A novel anti-inflammatory role for spleen-derived interleukin-10 in obesity-induced hypothalamic inflammation

Obesity can be associated with systemic low-grade inflammation that contributes to obesity-related metabolic disorders. Recent studies raise the possibility that hypothalamic inflammation contributes to the pathogenesis of diet-induced obesity (DIO), while another study reported that obesity decreases the expression of pro-inflammatory cytokines in spleen. The following study examines the hypothesis that obesity suppresses the splenic synthesis of the anti-inflammatory cytokine, interleukin (IL)-10, thereby resulting in chronic hypothalamic inflammation. The results showed that due to oxidative stress or apoptosis, the synthesis of splenic IL-10 was decreased in DIO when compared with non-obesity rats. Splenectomy (SPX) accelerated DIO-induced inflammatory responses in the hypothalamus. Interestingly, SPX suppressed the DIO-induced increases in food intake and body weight and led to a hypothalamic pro-inflammatory state that was similar to that produced by DIO, indicating that hypothalamic inflammation exerts a dual effect on energy metabolism. These SPX-induced changes were inhibited by the systemic administration of IL-10. Moreover, SPX had no effect on hypothalamic inflammatory responses in IL-10-deficient mice. These data suggest that spleen-derived IL-10 plays an important role in the prevention of hypothalamic inflammation and may be a therapeutic target for the treatment of obesity and hypothalamic inflammation.     

Purification and identification of two carnosine-cleaving enzymes, carnosine dipeptidase I and Xaa-methyl-His dipeptidase, from Japanese eel (Anguilla japonica)

Three enzymes, carnosine dipeptidase I (EC 3.4.13.20, CNDP1), carnosine dipeptidase II (EC 3.4.13.18, CNDP2), and Xaa-methyl-His dipeptidase (or anserinase: EC 3.4.13.5, ANSN), are known to be capable of catalyzing the hydrolysis of carnosine (β-alanyl-l-histidine), in vertebrates. Here we report the purification and identification of two unidentified carnosine-cleaving enzymes from Japanese eel (Anguilla japonica). Two different dipeptidases were successfully purified to homogeneity from the skeletal muscle; one exhibited a broad substrate specificity, while the other a narrow specificity. N-terminal amino-acid sequencing, deglycosylation analysis, and genetic analysis clearly revealed that the former is a homodimer of glycosylated subunits, encoded by ANSN, and the latter is another homodimer of glycosylated subunits, encoded by CNDP1; that is, Xaa-methyl-His dipeptidase, and carnosine dipeptidase I respectively. This is the first report on the identification of carnosine dipeptidase I from a non-mammal. Database search revealed presence of a CNDP1 ortholog only from salmonid fishes, including Atlantic salmon and rainbow trout, but not from other ray-finned fish species, such as zebrafish, fugu, and medaka whose genomes have been completely sequenced. The mRNAs of CNDP1 and ANSN are strongly expressed in the liver of Japanese eel, compared with other tissues, while that of CNDP2 is widely distributed in all tissues tested.     

In vitro synthesis of polyhydroxyalkanoate (PHA) incorporating lactate (LA) with a block sequence by using a newly engineered thermostable PHA synthase from <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. SG4502 with acquired LA-polymerizing activity

Recently, we succeeded in isolating a thermotolerant bacterium, Pseudomonas sp. SG4502, which is capable of accumulating polyhydroxyalkanoate (PHA) even at 55 °C, as a source of thermostable enzymes. In this study, we cloned a pha locus from the bacterium and identified two genes encoding PHA synthases (PhaC1_SG and PhaC2_SG). Two mutations, Ser324Thr and Gln480Lys, corresponding to those of a lactate (LA)-polymerizing enzyme (LPE) from mesophilic Pseudomonas sp. 61-3 were introduced into PhaC1_SG to evaluate the potential of the resulting protein as a “thermostable LPE”. The mutated PhaC1_SG [PhaC1_SG(STQK)] showed high thermal stability in synthesizing P(LA-co-3HB) in an in vitro reaction system under a range of high temperatures. Requirement of 3HBCoA as a priming unit for LA polymerization by the LPE has been suggested in both of the in vitro and in vivo experiments. Based on the finding, the PhaC1_SG(STQK)-mediated synthesis of a LA-based copolymer with a block sequence was achieved in the in vitro system by sequential feeding of the corresponding two substrates. This in vitro reaction system using the thermostable LPE provides us with a versatile way to synthesize the various types of LA-based copolymers with desired sequence patterns, random or block, depending on the way of supplying hydroxyalkanoates (mixed or sequential feeding).     

