or area under the plasma concentration-time curve (AUC). Similarly, dapagliflozin did not affect the C(max) or AUC for the co-administered drug, except for slight extensions of the 90% CI for the ratio of geometric means for glimepiride AUC (upper limit 1.29) and pioglitazone C(max) (lower limit 0.75). All monotherapies and combination therapies were well tolerated. CONCLUSION: Dapagliflozin can be co-administered with pioglitazone, metformin, glimepiride or sitagliptin without dose adjustment of either drug.

Homozygosity mapping places the acrodermatitis enteropathica gene on chromosomal region 8q24.3.

Acrodermatitis enteropathica (AE) is a rare autosomal recessive pediatric disease characterized by dermatitis, diarrhea, alopecia, and growth failure. The disease results from insufficient uptake of zinc by the intestine and can be fatal unless the diet is supplemented with zinc. To map the gene responsible for AE, a genomewide screen was performed on 17 individuals, including 4 affected individuals, in a consanguineous Jordanian family. Three markers-D8S373, D10S212, and D6S1021-had a pattern consistent with tight linkage to a recessive disease: one allele in the affected sibs and multiple alleles in unaffected sibs and parents. Two-point parametric linkage analysis using FASTLINK identified one region, D8S373, with a maximum LOD score >1.5 (1.94 at D8S373: recombination fraction.001). Twelve additional markers flanking D8S373 were used to genotype the extended family, to fine-map the AE gene. All five affected individuals-including one who was not genotyped in the genomewide screen-were found to be homozygous for a common haplotype, spanning approximately 3.5 cM, defined by markers D8S1713 and D8S2334 on chromosomal region 8q24.3. To support these mapping data, seven consanguineous Egyptian families with eight patients with AE were genotyped using these markers, and six patients from five families were found to be homozygous in this region. Multipoint analysis with all consanguineous families, by Mapmaker/Homoz, resulted in a maximum LOD score of 3.89 between D8S1713 and D8S373. Sliding three-point analysis resulted in a maximum LOD score of 5.16 between markers D8S1727 and D8S1744.

A genetic defect in beta-cell gene expression segregates independently from the fa locus in the ZDF rat.

Type 2 diabetes is a strongly genetic disorder resulting from inadequate compensatory insulin secretion in the face of insulin resistance. The Zucker diabetic fatty (ZDF) rat is a model of type 2 diabetes and, like the human disease, has both insulin resistance (from a mutant leptin receptor causing obesity) and inadequate beta-cell compensation. To test for an independently inherited beta-cell defect, we examined beta-cell function in fetuses of ZDF-lean rats, which have wild-type leptin receptors. beta-Cell number and insulin content do not differ among wild-type, heterozygous, and homozygous ZDF-lean fetuses. However, insulin promoter activity is reduced 30-50% in homozygous ZDF-lean fetal islets, and insulin mRNA levels are similarly reduced by 45%. This is not a generalized defect in gene expression nor an altered transfection efficiency, because the islet amyloid polypeptide promoter and viral promoters are unaffected. Insulin promoter mapping studies suggest that the defect involves the critical A2-C1-E1 region. This study demonstrates that the ZDF rat carries a genetic defect in beta-cell transcription that is inherited independently from the leptin receptor mutation and insulin resistance. The genetic reduction in beta-cell gene transcription in homozygous animals likely contributes to the development of diabetes in the setting of insulin resistance.

A novel glucose-responsive element in the human insulin gene functions uniquely in primary cultured islets.

Insulin gene transcription is limited to the beta cells within the mammalian pancreas and, like insulin secretion, is regulated by glucose. Our previous studies in primary cultured beta cells suggested the presence of a strong glucose-responsive enhancer element between base pairs -341 and -260 of the human insulin promoter, the same region in which a transcriptional repressor had been identified in beta-cell tumor lines. In an attempt to map these promoter activities and resolve these conflicting data, we designed minienhancer constructs spanning this region, and tested them in primary cultured and immortalized cells. One sequence, the Z element (base pairs -292 to -243), functions as both a potent glucose-responsive transcriptional enhancer in primary cultured islet cells and as a transcriptional repressor in immortalized beta and nonbeta cells and in primary fibroblasts. In addition, the Z element binds a novel glucose-responsive protein complex that is found in the nuclei of primary cultured islet cells, but not in the nuclei of tumor cells or primary cultured fibroblasts. These data demonstrate a critical role for the Z element in human insulin gene transcription and its regulation by glucose.

