N-acetylaspartate catabolism determines cytosolic acetyl-CoA levels and histone acetylation in brown adipocytes.

Histone acetylation depends on the abundance of nucleo-cytoplasmic acetyl-CoA. Here, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. N-acetylaspartate (NAA) is a highly abundant brain metabolite catabolized by aspartoacylase yielding aspartate and acetate. The latter can be further used for acetyl-CoA production. Prior to this work, the presence of NAA has not been described in adipocytes. Here, we show that accumulation of NAA decreases the brown adipocyte phenotype. We increased intracellular NAA concentrations in brown adipocytes via media supplementation or knock-down of aspartoacylase and measured reduced lipolysis, thermogenic gene expression, and oxygen consumption. Combinations of approaches to increase intracellular NAA levels showed additive effects on lipolysis and gene repression, nearly abolishing the expression of Ucp1, Cidea, Prdm16, and Ppara. Transcriptome analyses of aspartoacylase knock-down cells indicate deficiencies in acetyl-CoA and lipid metabolism. Concordantly, cytoplasmic acetyl-CoA levels and global histone H3 acetylation were decreased. Further, activating histone marks (H3K27ac and H3K9ac) in promoters/enhancers of brown marker genes showed reduced acetylation status. Taken together, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. Thereby, we mechanistically connect the NAA pathway to the epigenomic regulation of gene expression, modulating the phenotype of brown adipocytes.

Toll-like receptor 2-deficient mice are protected from insulin resistance and beta cell dysfunction induced by a high-fat diet.

AIMS/HYPOTHESIS: Inflammation contributes to both insulin resistance and pancreatic beta cell failure in human type 2 diabetes. Toll-like receptors (TLRs) are highly conserved pattern recognition receptors that coordinate the innate inflammatory response to numerous substances, including NEFAs. Here we investigated a potential contribution of TLR2 to the metabolic dysregulation induced by high-fat diet (HFD) feeding in mice. METHODS: Male and female littermate Tlr2(+/+) and Tlr2(-/-) mice were analysed with respect to glucose tolerance, insulin sensitivity, insulin secretion and energy metabolism on chow and HFD. Adipose, liver, muscle and islet pathology and inflammation were examined using molecular approaches. Macrophages and dendritic immune cells, in addition to pancreatic islets were investigated in vitro with respect to NEFA-induced cytokine production. RESULTS: While not showing any differences in glucose homeostasis on chow diet, both male and female Tlr2(-/-) mice were protected from the adverse effects of HFD compared with Tlr2(+/+) littermate controls. Female Tlr2(-/-) mice showed pronounced improvements in glucose tolerance, insulin sensitivity, and insulin secretion following 20 weeks of HFD feeding. These effects were associated with an increased capacity of Tlr2(-/-) mice to preferentially burn fat, combined with reduced tissue inflammation. Bone-marrow-derived dendritic cells and pancreatic islets from Tlr2(-/-) mice did not increase IL-1beta expression in response to a NEFA mixture, whereas Tlr2(+/+) control tissues did. CONCLUSION/INTERPRETATION: These data suggest that TLR2 is a molecular link between increased dietary lipid intake and the regulation of glucose homeostasis, via regulation of energy substrate utilisation and tissue inflammation.

The molecular archaeology of a mitochondrial death effector: AIF in Drosophila.

