Separation of the glucose-stimulated cytoplasmic and mitochondrial NAD(P)H responses in pancreatic islet β cells

Two-photon excitation microscopy was used to image and quantify NAD(P)H autofluorescence from intact pancreatic islets under glucose stimulation. At maximal glucose stimulation, the rise in whole-cell NAD(P)H levels was estimated to be ≈30 μM. However, because glucose-stimulated insulin secretion involves both glycolytic and Kreb's cycle metabolism, islets were cultured on extracellular matrix that promotes cell spreading and allows spatial resolution of the NAD(P)H signals from the cytoplasm and mitochondria. The metabolic responses in these two compartments are shown to be differentially stimulated by various nutrient applications. The glucose-stimulated increase of NAD(P)H fluorescence within the cytoplasmic domain is estimated to be ≈7 μM. Likewise, the NAD(P)H increase of the mitochondrial domain is ≈60 μM and is delayed with respect to the change in cytoplasmic NAD(P)H by ≈20 sec. The large mitochondrial change in glucose-stimulated NAD(P)H thus dominates the total signal but may depend on the smaller but more rapid cytoplasmic increase.

Plant growth in elevated CO2 alters mitochondrial number and chloroplast fine structure

With increasing interest in the effects of elevated atmospheric CO2 on plant growth and the global carbon balance, there is a need for greater understanding of how plants respond to variations in atmospheric partial pressure of CO2. Our research shows that elevated CO2 produces significant fine structural changes in major cellular organelles that appear to be an important component of the metabolic responses of plants to this global change. Nine species (representing seven plant families) in several experimental facilities with different CO2-dosing technologies were examined. Growth in elevated CO2 increased numbers of mitochondria per unit cell area by 1.3–2.4 times the number in control plants grown in lower CO2 and produced a statistically significant increase in the amount of chloroplast stroma (nonappressed) thylakoid membranes compared with those in lower CO2 treatments. There was no observable change in size of the mitochondria. However, in contrast to the CO2 effect on mitochondrial number, elevated CO2 promoted a decrease in the rate of mass-based dark respiration. These changes may reflect a major shift in plant metabolism and energy balance that may help to explain enhanced plant productivity in response to elevated atmospheric CO2 concentrations.

Nitrosative stress: Metabolic pathway involving the flavohemoglobin

Nitric oxide (NO) biology has focused on the tightly regulated enzymatic mechanism that transforms l-arginine into a family of molecules, which serve both signaling and defense functions. However, very little is known of the pathways that metabolize these molecules or turn off the signals. The paradigm is well exemplified in bacteria where S-nitrosothiols (SNO)—compounds identified with antimicrobial activities of NO synthase—elicit responses that mediate bacterial resistance by unknown mechanisms. Here we show that Escherichia coli possess both constitutive and inducible elements for SNO metabolism. Constitutive enzyme(s) cleave SNO to NO whereas bacterial hemoglobin, a widely distributed flavohemoglobin of poorly understood function, is central to the inducible response. Remarkably, the protein has evolved a novel heme-detoxification mechanism for NO. Specifically, the heme serves a dioxygenase function that produces mainly nitrate. These studies thus provide new insights into SNO and NO metabolism and identify enzymes with reactions that were thought to occur only by chemical means. Our results also emphasize that the reactions of SNO and NO with hemoglobins are evolutionary conserved, but have been adapted for cell-specific function.

11β-Hydroxysteroid dehydrogenase type 1 knockout mice show attenuated glucocorticoid-inducible responses and resist hyperglycemia on obesity or stress

Glucocorticoid hormones, acting via nuclear receptors, regulate many metabolic processes, including hepatic gluconeogenesis. It recently has been recognized that intracellular glucocorticoid concentrations are determined not only by plasma hormone levels, but also by intracellular 11β-hydroxysteroid dehydrogenases (11β-HSDs), which interconvert active corticosterone (cortisol in humans) and inert 11-dehydrocorticosterone (cortisone in humans). 11β-HSD type 2, a dehydrogenase, thus excludes glucocorticoids from otherwise nonselective mineralocorticoid receptors in the kidney. Recent data suggest the type 1 isozyme (11β-HSD-1) may function as an 11β-reductase, regenerating active glucocorticoids from circulating inert 11-keto forms in specific tissues, notably the liver. To examine the importance of this enzyme isoform in vivo, mice were produced with targeted disruption of the 11β-HSD-1 gene. These mice were unable to convert inert 11-dehydrocorticosterone to corticosterone in vivo. Despite compensatory adrenal hyperplasia and increased adrenal secretion of corticosterone, on starvation homozygous mutants had attenuated activation of the key hepatic gluconeogenic enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, presumably, because of relative intrahepatic glucocorticoid deficiency. The 11β-HSD-1 −/− mice were found to resist hyperglycamia provoked by obesity or stress. Attenuation of hepatic 11β-HSD-1 may provide a novel approach to the regulation of gluconeogenesis.