Maria Cristina Aisa

Early Improvement of Glucose Tolerance after Ileal Transposition in a Non-obese Type 2 Diabetes Rat Model

Background: Surgical operations which shorten the intestinal tract between the stomach and the terminal ileum result in an early improvement in type 2 diabetes, and one possible explanation is the arrival of undigested food in the terminal ileum. This study was designed to evaluate the role of the distal ileum in the improvement of glucose control in type 2 diabetic patients who underwent bariatric surgery. Methods: An ileal transposition (IT) to the jejunum was performed in lean diabetic Goto-Kakizaki (GK) rats. The IT was compared to sham-operated diabetic rats and a control group of diabetic rats. Non-diabetic controls were age-matched Sprague-Dawley (SD) rats, which underwent IT and no operation. Food intake and body weight were measured. An oral glucose tolerance test (OGTT) was performed 10 days before the operation and 10 days, 30 days and 45 days after the surgery. GLP-1 and insulin were measured during the OGTT 45 days after surgery. An insulin tolerance test (ITT) was performed 50 days after surgery. Results: Glucose tolerance improved in the IT diabetic group compared with both the sham-operated animals and control diabetic group 30 days and 45 days after surgery (P=0.029 and P=0.023, respectively). Insulin sensitivity, as measured by an ITT, was not significantly different between diabetic groups and the normal groups respectively after surgery. No differences in basal glucose and glucose tolerance were noted between non-diabetic operated animals and control non-diabetic rats. No differences were recorded between the diabetic rat groups and the non-diabetic rats in terms of weight and food intake. GLP-1 levels were significantly higher in the IT diabetic group compared with the sham-operated rats (P=0.05). Conclusions: Ileal transposition is effective in inducing an improvement in glucose tolerance in lean diabetic rats without affecting weight and food intake. The possible mechanism underlying the early improvement of diabetes after bariatric surgery may be due to the action of the terminal ileum through an insulin-independent action.

S‐100a0 protein stimulates Ca2+ ‐induced Ca2+ release from isolated sarcoplasmic reticulum vesicles

S‐100a0 protein, the αα‐isoform of the S‐100 family, stimulates Ca2+ ‐induced Ca2+ release from terminal cisternae isolated from rat skeletal muscle cells. The stimulatory effect of S‐100a0 is maximal at ∼5 μM S‐100a0 and half maximal at ∼0.1 μM S‐100a0, at 1.8 μM free Ca2+ in the presence of 5 mM Mg2+ plus 0.1 M KCl. The effect of the protein on Ca2+ ‐induced Ca2+ release is completely inhibted by the calcium release blocker, ruthenium red.

Cardiac S-100a0 protein: purification by a simple procedure and related immunocytochemical and immunochemical studies

A simple procedure is described for the purification of the alpha alpha isoform of S-100 proteins (S-100a0) from porcine heart. Purification steps include the following: i) extraction of the tissue with a hypotonic medium containing EDTA; ii) ammonium sulfate fractionation (0-50%) of the extract; iii) Ca2+-dependent affinity chromatography of the supernatant obtained through the preceding step on phenyl-sepharose and elution of absorbed proteins through a two-chamber gradient of 1.0-0.0 mM CaCl2 and 0.0--1.0 mM EGTA, respectively; and iv) chromatography of the resultant S-100-containing fractions on Sephadex G-200. The yield is 20 mg S-100a0/kg porcine heart. The whole procedure takes five days and is highly reproducible. Data obtained from the phenyl-sepharose step suggest that the affinity of Ca2+ for S-100a0 increases by several orders of magnitude once the protein had interacted with that matrix. This observation is discussed in relation to the role of S-100 proteins in amplification of the Ca2+ signal. Immunocytochemical and immunoblotting analyses indicate that S-100a0 is exclusively found at the level of the sarcolemmal membranes, the membranes of the sarcoplasmic reticulum, the external mitochondrial membranes, and in the adjacent sarcoplasm. No evidence of S-100a0 being associated with the nuclei or with myofibrils has been obtained. Finally, the cardiac tissue does not contain the Triton X-100-extractable fraction of S-100 normally detected in the brain and in adipocytes. Our data suggest that S-100a0 behaves as a peripheral membrane protein in cardiac tissue.

