Glucagon signalling in the dorsal vagal complex is sufficient and necessary for high‐protein feeding to regulate glucose homeostasis in vivo

High‐protein feeding acutely lowers postprandial glucose concentration compared to low‐protein feeding, despite a dichotomous rise of circulating glucagon levels. The physiological role of this glucagon rise has been largely overlooked. We here first report that glucagon signalling in the dorsal vagal complex (DVC) of the brain is sufficient to lower glucose production by activating a Gcgr–PKA–ERK–K_ATP channel signalling cascade in the DVC of rats in vivo. We further demonstrate that direct blockade of DVC Gcgr signalling negates the acute ability of high‐ vs. low‐protein feeding to reduce plasma glucose concentration, indicating that the elevated circulating glucagon during high‐protein feeding acts in the brain to lower plasma glucose levels. These data revise the physiological role of glucagon and argue that brain glucagon signalling contributes to glucose homeostasis during dietary protein intake.     

Structural and immunological studies of a pectin and a pectic arabinogalactan from Vernonia kotschyana Sch. Bip. ex Walp. (Asteraceae)

Two polysaccharides, a pectin (Vk100A2b) and a pectic arabinogalactan (Vk100A2a) with mean Mw 2 x 10(4) and 1.15 x 10(6)Da, respectively, were isolated from the dried powdered roots of Vernonia kotschyana Sch. Bip. ex Walp. by hot water extraction followed by fractionation on DEAE-Sepharose fast flow and Sephacryl S-400 HR. The pectin showed low-complement fixation activity and no influence on proliferation of B or T cells, while the pectic arabinogalactan showed a potent, dose-dependent complement fixation activity and a T cell independent induction of B-cell proliferation. Both polysaccharides induced chemotaxis of human macrophages, T cells and NK cells. exo-alpha-L-arabinofuranosidase and exo-beta-D-galactosidase digestion followed by component sugar and methylation analysis indicated that Vk100A2a consisted of a highly branched rhamnogalacturonan core with approximately 50% of the rhamnose 1,2,4-substituted, side chains rich in terminal-, 1,5-linked and 1,3,5-branched arabinose and terminal-, 1,4-, 1,6-linked and 1,3,6-branched galactose. The enzyme resistant part of Vk100A2a still showed strong complement fixating activity, suggesting that this activity may at least in part be expressed by carbohydrate structures present in the enzyme resistant, inner portion of the polymer.     

Differential utilization of cyclic ADP-ribose pathway by chemokines to induce the mobilization of intracellular calcium in NK cells.

We show here that cyclic adenosine diphosphate-ribose (cADPR) may be a second messenger for chemokines. Extracts collected from NK cells stimulated with IL-8 for 2 min were incubated with beta-NAD for an additional 2 min (designated as IL-8 extracts). This mixture elevated the mobilization of (Ca(2+))(i) in alpha-toxin permeabilized NK cells. This activity was inhibited upon prior incubation of these cells with ruthenium red but not with heparin. Purified cADPR and not Ins 1,4,5 P(3) desensitized NK cells to the calcium mobilization effect of IL-8 extracts. Further analysis showed that ruthenium red and heparin differentially inhibit RANTES-, SDF-1alpha-, or MDC-induced calcium mobilization in IL-2-activated NK cells. Also, introduction of anti-ryanodine receptor antibody inside streptolysin O-permeabilized NK cells resulted in complete inhibition of MDC, and only partial inhibition of RANTES and SDF-1alpha-induced calcium fluxes in NK cells. Collectively, these results suggest that chemokines may utilize the cADPR/ryanodine receptor pathway as well as the Ins 1,4,5 P(3)/Ins 1,4,5 P(3) receptor signaling pathway to induce the accumulation of calcium in NK cells.     