A model for the evolution of polyubiquitin genes from the study of Arabidopsis thaliana ecotypes.

Polyubiquitin genes encode the highly conserved 76-amino acid protein ubiquitin that is covalently attached to substrate proteins targeting most for degradation. Polyubiquitin genes are characterized by the presence of tandem repeats of the 228 bp that encode a ubiquitin monomer. Five polyubiquitin genes UBQ3, UBQ4, UBQ10, UBQ11, and UBQ14, previously isolated from Arabidopsis thaliana ecotype Columbia [10] encode identical mature ubiquitin proteins, but differ in synonymous substitutions, nature of amino acids terminating the open reading frame, and in the number of ubiquitin repeats. The presence of these five genes in nine other Arabidopsis ecotypes was verified by polymerase chain reaction (PCR). Size differences in UBQ3 and UBQ11 amplified products from several ecotypes were observed, suggesting that alleles differ in ubiquitin repeat number. DNA sequence of UBQ11 alleles from each size class (ecotypes Be-0, Ler. and Rld-0) verified that PCR product size differences resulted from changes in the number of ubiquitin repeats. Nucleotide sequence between two UBQ11 alleles containing the same number of repeats was identical. Transcript size differences for UBQ3 and UBQ11 mRNAs between ecotypes Columbia and Landsberg indicated that repeat number changes did not inactivate these genes. Nucleotide sequence comparisons between UBQ11 repeats from different ecotypes suggest that first repeats are related to each other and last repeats are related to each other. We hypothesize that changes in UBQ11 ubiquitin repeat number occurred via the contraction and/or expansion of specific internal repeats or portions thereof by misalignment of alleles and recombination, most likely via unequal crossing-over events.

Vasopressin response to haemorrhage in rats: effect of hypoxia and water restriction.

1. The aim of the present study was to determine the effect of water restriction and/or hypoxia on the vasopressin response to haemorrhage in conscious rats. 2. Male, Long-Evans rats (n = 39) were prepared with chronically indwelling femoral artery and vein catheters and exposed to 24 h of one of the following: normoxia with ad lib drinking water (N + W); normoxia with water restriction (N - W); hypoxia with ad lib drinking water (H + W); and hypoxia with water restriction (H - W). At the end of 24 h, a 15 mL/kg arterial haemorrhage was performed. 3. Water restricted rats had elevated pre-haemorrhage vasopressin levels. Haemorrhage induced an increase in vasopressin in all groups. Water restriction (N - W) or hypoxia (H + W) each augmented the vasopressin response to haemorrhage. However, the combination of hypoxia and water restriction (H - W) failed to augment the vasopressin response to haemorrhage as compared to normoxic, water replete (N + W) rats. 4. Hypoxia or water restriction per se augment the vasopressin response to haemorrhage. This augmented vasopressin response to haemorrhage is not maintained when hypoxia and water restriction are combined.

Vasopressin responses to hypoxia in conscious rats: interaction with water restriction.

The purpose of this study was to determine the effect of water restriction on the vasopressin response to hypoxia in conscious Long-Evans rats. Rats were prepared with chronic indwelling femoral artery and vein catheters 1 week before experimentation. At 24 h before the first blood sample, the supply of drinking water was maintained ad libitum (water replete) or removed (water deplete). At 24 h, a control blood sample was taken and then normoxia (21% O2) was maintained or hypoxia (10% O2) induced. Additional blood samples were taken at 1, 18 and 24 h. All blood samples (2.5 ml) were simultaneously replaced with donor blood to maintain isovolaemia. Hypoxia led to a very small and transient increase in vasopressin in the water-replete rats. The combination of hypoxia and water restriction led to a greatly augmented vasopressin response at 1 h (60 +/- 16 pmol/l); this response was also not sustained. Additional non-cannulated rats were exposed to 24 h of normoxia or hypoxia with or without water available ad libitum and posterior pituitaries were collected after decapitation for measurement of vasopressin content. Water restriction, hypoxia and water restriction plus hypoxia all led to decreased pituitary vasopressin content. We conclude that the vasopressin response to hypoxia in conscious rats is small and transient, and that concomitant water restriction augments the vasopressin response to acute but not chronic hypoxia.