Apoptosis-inducing factor (AIF) is a phylogenetically conserved redox-active flavoprotein that contributes to cell death and oxidative phosphorylation in Saccharomyces cerevisiae, Caenorhabditis elegans, mouse and humans. AIF has been characterized as a caspase-independent death effector that is activated by its translocation from mitochondria to the cytosol and nucleus. Here, we report the molecular characterization of AIF in Drosophila melanogaster, a species in which most cell deaths occur in a caspase-dependent manner. Interestingly, knockout of zygotic D. melanogaster AIF (DmAIF) expression using gene targeting resulted in decreased embryonic cell death and the persistence of differentiated neuronal cells at late embryonic stages. Although knockout embryos hatch, they undergo growth arrest at early larval stages, accompanied by mitochondrial respiratory dysfunction. Transgenic expression of DmAIF misdirected to the extramitochondrial compartment (DeltaN-DmAIF), but not wild-type DmAIF, triggered ectopic caspase activation and cell death. DeltaN-DmAIF-induced death was not blocked by removal of caspase activator Dark or transgenic expression of baculoviral caspase inhibitor p35, but was partially inhibited by Diap1 overexpression. Knockdown studies revealed that DeltaN-DmAIF interacts genetically with the redox protein thioredoxin-2. In conclusion, we show that Drosophila AIF is a mitochondrial effector of cell death that plays roles in developmentally regulated cell death and normal mitochondrial function.

Long-term treatment with the dipeptidyl peptidase IV inhibitor P32/98 causes sustained improvements in glucose tolerance, insulin sensitivity, hyperinsulinemia, and beta-cell glucose responsiveness in VDF (fa/fa) Zucker rats.

The incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are responsible for >50% of nutrient-stimulated insulin secretion. After being released into the circulation, GIP and GLP-1 are rapidly inactivated by the circulating enzyme dipeptidyl peptidase IV (DP IV). The use of DP IV inhibitors to enhance these insulinotropic hormonal axes has proven effective on an acute scale in both animals and humans; however, the long-term effects of these compounds have yet to be determined. Therefore, we carried out the following study: two groups of fa/fa Zucker rats (n = 6 each) were treated twice daily for 3 months with the DP IV inhibitor P32/98 (20 mg.kg(-1).day(-1), p.o.). Monthly oral glucose tolerance tests (OGTTs), performed after drug washout, revealed a progressive and sustained improvement in glucose tolerance in the treated animals. After 12 weeks of treatment, peak OGTT blood glucose values in the treated animals averaged 8.5 mmol/l less than in the controls (12.0 +/- 0.7 vs. 20.5 +/- 1.3 mmol/l, respectively). Concomitant insulin determinations showed an increased early-phase insulin response in the treated group (43% increase). Furthermore, in response to an 8.8 mmol/l glucose perfusion, pancreata from controls showed no increase in insulin secretion, whereas pancreata from treated animals exhibited a 3.2-fold rise in insulin secretion, indicating enhanced beta-cell glucose responsiveness. Also, both basal and insulin-stimulated glucose uptake were increased in soleus muscle strips from the treated group (by 20 and 50%, respectively), providing direct evidence for an improvement in peripheral insulin sensitivity. In summary, long-term DP IV inhibitor treatment was shown to cause sustained improvements in glucose tolerance, insulinemia, beta-cell glucose responsiveness, and peripheral insulin sensitivity, novel effects that provide further support for the use of DP IV inhibitors in the treatment of diabetes.

Metabolism of glucagon by dipeptidyl peptidase IV (CD26).

Glucagon is a 29-amino acid polypeptide released from pancreatic islet alpha-cells that acts to maintain euglycemia by stimulating hepatic glycogenolysis and gluconeogenesis. Despite its importance, there remains controversy about the mechanisms responsible for glucagon clearance in the body. In the current study, enzymatic metabolism of glucagon was assessed using sensitive mass spectrometric techniques to identify the molecular products. Incubation of glucagon with purified porcine dipeptidyl peptidase IV (DP IV) yielded sequential production of glucagon(3-29) and glucagon(5-29). In human serum, degradation to glucagon(3-29) was rapidly followed by N-terminal cyclization of glucagon, preventing further DP IV-mediated hydrolysis. Bioassay of glucagon, following incubation with purified DP IV or normal rat serum demonstrated a significant loss of hyperglycemic activity, while a similar incubation in DP IV-deficient rat serum did not show any loss of glucagon bioactivity. Degradation, monitored by mass spectrometry and bioassay, was blocked by the specific DP IV inhibitor, isoleucyl thiazolidine. These results identify DP IV as a primary enzyme involved in the degradation and inactivation of glucagon. These findings have important implications for the determination of glucagon levels in human plasma.

