| 1. |
- Burén, Jonas, et al.
(author)
-
High glucose and insulin in combination cause insulin receptor substrate-1 and -2 depletion and protein kinase B desensitisation in primary cultured rat adipocytes : possible implications for insulin resistance in type 2 diabetes.
- 2003
-
In: European Journal of Endocrinology. - : European Society of Endocrinology. - 0804-4643 .- 1479-683X. ; 148:1, s. 157-67
-
Journal article (peer-reviewed)abstract
- OBJECTIVE: The purpose of this study was to investigate the cellular effects of long-term exposure to high insulin and glucose levels on glucose transport and insulin signalling proteins.DESIGN AND METHODS: Rat adipocytes were cultured for 24 h in different glucose concentrations with 10(4) microU/ml of insulin or without insulin. After washing, (125)I-insulin binding, basal and acutely insulin-stimulated d-[(14)C]glucose uptake, and insulin signalling proteins and glucose transporter 4 (GLUT4) were assessed.RESULTS: High glucose (15 and 25 mmol/l) for 24 h induced a decrease in basal and insulin-stimulated glucose uptake compared with control cells incubated in low glucose (5 or 10 mmol/l). Twenty-four hours of insulin treatment decreased insulin binding capacity by approximately 40%, and shifted the dose-response curve for insulin's acute effect on glucose uptake 2- to 3-fold to the right. Twenty-four hours of insulin treatment reduced basal and insulin-stimulated glucose uptake only in the presence of high glucose (by approximately 30-50%). At high glucose, insulin receptor substrate-1 (IRS-1) expression was downregulated by approximately 20-50%, whereas IRS-2 was strongly upregulated by glucose levels of 10 mmol/l or more (by 100-400%). Insulin treatment amplified the suppression of IRS-1 when combined with high glucose and also IRS-2 expression was almost abolished. Twenty-four hours of treatment with high glucose or insulin, alone or in combination, shifted the dose-response curve for insulin's effect to acutely phosphorylate protein kinase B (PKB) to the right. Fifteen mmol/l glucose increased GLUT4 in cellular membranes (by approximately 140%) compared with 5 mmol/l but this was prevented by a high insulin concentration.CONCLUSIONS: Long-term exposure to high glucose per se decreases IRS-1 but increases IRS-2 content in rat adipocytes and it impairs glucose transport capacity. Treatment with high insulin downregulates insulin binding capacity and, when combined with high glucose, it produces a marked depletion of IRS-1 and -2 content together with an impaired sensitivity to insulin stimulation of PKB activity. These mechanisms may potentially contribute to insulin resistance in type 2 diabetes.
|
|
| 2. |
- Croxatto, Antony, et al.
(author)
-
VanT, a Homologue of Vibrio harveyi LuxR, Regulates Serine, Metalloprotease, Pigment, and Biofilm Production in Vibrio anguillarum
- 2002
-
In: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 184:6, s. 1617-1629
-
Journal article (peer-reviewed)abstract
- Vibrio anguillarum possesses at least two N-acylhomoserine lactone (AHL) quorum-sensing circuits, one of which is related to the luxMN system of Vibrio harveyi. In this study, we have cloned an additional gene of this circuit, vanT, encoding a V. harveyi LuxR-like transcriptional regulator. A V. anguillarum ΔvanT null mutation resulted in a significant decrease in total protease activity due to loss of expression of the metalloprotease EmpA, but no changes in either AHL production or virulence. Additional genes positively regulated by VanT were identified from a plasmid-based gene library fused to a promoterless lacZ. Three lacZ fusions (serA::lacZ, hpdA-hgdA::lacZ, and sat-vps73::lacZ) were identified which exhibited decreased expression in the ΔvanT strain. SerA is similar to 3-phosphoglycerate dehydrogenases and catalyzes the first step in the serine-glycine biosynthesis pathway. HgdA has identity with homogentisate dioxygenases, and HpdA is homologous to 4-hydroxyphenylpyruvate dioxygenases (HPPDs) involved in pigment production. V. anguillarum strains require an active VanT to produce high levels of an l-tyrosine-induced brown color via HPPD, suggesting that VanT controls pigment production. Vps73 and Sat are related to Vibrio cholerae proteins encoded within a DNA locus required for biofilm formation. A V. anguillarum ΔvanT mutant and a mutant carrying a polar mutation in the sat-vps73 DNA locus were shown to produce defective biofilms. Hence, a new member of the V. harveyi LuxR transcriptional activator family has been characterized in V. anguillarum that positively regulates serine, metalloprotease, pigment, and biofilm production.
|
|
| 3. |
- Croxatto, Antony, et al.
(author)
-
Vibrio anguillarum colonization of rainbow trout integument requires a DNA locus involved in exopolysaccharide transport and biosynthesis.