The influence of immunosuppression on peptic ulceration following renal transplantation and the role of endoscopy.

Peptic ulceration is common in patients undergoing renal transplantation. With a change in routine immunosuppression from azathioprine and steroids to cyclosporin, or cyclosporin and low-dose steroids and azathioprine, less peptic ulceration might be expected. This was investigated in two groups of patients undergoing renal transplantation. Group 1 (n = 90) received azathioprine and prednisolone; Group 2 (n = 44) received cyclosporin and low-dose azathioprine and prednisolone. Patients from both groups were endoscoped 7-14 days after operation, when peptic ulceration, oesophagitis, gastritis, and duodenitis were assessed. Whilst Group 2 differed significantly in receiving more methylprednisolone than Group 1 (P less than 0.05), there was no difference between the two groups with regard to the incidence of peptic ulceration or inflammatory lesions. In both groups there was a trend for those treated with methylprednisolone for rejection to develop ulcers or inflammatory lesions when a total dose of more than 2 g had been given by the time of endoscopy. Any possible reduction in the incidence of peptic ulceration after transplantation is outweighed by the continued use of high-dose methylprednisolone to treat episodes of rejection. The true incidence of peptic ulceration after transplantation is much higher than if presenting symptoms alone are the starting point for investigation. Regular post-transplantation endoscopy allows prompt diagnosis and treatment, with attainment of a very low mortality rate (0.75% overall, or 4% in those with peptic ulcers) from peptic ulceration in patients undergoing renal transplantation.

Insulin exerts metabolic and growth-promoting effects by a direct action on the liver in vivo: clarification of the functional significance of the portal vascular link between the beta cells of the pancreatic islets and the liver.

The functional significance of the portal vascular link between the beta cells of the pancreatic islets and the liver has not been established. Previous studies indicated that insulin does not acutely regulate glucose metabolism by a direct hepatic effect. More recent observations suggest that the role of insulin in regulating body growth may be mediated, at least in part, by the liver. Our experiments were designed to test whether insulin can promote body growth and regulate glucose metabolism by a direct hepatic action in vivo. Rats were made diabetic by injections of streptozotocin, and insulin or solvent was infused into the jugular vein (JV) or the hepatic portal vein (HPV) for 14 days using catheters that were attached to osmotic minipumps. Infusion of a low dose of insulin (2 units per kg per day) into the JV had no effects on the hyperglycemia, body weight gain, tail growth, tibial epiphysial cartilage plate thickness, or serum levels of somatomedin C in the diabetic rats. However, the same dose given into the HPV caused a 30% reduction of blood glucose and stimulated a significant degree of growth, as determined by all indices. Infusion of a higher dose of insulin (5 units per kg per day) into either vein caused full restoration of body weight gain and tail growth and it restored the glycemic status almost to normal. However, it did not increase the tibial epiphysial plate width or serum somatomedin C levels above those of the rats given the low dose of the hormone into the HPV. These results indicate that insulin can act directly on the liver to promote body growth and to regulate glucose metabolism. The significance of direct delivery of insulin from the pancreatic beta cells to the liver may be as much for growth control as for glucose homeostasis.

Rat cardiac muscle single cell automaticity responses to alpha- and beta-adrenergic agonists and antagonists.

Isolated cardiac muscle cells from neonatal rat ventricular myocardium have both alpha- and beta-adrenergic, positive chronotropic responses, with sensitivity to applied adrenergic agents more than 100 times greater than the intact neonatal heart. These highly sensitive isolated single cells and small groups of cells also reveal partial agonist activity of the alpha-adrenergic antagonist, phentolamine, and the beta-adrenergic antagonist, propranolol. The most likely explanation for the high sensitivity is the lack of a desensitization process that could involve both a desensitization substance and changes in receptors. These experiments suggest that all adrenergic antagonists could possibly show partial agonist activity.