T Cell Specific Adapter Protein (TSAd) Interacts with Tec Kinase ITK to Promote CXCL12 Induced Migration of Human and Murine T Cells

Background

The chemokine CXCL12/SDF-1α interacts with its G-protein coupled receptor CXCR4 to induce migration of lymphoid and endothelial cells. T cell specific adapter protein (TSAd) has been found to promote migration of Jurkat T cells through interaction with the G protein β subunit. However, the molecular mechanisms for how TSAd influences cellular migration have not been characterized in detail.

Principal Findings

We show that TSAd is required for tyrosine phosphorylation of the Lck substrate IL2-inducible T cell kinase (Itk). Presence of Itk Y511 was necessary to boost TSAd''s effect on CXCL12 induced migration of Jurkat T cells. In addition, TSAd''s ability to promote CXCL12-induced actin polymerization and migration of Jurkat T lymphocytes was dependent on the Itk-interaction site in the proline-rich region of TSAd. Furthermore, TSAd-deficient murine thymocytes failed to respond to CXCL12 with increased Itk phosphorylation, and displayed reduced actin polymerization and cell migration responses.

Conclusion

We propose that TSAd, through its interaction with both Itk and Lck, primes Itk for Lck mediated phosphorylation and thereby regulates CXCL12 induced T cell migration and actin cytoskeleton rearrangements.     

Phylogenetic and functional conservation of the NKR-P1F and NKR-P1G receptors in rat and mouse

Two clusters of rat Nkrp1 genes can be distinguished based on phylogenetic relationships and functional characteristics. The proximal (centromeric) cluster encodes the well-studied NKR-P1A and NKR-P1B receptors and the distal cluster, the largely uncharacterized, NKR-P1F and NKR-P1G receptors. The inhibitory NKR-P1G receptor is expressed only by the Ly49s3⁺ NK cell subset as detected by RT-PCR, while the activating NKR-P1F receptor is detected in both Ly49s3⁺ and NKR-P1B⁺ NK cells. The mouse NKR-P1G ortholog is expressed by both NKR-P1D⁻ and NKR-P1D⁺ NK cells in C57BL/6 mice. The rat and mouse NKR-P1F and NKR-P1G receptors demonstrate a striking, cross-species conservation of specificity for Clr ligands. NKR-P1F and NKR-P1G reporter cells reacted with overlapping panels of tumour cell lines and with cells transiently transfected with rat Clr2, Clr3, Clr4, Clr6 and Clr7 and mouse Clrc, Clrf, Clrg and Clrd/x, but not with Clr11 or Clrb, which serve as ligands for NKR-P1 from the proximal cluster. These data suggest that the conserved NKR-P1F and NKR-P1G receptors function as promiscuous receptors for a rapidly evolving family of Clr ligands in rodent NK cells.     

An immunomodulating pectic polymer from Glinus oppositifolius

An immunomodulating pectic polymer, GOA1, obtained from the aerial parts of the Malian medicinal plant Glinus oppositifolius (L.) Aug. DC. (Aizoaceae) has previously been reported to consist of arabinogalactans type I and II, probably linked to a rhamnogalacturonan backbone. To further elucidate the structure of the polymer GOA1, enzymatic degradation studies and weak acid hydrolysis were performed. Five different glycosidases were used, endo-alpha-D-(1-->4)-polygalacturonase, exo-alpha-L-arabinofuranosidase, endo-alpha-L-(1-->5)-arabinanase, endo-beta-D-(1-->4)-galactanase and exo-beta-D-galactosidase. It appears that GOA1 may contain a structural moiety consisting of a 1,3-linked galactopyranosyl (Galp) main chain with 1,6-linked Galp side chains attached to position 6 of the main chain. The 1,6-linked Galp side chain may be branched in position 3 with arabinofuranosyl (Araf) side chains. A 1,4-linked Galp backbone which might carry side chains or glycosyl units attached to position 3 is also a structural element in the polymer. We further show that GOA1 induce proliferation of B cells and the secretion of IL-1beta by macrophages, in addition to a marked increase of mRNA for IFN-gamma in NK-cells. To elucidate structure-activity relations the native polymer and the digested fractions were tested for complement fixing activity and intestinal immune stimulating activity. The partial removal of Araf residues after enzymatic degradations did not affect the bioactivities, while the acid hydrolysed fraction showed reduced complement fixing activity. A decrease in Araf units, 1,3,6-linked Galp units and a partial hydrolysed rhamnogalacturonan backbone, in addition to a reduction in molecular weight are factors that might have contributed to reduced bioactivity.     