- 2007
-
In: Environmental Microbiology. - : Wiley. - 1462-2912 .- 1462-2920. ; 9:2, s. 370-382
-
Journal article (peer-reviewed)abstract
- Vibrio anguillarum, part of the normal flora of the aquatic milieu, causes a fatal haemorrhagic septicaemia in marine fish. In this study, a rainbow trout model was used to characterize the colonization of fish skin by V. anguillarum. Within 5 h after infection, the bacterium penetrated the skin mucosal layer, attached to the scales within 12 h, and formed a biofilm by 24-48 h. Two divergently transcribed putative operons, orf1-wbfD-wbfC-wbfB and wza-wzb-wzc, were shown to play a role in skin colonization and virulence. The first operon encodes proteins of unknown function. The wza-wzb-wzc genes encode a secretin, tyrosine kinase and tyrosine phosphatase, respectively, which are similar to proteins in polysaccharide transport complexes. Compared with the wild type, polar mutations in wza, orf1 and wbfD caused a decrease in exopolysaccharide biosynthesis but not lipopolysaccharide biosynthesis. The wza and orf1 mutants did not attach to fish scales; whereas, the wbfD mutant had a wild-type phenotype. Moreover, the wza and orf1 mutants had decreased exoprotease activity, in particular the extracellular metalloprotease EmpA, as well as mucinase activity suggesting that these mutations also affect exoenzyme secretion. Thus, the exopolysaccharide transport system in V. anguillarum is required for attachment to fish skin, possibly preventing mechanical removal of bacteria via natural sloughing of mucus.
|
|
| 4. |
- Wang, Su-Yan, et al.
(author)
-
A ToxR homolog from Vibrio anguillarum serotype O1 regulates its own production, bile resistance, and biofilm formation
- 2002
-
In: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 184:6, s. 1630-1639
-
Journal article (peer-reviewed)abstract
- ToxR, a transmembrane regulatory protein, has been shown to respond to environmental stimuli. To better understand how the aquatic bacterium Vibrio anguillarum, a fish pathogen, responds to environmental signals that may be necessary for survival in the aquatic and fish environment, toxR and toxS from V. anguillarum serotype O1 were cloned. The deduced protein sequences were 59 and 67% identical to the Vibrio cholerae ToxR and ToxS proteins, respectively. Deletion mutations were made in each gene and functional analyses were done. Virulence analyses using a rainbow trout model showed that only the toxR mutant was slightly decreased in virulence, indicating that ToxR is not a major regulator of virulence factors. The toxR mutant but not the toxS mutant was 20% less motile than the wild type. Like many regulatory proteins, ToxR was shown to negatively regulate its own expression. Outer membrane protein (OMP) preparations from both mutants indicated that ToxR and ToxS positively regulate a 38-kDa OMP. The 38-kDa OMP was shown to be a major OMP, which cross-reacted with an antiserum to OmpU, an outer membrane porin from V. cholerae, and which has an amino terminus 75% identical to that of OmpU. ToxR and to a lesser extent ToxS enhanced resistance to bile. Bile in the growth medium increased expression of the 38-kDa OMP but did not affect expression of ToxR. Interestingly, a toxR mutant forms a better biofilm on a glass surface than the wild type, suggesting a new role for ToxR in the response to environmental stimuli.
|
|
| 5. |
- Wang, Su-Yan, et al.
(author)
-
Role for the major outer-membrane protein from Vibrio anguillarum in bile resistance and biofilm formation.
- 2003
-
In: Microbiology. - : Microbiology Society. - 1350-0872 .- 1465-2080. ; 149:Pt 4, s. 1061-1071
-
Journal article (peer-reviewed)abstract
- Vibrio anguillarum, a fish pathogen, produces a 38 kDa major outer-membrane porin, which may be involved in environmental adaptation. The gene encoding the 38 kDa porin was cloned and deleted. The deduced protein sequence was 75 % identical to that of the major outer-membrane protein (OMP), OmpU, from Vibrio cholerae. LacZ expression from an ompU : : lacZ transcriptional gene fusion was increased 1.5-fold in the presence of bile salts and was decreased 50- to 100-fold in a toxR mutant compared to that in the wild-type, showing that ompU expression is positively regulated by ToxR and induced by bile salts. Similar to a toxR mutant, an ompU mutant showed a slight decrease in motility, an increased sensitivity to bile salts and a thicker biofilm with better surface area coverage compared to that of the wild-type. When ompU was expressed under a ToxR-independent promoter in the toxR mutant, the phenotypes for bile resistance and biofilm formation, but not motility were complemented to that of the wild-type. In rainbow trout, the ompU mutant showed wild-type virulence via immersion into infected seawater and intraperitoneal injection. The ompU mutant produced two colony morphologies: opaque, which did not grow at 0.2 % bile, and translucent, which grew at 2 % bile. The translucent ompU mutant strain produced a second major OMP that was induced by bile. All ompU mutants showed variations in the amount and length of smooth LPS. In V. anguillarum, OmpU is not required for virulence, possibly due to a second OMP also critical for resistance to bile; however, outside of the fish host, OmpU limits the progression of biofilm formation.
|
|