Nitroblue tetrazolium blocks BK channels in cerebrovascular smooth muscle cell membranes.

The effects of p-nitroblue tetrazolium (NBT) on large conductance, calcium-activated potassium channels (BK channels) in enzymatically dispersed rat cerebrovascular smooth muscle cells (CVSMCs) were examined. Patch clamp methods were employed to record single BK channel currents from inside-out patches of CVMC membrane maintained at 21 - 23 degrees C. When applied to the cytoplasmic face of inside-out membrane patches (internally applied NBT), micromolar concentrations of NBT reversible reduced the mean open time of BK channels, without changing channel conductance. NBT altered the frequency distribution of BK channel open times from a two exponential to a single exponential form. In the absence of NBT, mean channel open time increased on membrane depolarization. In the presence of internally applied NBT, mean channel open became essentially independent of membrane potential. Internally applied NBT also reduced the mean closed time of BK channels when measured at membrane potentials in the range -80 mV to +20 mV. The combined effects of internal NBT on mean open and closed times resulted in the suppression of BK channel open probability when measured at positive membrane potentials. When applied to the external membrane face, micromolar concentrations of NBT reduced mean channel open time progressively as the membrane was hyperpolarized, and also reduced open probability at negative membrane potentials. A model is proposed in which NBT alters channel gating by binding to a site at or near to the cytoplasmic membrane face. Externally applied NBT suppressed BK channel open probability at concentrations which also inhibit nitric oxide synthase (NOS). Therefore, the potential role of potassium channel block in NBT actions previously attributed to NOS inhibition is discussed.

Dipeptidyl peptidase IV (DPIV/CD26) degradation of glucagon. Characterization of glucagon degradation products and DPIV-resistant analogs.

Over the past decade, numerous studies have been targeted at defining structure-activity relationships of glucagon. Recently, we have found that glucagon(1-29) is hydrolyzed by dipeptidyl peptidase IV (DPIV) to produce glucagon(3-29) and glucagon(5-29); in human serum, [pyroglutamyl (pGlu)(3)]glucagon(3-29) is formed from glucagon(3-29), and this prevents further hydrolysis of glucagon by DPIV (H.-U. Demuth, K. Glund, U. Heiser, J. Pospisilik, S. Hinke, T. Hoffmann, F. Rosche, D. Schlenzig, M. Wermann, C. McIntosh, and R. Pederson, manuscript in preparation). In the current study, the biological activity of these peptides was examined in vitro. The amino-terminally truncated peptides all behaved as partial agonists in cyclic AMP stimulation assays, with Chinese hamster ovary K1 cells overexpressing the human glucagon receptor (potency: glucagon(1-29) > [pGlu(3)]glu- cagon(3-29) > glucagon(3-29) > glucagon(5-29) > [Glu(9)]glu- cagon(2-29)). In competition binding experiments, [pGlu(3)]glucagon(3-29) and glucagon(5-29) both demonstrated 5-fold lower affinity for the receptor than glucagon(1-29), whereas glucagon(3-29) exhibited 18-fold lower affinity. Of the peptides tested, only glucagon(5-29) showed antagonist activity, and this was weak compared with the classical glucagon antagonist, [Glu(9)]glucagon(2-29). Hence, DPIV hydrolysis of glucagon yields low affinity agonists of the glucagon receptor. As a corollary to evidence indicating that DPIV degrades glucagon (Demuth, et al., manuscript in preparation), DPIV-resistant analogs were synthesized. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry was used to assess DPIV resistance, and it allowed kinetic analysis of degradation. Of several analogs generated, only [D-Ser(2)] and [Gly(2)]glucagon retained high affinity binding and biological potency, similar to native glucagon in vitro. [D-Ser(2)]Glucagon exhibited enhanced hyperglycemic activity in a bioassay, whereas [Gly(2)]glucagon was not completely resistant to DPIV degradation.