Glucose-dependent insulinotropic polypeptide prevents the progression of macrophage-driven atherosclerosis in diabetic apolipoprotein E-null mice

Aim

We recently reported that glucose-dependent insulinotropic polypeptide (GIP) prevents the development of atherosclerosis in apolipoprotein E-null (Apoe ^−/−) mice. GIP receptors (GIPRs) are found to be severely down-regulated in diabetic animals. We examined whether GIP can exert anti-atherogenic effects in diabetes.

Methods

Nondiabetic Apoe ^−/− mice, streptozotocin-induced diabetic Apoe ^−/− mice, and db/db mice were administered GIP (25 nmol/kg/day) or saline (vehicle) through osmotic mini-pumps for 4 weeks. The animals were assessed for aortic atherosclerosis and for oxidized low-density lipoprotein-induced foam cell formation in exudate peritoneal macrophages.

Results

Diabetic Apoe ^−/− mice of 21 weeks of age exhibited more advanced atherosclerosis than nondiabetic Apoe ^−/− mice of the same age. GIP infusion in diabetic Apoe ^−/− mice increased plasma total GIP levels by 4-fold without improving plasma insulin, glucose, or lipid profiles. GIP infusion significantly suppressed macrophage-driven atherosclerotic lesions, but this effect was abolished by co-infusions with [Pro³]GIP, a GIPR antagonist. Foam cell formation was stimulated by 3-fold in diabetic Apoe ^−/− mice compared with their nondiabetic counterparts, but this effect was halved by GIP infusion. GIP infusion also attenuated the foam cell formation in db/db mice. In vitro treatment with GIP (1 nM) reduced foam cell formation by 15% in macrophages from diabetic Apoe ^−/− mice, and this attenuating effect was weaker than that attained by the same treatment of macrophages from nondiabetic counterparts (35%). While GIPR expression was reduced by only about a half in macrophages from diabetic mice, it was reduced much more dramatically in pancreatic islets from the same animals. Incubation with high glucose (500 mg/dl) for 9–10 days markedly reduced GIPR expression in pancreatic islet cells, but not in macrophages.

Conclusions

Long-term infusion of GIP conferred significant anti-atherogenic effects in diabetic mice even though the GIPR expression in macrophages was mildly down-regulated in the diabetic state.     

Electrophysiological studies of salty taste modification by organic acids in the labellar taste cell of the blowfly.

Using the labellar salt receptor cells of the blowfly, Phormia regina, we electrophysiologically showed that the response to NaCl and KCl aqueous solutions was enhanced and depressed by acetic, succinic and citric acids. The organic acid concentrations at which the most enhanced salt response (MESR) was obtained were found to be different: 0.05-1 mM citric acid, 0.5-2 mM succinic acid and 5-50 mM acetic acid. Moreover, the degree of the salt response was not always dependent on the pH values of the stimulating solutions. The salt response was also enhanced by HCl (pH 3.5-3.0) only when the NaCl concentration was greater than the threshold, indicating that the salty taste would be enhanced by the comparatively lower concentrations of hydrogen ions. Another explanation for the enhancement is that the salty taste may also be enhanced by undissociated molecules of the organic acids, because the MESRs were obtained at the pH values lower than the pKa(1) or pKa(2) values of these organic acids. On the other hand, the salty taste could be depressed by both the lower pH range (pH 2.5-2.0) and the dissociated organic anions from organic acid molecules with at least two carboxyl groups.     

Purification, crystallization, and preliminary X-ray crystallographic analysis of thermus thermophilus V(1)-ATPase B subunit.

The gene of V(1)-ATPase B subunit from the thermophilic eubacterium Thermus thermophilus has been cloned and the protein overproduced in Escherichia coli. The purified protein, with a molecular weight of 53.2 kDa, was crystallized from 10% (w/v) polyethylene glycol 1000, 120 mM magnesium chloride, and 100 mM Na-tricine, pH 8.0, by the vapor diffusion method. The crystals diffracted X-rays beyond 3.5 A on a synchrotron radiation source. The crystals belong to the monoclinic space group C2, with unit cell dimensions of a = 153.1 A, b = 129.6 A, c = 92.7 A, and beta = 100.3 degrees. Assuming that three or four molecules are contained in an asymmetric unit, the V(M) value is calculated as 2.8 or 2.1 A (3)/Da, respectively.     

Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae

Summary The galactose analogue 2-deoxygalactose was found to inhibit the growth of a mutant strain of Saccharomyces cerevisiae constitutively producing the set of galactose utilization enzymes. Based on this fact, the yeast GAL80 gene negatively regulating the expression of the genes encoding those enzymes was isolated for its ability to confer 2-deoxygalactose resistance on a strain carrying a recessive mutation in that gene. The GAL80 gene was located within a 3.0 kb fragment in the cloned DNA. When the isolated gene was incorporated into a multi-copy plasmid, the induced level of three enzymes encoded by the gene cluster GAL7-GAL10-GAL1 in the host chromosome was lowered. Such a gene dosage effect of GAL80 was further pronounced if sucrose, a sugar causing catabolite repression, was added to the growth medium. The ratio of the enzyme activity of the yeast bearing multiple copies of GAL80 to that of the yeast bearing its single copy significantly varied with the enzyme. From these results we suggest that the intracellular inducer interacts with the GAL80 product and that GAL80 molecules directly bind the GAL cluster genes with an affinity different from one gene to another.The first article of this series is in Mol Gen Genet 191:31–38On a leave absence from Nikka Whisky Co.     

