Studies on secretory glycoproteins in the rat exocrine pancreas

Summary A fetuin, fucosyl transferase has been identified in the smooth microsomal fraction from the rat exocrine pancreas. This enzyme is involved in the glycosylation of secretory proteins and is bound to membranes, predominantly of the Golgi complex. Optimal in vitro conditions for the assay of the enzyme activity were established: a pH of 5.5–6.0, a temperature of 21° C and concentrations of Mg^{+ +} at 5.0 mM and ATP at 2.0 mM.Supported by a grant from the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (Ke 113/10). Dedicated to Professor Helmut Ferner, Vienna, on the occasion of his 65^th birthday.     

Design of peptides with high affinities for heparin and endothelial cell proteoglycans

Proteoglycan-binding peptides were designed based on consensus sequences in heparin-binding proteins: XBBXBX and XBBBXXBX, where X and B are hydropathic and basic residues, respectively. Initial peptide constructs included (AKKARA)(n) and (ARKKAAKA)(n) (n = 1-6). Affinity coelectrophoresis revealed that low M(r) peptides (600-1,300) had no affinities for low M(r) heparin, but higher M(r) peptides (2,000-3,500) exhibited significant affinities (K(d) congruent with 50-150 nM), which increased with peptide M(r). Affinity was strongest when sequence arrays were contiguous and alanines and arginines occupied hydropathic and basic positions, but inclusion of prolines was disruptive. A peptide including a single consensus sequence of the serglycin proteoglycan core protein bound heparin strongly (K(d) congruent with 200 nM), likely owing to dimerization through cysteine-cysteine linkages. Circular dichroism showed that high affinity heparin-binding peptides converted from a charged coil to an alpha-helix upon heparin addition, whereas weak heparin-binding peptides did not. Higher M(r) peptides exhibited high affinities for total endothelial cell proteoglycans (K(d) congruent with 300 nM), and approximately 4-fold weaker affinities for their free glycosaminoglycan chains. Thus, peptides including concatamers of heparin-binding consensus sequences may exhibit strong affinities for heparin and proteoglycans. Such peptides may be applicable in promoting cell-substratum adhesion or in the design of drugs targeted to proteoglycan-containing cell surfaces and extracellular matrices.     

The Unrecognized Effects of Phosphodiesterase 4 on Epithelial Cells in Pulmonary Inflammation

Acute pulmonary inflammation is characterized by migration of polymorphonuclear neutrophils (PMNs) into the different compartments of the lung, passing an endothelial and epithelial barrier. Recent studies showed evidence that phosphodiesterase (PDE)4-inhibitors stabilized endothelial cells. PDE4B and PDE4D subtypes play a pivotal role in inflammation, whereas blocking PDE4D is suspected to cause gastrointestinal side effects. We thought to investigate the particular role of the PDE4-inhibitors roflumilast and rolipram on lung epithelium. Acute pulmonary inflammation was induced by inhalation of LPS. PDE4-inhibitors were administered i.p. or nebulized after inflammation. The impact of PDE4-inhibitors on PMN migration was evaluated in vivo and in vitro. Microvascular permeability, cytokine levels, and PDE4B and PDE4D expression were analyzed. In vivo, both PDE4-inhibitors decreased transendothelial and transepithelial migration even when administered after inflammation, whereas roflumilast showed a superior effect compared to rolipram on the epithelium. Both inhibitors decreased TNFα, IL6, and CXCL2/3. CXCL1, the strong PMN chemoattractant secreted by the epithelium, was significantly more reduced by roflumilast. In vitro assays with human epithelium also emphasized the pivotal role of roflumilast on the epithelium. Additionally, LPS-induced stress fibers, an essential requirement for a direct migration of PMNs into the alveolar space, were predominantly reduced by roflumilast. Expression of PDE4B and PDE4D were both increased in the lungs by LPS, PDE4-inhibitors decreased mainly PDE4B. The topical administration of PDE4-inhibitors was also effective in curbing down PMN migration, further highlighting the clinical potential of these compounds. In pulmonary epithelial cells, both subtypes were found coexistent around the nucleus and the cytoplasm. In these epithelial cells, LPS increased PDE4B and, to a lesser extend, PDE4D, whereas the effect of the inhibitors was prominent on the PDE4B subtype. In conclusion, we determined the pivotal role of the PDE4-inhibitor roflumilast on lung epithelium and emphasized its main effect on PDE4B in hyperinflammation.     