P253R fibroblast growth factor receptor‐2 mutation induces RUNX2 transcript variants and calvarial osteoblast differentiation

Unregulated fibroblast growth factor 2 (FGF2) signaling caused by mutations in the fibroblast growth factor receptor (FGFR2) leads to human craniosynostosis such as the Apert syndrome. In an in vitro control model of calvarial osteoblasts from Apert patients carrying the FGFR2 P253R mutation, we studied the changes in cellular phenotype and evaluated the effects of FGF2. Compared with wild‐type controls, osteocalcin mRNA was down‐regulated in Apert osteoblasts, Runt‐related transcription factor‐2 (RUNX2) mRNA was differentially spliced, and FGF2 secretion was greater. Total protein synthesis, fibronectin and type I collagen secretion were up‐regulated, while protease and glycosidase activities and matrix metalloproteinase‐13 (MMP‐13) transcription were decreased, suggesting an altered ECM turnover. Adding FGF2 increased protease and glycosidase activities and down‐regulated fibronectin and type I collagen secretion in Apert osteoblasts. High affinity FGF2 receptors were up‐regulated in Apert osteoblasts and analysis of signal transduction showed elevated levels of Grb2 tyrosine phosphorylation and the Grb2‐p85 beta association, which FGF2 stimulation strongly reduced. All together these findings suggest increased constitutive receptor activity in Apert mutant osteoblasts and an autocrine loop involving the FGF2 pathway in modulation of Apert osteoblast behavior.

S-100a0 protein stimulates the basal (Mg2+-activated) adenylate cyclase activity associated with skeletal muscle membranes

S‐100a0, protein, the αα isoform of the S‐100 family, stimulates basal (Mg2+‐activated) adenylate cyclase (AC) activity associated with the sarcolemma, longitudinal tubules and terminal cisternae of rat skeletal muscle cells. The stimulatory effect of S‐100a0 on AC activity is maximal around 5μM S‐100a0 and half‐maximal around 0.2 μM S‐100a0. Also, the stimulatory effect is greatest on the AC activity associated with the terminal cisternae than on the other membrane fractions studied. These data are discussed in relation to the subcellular localization of S‐100a0 in muscle cells.

Molecular Interaction of S‐100 Proteins with Microtubule Proteins In Vitro

Abstract: Several procedures were employed to examine the in vitro interaction between S‐100 proteins and microtubule proteins. Binding of S‐100 to τ factors was observed under all experimental conditions. S‐100 binding to microtubule‐associated protein 2 (MAP2) was best detected by exposing nitrocellulose‐immobilized MAP2 or MAPs to either 125I‐labeled S‐100 or biotinylated S‐100. S‐100 binding to tubulin was detected when the two protein fractions were first incubated with each other followed by exposure to the Afunctional cross‐linker disuccinimidylsuberate, and then separated by sodium dodecyl sulfate‐polyacrylamide gel [electrophoresis (SDS‐PAGE) and transfered onto nitrocellulose paper. By this procedure, complex formation between S‐100 and tubulin, as well as between S‐100 and a relatively low‐molecular‐weight MAP, was evidenced by immunoblotting using an anti‐S‐100 antiserum. Alternatively, complex formation between biotinylated S‐100 and either tubulin or MAPs was visualized by means of avidin‐peroxidase, after SDS‐PAGE of the complex mixtures and transfer of the separated proteins onto nitrocellulose. The interaction between S‐100 and tubulin was strictly Ca2+ dependent, and resistant to high concentrations of KC1, colchicine, or vinblastine.

S-100b protein regulates the activity of skeletal muscle adenylate cyclase in vitro.

We have investigated the effect of the b isoform of S‐100 proteins on adenylate cyclase activity of rat skeletal muscle. S‐100b inhibits the adenylate cyclase activity in the presence of Mg2+ (5.0–50 mM), while it activates the same enzyme in the presence of Ca2+ (0.1–1.0 mM) dose‐dependently in both cases. S‐100b counteracts the stimulatory effect of NaF on adenylate cyclase in the presence of Mg2+ and the inhibitory effect of RMI 12330 A in the presence of Ca2+.