Strain-specific lethal effect of the adenovirus E1a protein on Saccharomyces cerevisiae

Various adenovirus E1a proteins, including 13S protein, 12S protein and three other derivatives of 13S protein with deletions were expressed in Saccharomyces cerevisiae. Both the C-terminal 67 residues and the 13S unique domain are required for the nuclear targeting in yeast. The N-terminus containing multiple functional domains appears to be involved in the G1 arrest of diploid yeast and two other regions, the region containing amino acid residues between 122 and 139, and the 67 residues of the C-terminus are required for the lethal effect on haploid yeast. The latter effect, however, is dependent on strains. Thus, the yeast system may be utilized for functional dissection of E1a protein by further analyzing metabolic consequences.     

Interaction of super-repressible and dominant constitutive mutations for the synthesis of galactose pathway enzymes in Saccharomyces cerevisiae.

Two dominant uninducible mutant alleles in the gal80 locus were identified. The GAL80s-1 and GAL80s-2 mutants showed novel phenotypes in response to the newly isolated GAL81-1 mutant allele, a dominant constitutive mutation linked to the gal4 locus; the GAL80s-1 GAL81-1 strain was inducible and the GAL80s-2 GAL81-1 strain was uninducible. Many galactose positive revertants from the GAL80s-2 GAL81-1 strain were isolated. It was proved that each revertant was due to a secondary mutation either in the gal80 or GAL81 locus, whereas revertants due to mutation at the supposed controlling site for the structural gene cluster of the galactose-pathway enzymes have not been isolated.     

Marinobacter alkaliphilus sp. nov., a novel alkaliphilic bacterium isolated from subseafloor alkaline serpentine mud from Ocean Drilling Program Site 1200 at South Chamorro Seamount, Mariana Forearc

Novel alkaliphilic, mesophilic bacteria were isolated from subseafloor alkaline serpentine mud from the Ocean Drilling Program (ODP) Hole 1200D at a serpentine mud volcano, South Chamorro Seamount in the Mariana Forearc. The cells of type strain ODP1200D-1.5^T were motile rods with a single polar flagellum. Growth was observed between 10 and 45–50°C (optimum temperature: 30–35°C, 45-min doubling time), between pH 6.5 and 10.8–11.4 (optimum: pH 8.5–9.0), and between NaCl concentrations of 0 and 21% (w/v) (optimum NaCl concentration: 2.5–3.5%). The isolate was a facultatively anaerobic heterotroph utilizing various complex substrates, hydrocarbons, carbohydrates, organic acids, and amino acids. Nitrate or fumarate could serve as an electron acceptor to support growth under anaerobic conditions. The G+C content of the genomic DNA was 57.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belonged to the genus Marinobacter and was the most closely related to M. aquaeolei strain VT8^T and M. hydrocarbonoclasticus strain SP.17^T, while DNA–DNA hybridization demonstrated that the new isolate could be genetically differentiated from the previously described species of Marinobacter. Based on the physiological and molecular properties of the new isolate, we propose the name Marinobacter alkaliphilus sp. nov., type strain: ODP1200D-1.5^T (JCM12291^T and ATCC BAA-889^T).     

Hyper-production of an isomalto-dextranase of an Arthrobacter sp. by a proteases-deficient Bacillus subtilis: sequencing, properties, and crystallization of the recombinant enzyme

Arthrobacter globiformis T6 is unique in that it produces an enzyme yielding only isomaltose from dextran. In the present study, the organism was re-identified and its classification as a new species of the genus Arthrobacter, A. dextranlyticum, was proposed. The high G+C gene (66.8 mol%) for the isomalto-dextranase was sequenced. The deduced amino acid sequence, with a calculated molecular mass of 65,993 Da (603 amino acids), was confirmed by nanoscale capillary liquid chromatography coupled to tandem mass spectrometry, which covered 71.1% of the amino acid residues of the entire sequence. The enzyme was grouped into glycoside hydrolase family 27, and the C-terminal domain has homology to carbohydrate-binding module family 6. Hyper-exoproduction of the recombinant enzyme was achieved at a level corresponding to approximately 4.6 g l^–1 of culture broth when proteases-deficient Bacillus subtilis cells were used as the host. The purified enzyme (65.5 kDa) had an optimal pH and temperature for activity of 3.5 and 60°C, respectively. It was crystallized using the sitting-drop vapor-diffusion method at 293 K.