Buchbesprechungen

Ohne Zusammenfassung     

Variation in the Phosphoinositide 3-Kinase Gamma Gene Affects Plasma HDL-Cholesterol without Modification of Metabolic or Inflammatory Markers

Objective

Phosphoinositide 3-kinase γ (PI3Kγ) is a G-protein-coupled receptor-activated lipid kinase mainly expressed in leukocytes and cells of the cardiovascular system. PI3Kγ plays an important signaling role in inflammatory processes. Since subclinical inflammation is a hallmark of atherosclerosis, obesity-related insulin resistance, and pancreatic β-cell failure, we asked whether common genetic variation in the PI3Kγ gene (PIK3CG) contributes to body fat content/distribution, serum adipokine/cytokine concentrations, alterations in plasma lipid profiles, insulin sensitivity, insulin release, and glucose homeostasis.

Study Design

Using a tagging single nucleotide polymorphism (SNP) approach, we analyzed genotype-phenotype associations in 2,068 German subjects genotyped for 10 PIK3CG SNPs and characterized by oral glucose tolerance tests. In subgroups, data from hyperinsulinaemic-euglycaemic clamps, magnetic resonance spectroscopy of the liver, whole-body magnetic resonance imaging, and intravenous glucose tolerance tests were available, and peripheral blood mononuclear cells (PBMCs) were used for gene expression analysis.

Results

After appropriate adjustment, none of the PIK3CG tagging SNPs was significantly associated with body fat content/distribution, adipokine/cytokine concentrations, insulin sensitivity, insulin secretion, or blood glucose concentrations (p>0.0127, all; Bonferroni-corrected α-level: 0.0051). However, six non-linked SNPs displayed at least nominal associations with plasma HDL-cholesterol concentrations, two of them (rs4288294 and rs116697954) reaching the level of study-wide significance (p = 0.0003 and p = 0.0004, respectively). More precisely, rs4288294 and rs116697954 influenced HDL2-, but not HDL3-, cholesterol. With respect to the SNPs’ in vivo functionality, rs4288294 was significantly associated with PIK3CG mRNA expression in PBMCs.

Conclusions

We could demonstrate that common genetic variation in the PIK3CG locus, possibly via altered PIK3CG gene expression, determines plasma HDL-cholesterol concentrations. Since HDL2-, but not HDL3-, cholesterol is influenced by PIK3CG variants, PI3Kγ may play a role in HDL clearance rather than in HDL biogenesis. Even though the molecular pathways connecting PI3Kγ and HDL metabolism remain to be further elucidated, this finding could add a novel aspect to the pathophysiological role of PI3Kγ in atherogenesis.     

Separation of fast from slow anabolism by site-specific PEGylation of insulin-like growth factor I (IGF-I)

Insulin-like growth factor I (IGF-I) has important anabolic and homeostatic functions in tissues like skeletal muscle, and a decline in circulating levels is linked with catabolic conditions. Whereas IGF-I therapies for musculoskeletal disorders have been postulated, dosing issues and disruptions of the homeostasis have so far precluded clinical application. We have developed a novel IGF-I variant by site-specific addition of polyethylene glycol (PEG) to lysine 68 (PEG-IGF-I). In vitro, this modification decreased the affinity for the IGF-I and insulin receptors, presumably through decreased association rates, and slowed down the association to IGF-I-binding proteins, selectively limiting fast but maintaining sustained anabolic activity. Desirable in vivo effects of PEG-IGF-I included increased half-life and recruitment of IGF-binding proteins, thereby reducing risk of hypoglycemia. PEG-IGF-I was equipotent to IGF-I in ameliorating contraction-induced muscle injury in vivo without affecting muscle metabolism as IGF-I did. The data provide an important step in understanding the differences of IGF-I and insulin receptor contribution to the in vivo activity of IGF-I. In addition, PEG-IGF-I presents an innovative concept for IGF-I therapy in diseases with indicated muscle dysfunction.