Mitochondrial diabetes in mice expressing a dominant-negative allele of nuclear respiratory factor-1 ( Nrf1 ) in pancreatic ß-cells.

Genetic polymorphisms in nuclear respiratory factor-1 ( NRF1 ), a key transcriptional regulator of nuclear-encoded mitochondrial proteins, have been linked to diabetes. Homozygous deletion of Nrf1 is embryonic lethal in mice. Our goal was to generate mice with ß-cell-specific reduction in NRF1 function to investigate the relationship between NRF1 and diabetes. We report the generation of mice expressing a dominant-negative allele of Nrf1 (DNNRF1) in pancreatic ß-cells. Heterozygous transgenic mice had high fed blood glucose levels detected at 3 wks of age, which persisted through adulthood. Plasma insulin levels in DNNRF1 transgenic mice were reduced, while insulin sensitivity remained intact in young animals. Islet size was reduced with increased numbers of apoptotic cells, and insulin content in islets by immunohistochemistry was low. Glucose-stimulated insulin secretion in isolated islets was reduced in DNNRF1-mice, but partially rescued by KCl, suggesting that decreased mitochondrial function contributed to the insulin secretory defect. Electron micrographs demonstrated abnormal mitochondrial morphology in ß- cells. Expression of NRF1 target genes Tfam , T@1m and T@2m , and islet cytochrome c oxidase and succinate dehydrogenase activities were reduced in DNNRF1-mice. Rescue of mitochondrial function with low level activation of transgenic c-Myc in ß-cells was sufficient to restore ß-cell mass and prevent diabetes. This study demonstrates that reduced NRF1 function can lead to loss of ß-cell function and establishes a model to study the interplay between regulators of bi- genomic gene transcription in diabetes.

Reduced cytochrome C is an essential regulator of sustained insulin secretion by pancreatic islets.

Influx of calcium is an essential but insufficient signal in sustained nutrient-stimulated insulin secretion, and increased metabolic rate of the beta cell is also required. The aim of the study was to test the hypothesis that the reduced state of cytochrome c is a metabolic co-factor necessary for insulin secretion, over and above its participation in the ATP-generating function of electron transport/oxidative phosphorylation. We found that nutrient stimulation of insulin secretion by isolated rat islets was strongly correlated with reduced cytochrome c, and agents that acutely and specifically reduced cytochrome c led to increased insulin secretion, even in the face of decreased oxygen consumption and calcium influx. In contrast, neither sites 1 nor 4 of the electron transport chain were both necessary and essential for the stimulation of insulin secretion to occur. Importantly, stimulation of islets with glucose, α-ketoisocaproate, or glyceraldehyde resulted in the appearance of cytochrome c in the cytosol, suggesting a pathway for the regulation of exocytotic machinery by reduction of cytochrome c. The data suggest that the metabolic factor essential for sustained calcium-stimulated insulin secretion to occur is linked to reduction and translocation of cytochrome c.

Roles of glucose in photoreceptor survival.

Vertebrate photoreceptor neurons have a high demand for metabolic energy, and their viability is very sensitive to genetic and environmental perturbations. We investigated the relationship between energy metabolism and cell death by evaluating the metabolic effects of glucose deprivation on mouse photoreceptors. Oxygen consumption, lactate production, ATP, NADH/NAD(+), TCA cycle intermediates, morphological changes, autophagy, and viability were evaluated. We compared retinas incubated with glucose to retinas deprived of glucose or retinas treated with a mixture of mitochondrion-specific fuels. Rapid and slow phases of cell death were identified. The rapid phase is linked to reduced mitochondrial activity, and the slower phase reflects a need for substrates for cell maintenance and repair.

Palmitate is not an effective fuel for pancreatic islets and amplifies insulin secretion independent of calcium release from endoplasmic reticulum.

The aim of the study was to determine the acute contribution of fuel oxidation in mediating the increase in insulin secretion rate (ISR) in response to fatty acids. Measures of mitochondrial metabolism, as reflected by oxygen consumption rate (OCR) and cytochrome c reduction, calcium signaling, and ISR by rat islets were used to evaluate processes stimulated by acute exposure to palmitic acid (PA). The contribution of mitochondrial oxidation of PA was determined in the presence and absence of a blocker of mitochondrial transport of fatty acids (etomoxir) at different glucose concentrations. Subsequent to increasing glucose from 3 to 20 mM, PA caused small increases in OCR and cytosolic calcium (about 20% of the effect of glucose). In contrast, the effect of PA on ISR was almost 3 times that by glucose, suggesting that the metabolism of PA is not the dominant mechanism mediating PA's effect on ISR. This was further supported by lack of inhibition of PA-stimulated OCR and ISR when blocking entry of PA into mitochondria (with etomoxir), and PA's lack of stimulation of reduced cytochrome c in the presence of high glucose. Consistent with the lack of metabolic stimulation by PA, an inhibitor of calcium release from the endoplasmic reticulum, but not a blocker of L-type calcium channels, abolished the PA-induced elevation of cytosolic calcium. Notably, ISR was unaffected by thapsigargin showing the dissociation of endoplasmic reticulum calcium release and second phase insulin secretion. In conclusion, stimulation of ISR by PA was mediated by mechanisms largely independent of the oxidation of the fuel.

Development of a high-throughput cell-based assay for identification of IL-17 inhibitors.

Human interleukin 17 (IL-17) is a proinflammatory cytokine derived mainly from activated T cells. Extensive evidence points to a significant role of IL-17 in many autoimmune and infectious diseases, as well as tumorigenesis and transplant rejection, and suggests that targeting IL-17 could be a promising therapeutic strategy. Robust cell-based assays would thus be essential for lead identification and the optimization of therapeutic candidates. Herein, we report a well-characterized two-step assay, consisting of (a) in vitro activation and stimulation of CD4(+) T lymphocytes by a defined complex of antibodies and cytokines, leading to T helper 17 (Th17) cell differentiation and IL-17 production, and (b) IL-17 quantification in cell supernatants using a homogeneous time-resolved fluorescence (HTRF) assay. The system was optimized for and shown to be reliable in high-throughput compatible 96- and 384-well plate formats. The assay is robust (Z' > 0.5) and simple to perform, yields a stable response, and allows for sufficient discrimination of positive (IL-17-producing cells) and negative controls (uninduced cells). The assay was validated by performing dose-response testing of rapamycin and cyclosporine A, which had previously been reported to inhibit IL-17, and determining, for the first time, their in vitro potencies (IC(50)s of 80 ± 23 pM and 223 ± 52 nM, respectively). Also, IKK 16, a selective small-molecule inhibitor of IκB kinase, was found to inhibit IL-17 production, with an IC(50) of 315 ± 79 nM.