RAGE mediates vascular injury and inflammation after global cerebral ischemia

The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor involved in a diverse range of pathological conditions. To analyze the roles of RAGE and its decoy receptor, endogenous secretory RAGE (esRAGE), in the global cerebral ischemia, three different mouse cohorts, wild-type, RAGE^−/−, and esRAGE transgenic (Tg) mice were subjected to bilateral common carotid artery occlusion (BCCAO). RT-PCR and immunohistochemical analysis revealed that expression of RAGE was induced in the vascular cells at 12 h, and then in the neurons and glia from 3 to 7 days in the hippocampus after BCCAO. The numbers of surviving neurons in the hippocampal CA1 region were significantly higher in RAGE^−/− and esRAGE Tg mice than those in wild-type mice in the periods between 24 h and 7 days after BCCAO. Lower levels of 3-nitrotyrosine (3-NT) and higher levels of endothelial nitric oxide synthase (eNOS), together with enlarged vascular areas were observed in RAGE^−/− and esRAGE Tg mice at 12 h after BCCAO. In the later periods, expressions of glia-derived inflammatory mediators TNFα and inducible nitric oxide synthase (iNOS) were reduced in RAGE^−/− and esRAGE Tg mice. These results suggest that RAGE may contribute to delayed neuronal death after global cerebral ischemia by enhancing vascular injury and deleterious glia-mediated inflammation.     

A novel function of Noc2 in agonist-induced intracellular Ca2+ increase during zymogen-granule exocytosis in pancreatic acinar cells

Noc2, a putative Rab effector, contributes to secretory-granule exocytosis in neuroendocrine and exocrine cells. Here, using two-photon excitation live-cell imaging, we investigated its role in Ca(2+)-dependent zymogen granule (ZG) exocytosis in pancreatic acinar cells from wild-type (WT) and Noc2-knockout (KO) mice. Imaging of a KO acinar cell revealed an expanded granular area, indicating ZG accumulation. In our spatiotemporal analysis of the ZG exocytosis induced by agonist (cholecystokinin or acetylcholine) stimulation, the location and rate of progress of ZG exocytosis did not differ significantly between the two strains. ZG exocytosis from KO acinar cells was seldom observed at physiological concentrations of agonists, but was normal (vs. WT) at high concentrations. Flash photolysis of a caged calcium compound confirmed the integrity of the fusion step of ZG exocytosis in KO acinar cells. The decreased ZG exocytosis present at physiological concentrations of agonists raised the possibility of impaired elicitation of calcium spikes. When calcium spikes were evoked in KO acinar cells by a high agonist concentration: (a) they always started at the apical portion and traveled to the basal portion, and (b) calcium oscillations over the 10 μM level were observed, as in WT acinar cells. At physiological concentrations of agonists, however, sufficient calcium spikes were not observed, suggesting an impaired [Ca(2+)](i)-increase mechanism in KO acinar cells. We propose that in pancreatic acinar cells, Noc2 is not indispensable for the membrane fusion of ZG per se, but instead performs a novel function favoring agonist-induced physiological [Ca(2+)](i) increases.     

BmEts upregulates promoter activity of lebocin in Bombyx mori

The Ets family protein BmEts is assumed to be implicated in determination of diapause in the embryogenesis of Bombyx mori. In this study, we found that expression of BmEts was increased in the fat body and other tissues of the 5th instar larvae in response to Escherichia coli injection. Cotransfection experiments using a silkworm cell line revealed that overexpression of BmEts significantly elevated the activity of lebocin promoter but not of cecropin B1, cecropin D, attacin, and moricin promoters. Activation of the lebocin promoter by BmEts was dependent on at least two κB elements and the most proximal GGAA/T motif located on the 5'-upstream region. BmEts further synergistically enhanced E. coli or BmRelish1-d2 (active form)-stimulated lebocin promoter activation. Two κB elements were also found to be involved in promoter activation by BmRelish1-d2 and in synergistic promoter activation by BmEts and BmRelish1-d2 in the silkworm cells. Specific binding of recombinant BmEts to the proximal κB element and the most proximal GGAA/T motif and interaction between BmEts and BmRelish1 were also observed. To our knowledge, this is the first report of an Ets family protein directly regulating immune-related genes in invertebrates.     

Synthesis and biological evaluation of truncated α-galactosylceramide derivatives focusing on cytokine induction profile

A series of truncated analogs of α-galactosylceramide with altered ceramide moiety was prepared, and evaluated for Th2-biased response in the context of IL-4/IFN-γ ratio. Phytosphingosine-modified analogs including cyclic, aromatic and ethereal compounds as well as the C-glycoside analog of OCH (2) with their cytokine inducing profile are disclosed.