Cathepsin B in osteoblasts

Active cathepsin B has been found in cell extract and medium of human osteoblast-like cells and MG-63 cells. The released form is stable at neutral and alkaline pH and, in both cell types, intracellular and extracellular cathepsin B activities are increased by interleukin-1 beta (IL-1beta) and parathyroid hormone (PTH). To evaluate the physiological role of cathepsin B in osteoblasts, we investigated the production and secretion of this enzyme in normal human synovial fibroblasts and modulation by IL-1beta and PTH. Lactate secretion concurrent with release of cathepsin B and comparable responses in osteoblasts were also examined. Our data show that synovial fibroblasts respond differently to treatment with the two agents, suggesting a cell-specific regulation of cathepsin B and possible involvement in osteoblast physiology. Cathepsin B involvement was then evaluated in the activation of plasminogen activator (PA) in MG-63 cells using two specific inhibitors of cathepsin B, CA074 and CA074-Me, in constitutive conditions and after treatment with IL-1beta. As results of PA activity obtained in the presence of IL-beta were in contrast with previous reports, we examined the activities of PA, pro-PA activated with trypsin, and plasmin in cell extract and media of MG-63 cells after 24-h treatment with IL-1beta. Results show that in normal conditions and in the presence of IL-1beta, cathepsin B is involved in the activation of PA. Moreover, IL-1beta stimulates PA, pro-PA activated by trypsin, and plasmin activity in medium, whereas in cell extract it stimulates pro-PA activated by trypsin and plasmin activity. IL-1beta has no effect on cell extract-associated PA.

Identification of S-100 proteins and S- 100-binding proteins in a detergent-resistant EDTA/KCl-extractable fraction from bovine brain membranes

The Triton X‐100‐resistant residue of brain membranes contains appreciable amounts of S‐100 proteins. This fraction of S‐100 can be solubilized by high concentrations of EDTA plus or minus high concentrations of KCl. Whereas KCl (0.6 M) extracts the detergent‐resistant S‐100, NACl (1 M) does not. Endogenous Ca2+ is required and is sufficient for S‐100 to remain associated with the detergent‐resistant residue. However, 0.6 M KCl extracts a further fraction of Triton X‐100‐resistant S‐100. In contrast, the Triton X‐100‐extractable fraction of S‐100 resists the action of EDTA. These data suggest that Ca2+ regulates the extent of association of S‐100 with Triton X‐100‐resistant components in brain membranes, whereas the association of S‐100 with the lipid bilayer of brain membranes and/or with some intrinsic membrane proteins is less Ca2+‐regulated. Several S‐100‐binding proteins are identified in the detergent‐resistant residue of brain membranes by an overlay procedure.

Basic fibroblast growth factor: effects on matrix remodeling, receptor expression, and transduction pathway in human periosteal fibroblasts with FGFR2 gene mutation.

The Crouzon syndrome, which is associated with fibroblast growth factor receptor (FGFR2) mutations, is characterized by premature fusion of cranial sutures. We used an in vitro model of cultured periosteal fibroblasts from normal subjects and from Crouzon patients with FGFR2 mutation. We analyzed the matrix turnover rate and the effects of adding FGF2 by evaluating fibronectin synthesis and the activity of some proteolytic enzymes. To assess the role of some FGF signaling molecules involved in FGFR2 regulation, we studied Grb2 tyrosine phosphorylation and the phosphotyrosine proteins associated with Grb2. The iodinate FGF binding assay was performed to quantify FGFR expression. Compared with normal fibroblasts, fibronectin synthesis was decreased in Crouzon fibroblasts, and protease activities in cells and medium were enhanced, suggesting that excess fibronectin catabolism is present. Differences were more marked when FGF2 was added. Very few phosphoproteins were visible in anti-Grb2 immunoprecipitations from Crouzon fibroblasts, which showed a significant increase in the number of high-affinity and low-affinity FGF2 receptors. These results suggest that the abnormal genotype and the Crouzon cellular phenotype are related. To compensate the low levels of tyrosine phosphorylation, Crouzon cells might increase the numbers of FGFR2, thus increasing the cell surface binding sites for FGF2.