Pectic polysaccharides from Biophytum petersianum Klotzsch, and their activation of macrophages and dendritic cells

The Malian medicinal plant Biophytum petersianum Klotzsch (Oxalidaceae) is used as a treatment against various types of illnesses related to the immune system, such as joint pains, inflammations, fever, malaria, and wounds. A pectic polysaccharide obtained from a hot water extract of the aerial parts of B. petersianum has previously been reported to consist of arabinogalactans types I and II (AG-I and AG-II), probably linked to a rhamnogalacturonan backbone. We describe here further structural characteristics of the main polysaccharide fraction (BP1002) and fractions obtained by enzymatic degradations using endo-alpha-d-(1-->4)-polygalacturonase (BP1002-I to IV). The results indicate that in addition to previously reported structures, rhamnogalacturan type II and xylogalacturonan areas appear to be present in the pectic polymer isolated from the plant. Atomic force microscopy confirmed the presence of branched structures, as well as a polydisperse nature. We further tested whether the BP1002 main fraction or the enzymatically degraded products could induce immunomodulating activity through stimulation of subsets of leukocytes. We found that macrophages and dendritic cells were activated by BP1002 fractions, while there was little response of T cells, B cells, and NK cells. The enzymatic treatment of the BP1002 main fraction gave important information on the structure-activity relations. It seems that the presence of rhamnogalacturonan type I is important for the bioactivity, as the bioactivity decreases with the decreased amounts of rhamnose, galactose, and arabinose. The demonstration of bioactivity by the plant extracts might indicate the mechanisms behind the traditional medical use of the plant.     

A scaffoldless technique for self-generation of three-dimensional keratinospheroids on liquid crystal surfaces

We describe a new scaffold-free three-dimensional (3D) cell culture model using cholesteryl ester based lyotropic liquid crystal (LC) substrates. Keratinocytes were deposited randomly on the LC surface where they self-assembled into 3D microtissues or keratinospheroids. The cell density required to form spheroids was optimized. We investigated cell viability using dead/live cell assays. The adhesion characteristics of cells within the microtissues were determined using histological sectioning and immunofluorescence staining. Fourier transform infrared spectroscopy (FTIR) was used to characterize the biochemistry of the keratinospheroids. We found that both cells and microtissues could migrate on the LC surface. The viability study indicated approximately 80% viability of cells in the microtissues up to 20 days of culture. Strong intercellular adhesion was observed in the stratification of the multi-layered microspheroids using field emission-scanning electron microscopy (FE-SEM) and histochemical staining. The cytoskeleton and vinculins of the cells in the microtissues were expressed diffusely, but the microtissues were enriched with lipids and nucleic acids, which indicates close resemblance to the conditions in vivo. The basic 3D culture model based on LC may be used for cell and microtissue migration studies in response to cytochemical treatment.     

Eicosapentaenoic acid and docosahexaenoic acid reduce interleukin-1β-mediated cartilage degradation

Introduction  

In inflammatory joint disease, such as osteoarthritis (OA), there is an increased level of proinflammatory cytokines, such as interleukin (IL)-1β. These cytokines stimulate the production of matrix metalloproteinases (MMPs), which leads to the degradation of the cartilage extracellular matrix and the loss of key structural components such as sulphated glycosaminoglycan (sGAG) and collagen II. The aim of this study was to examine the therapeutic potential of n-3 polyunsaturated fatty acids (PUFAs) in an in vitro model of cartilage inflammation.