AMPK activation through mitochondrial regulation results in increased substrate oxidation and improved metabolic parameters in models of diabetes.

Modulation of mitochondrial function through inhibiting respiratory complex I activates a key sensor of cellular energy status, the 5'-AMP-activated protein kinase (AMPK). Activation of AMPK results in the mobilization of nutrient uptake and catabolism for mitochondrial ATP generation to restore energy homeostasis. How these nutrient pathways are affected in the presence of a potent modulator of mitochondrial function and the role of AMPK activation in these effects remain unclear. We have identified a molecule, named R419, that activates AMPK in vitro via complex I inhibition at much lower concentrations than metformin (IC50 100 nM vs 27 mM, respectively). R419 potently increased myocyte glucose uptake that was dependent on AMPK activation, while its ability to suppress hepatic glucose production in vitro was not. In addition, R419 treatment of mouse primary hepatocytes increased fatty acid oxidation and inhibited lipogenesis in an AMPK-dependent fashion. We have performed an extensive metabolic characterization of its effects in the db/db mouse diabetes model. In vivo metabolite profiling of R419-treated db/db mice showed a clear upregulation of fatty acid oxidation and catabolism of branched chain amino acids. Additionally, analyses performed using both (13)C-palmitate and (13)C-glucose tracers revealed that R419 induces complete oxidation of both glucose and palmitate to CO2 in skeletal muscle, liver, and adipose tissue, confirming that the compound increases mitochondrial function in vivo. Taken together, our results show that R419 is a potent inhibitor of complex I and modulates mitochondrial function in vitro and in diabetic animals in vivo. R419 may serve as a valuable molecular tool for investigating the impact of modulating mitochondrial function on nutrient metabolism in multiple tissues and on glucose and lipid homeostasis in diabetic animal models.

Drak2 regulates the survival of activated T cells and is required for organ-specific autoimmune disease.

Drak2 is a serine/threonine kinase expressed in T and B cells. The absence of Drak2 renders T cells hypersensitive to suboptimal stimulation, yet Drak2(-/-) mice are enigmatically resistant to experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. We show in this study that Drak2(-/-) mice were also completely resistant to type 1 diabetes when bred to the NOD strain of mice that spontaneously develop autoimmune diabetes. However, there was not a generalized suppression of the immune system, because Drak2(-/-) mice remained susceptible to other models of autoimmunity. Adoptive transfer experiments revealed that resistance to disease was intrinsic to the T cells and was due to a loss of T cell survival under conditions of chronic autoimmune stimulation. Importantly, the absence of Drak2 did not alter the survival of naive T cells, memory T cells, or T cells responding to an acute viral infection. These experiments reveal a distinction between the immune response to persistent self-encoded molecules and transiently present infectious agents. We present a model whereby T cell survival depends on a balance of TCR and costimulatory signals to explain how the absence of Drak2 affects autoimmune disease without generalized suppression of the immune system.

Salmonella enterica serovar Typhi strains from which SPI7, a 134-kilobase island with genes for Vi exopolysaccharide and other functions, has been deleted.

Salmonella enterica serovar Typhi has a 134-kb island of DNA identified as salmonella pathogenicity island 7 (SPI7), inserted between pheU and 'pheU (truncated), two genes for tRNA(Phe). SPI7 has genes for Vi exopolysaccharide, for type IVB pili, for putative conjugal transfer, and for sopE bacteriophage. Pulsed-field gel electrophoresis following digestion with the endonuclease I-CeuI, using DNA from a set of 120 wild-type strains of serovar Typhi assembled from several sources, identified eight strains in which the I-CeuI G fragment, which contains SPI7, had a large deletion. In addition, agglutination tests with Vi antiserum and phage typing with Vi phages show that all eight strains are Vi negative. We therefore tested these strains for deletion of SPI7 by multiplex PCR, by microarray analysis, and by sequencing of PCR amplicons. Data show that seven of the eight strains are precise deletions of SPI7: a primer pair flanking SPI7 results in a PCR amplicon containing a single pheU gene; microarrays show that all SPI7 genes are deleted. Two of the strains produce amplicons which have A derived from pheU at bp 27, while five have C derived from 'pheU at this position; thus, the position of the crossover which results in the deletion can be inferred. The deletion in the eighth strain, TYT1669, removes 175 kb with junction points in genes STY4465 and STY4664; the left junction of SPI7 and adjacent genes, as well as part of SPI7 including the viaB operon for Vi exopolysaccharide, was removed, while the right junction of SPI7 was retained. We propose that these deletions occurred during storage following isolation.