Apolipoprotein CIII links islet insulin resistance to ß-cell failure in diabetes.

Insulin resistance and ß-cell failure are the major defects in type 2 diabetes mellitus. However, the molecular mechanisms linking these two defects remain unknown. Elevated levels of apolipoprotein CIII (apoCIII) are associated not only with insulin resistance but also with cardiovascular disorders and inflammation. We now demonstrate that local apoCIII production is connected to pancreatic islet insulin resistance and ß-cell failure. An increase in islet apoCIII causes promotion of a local inflammatory milieu, increased mitochondrial metabolism, deranged regulation of ß-cell cytoplasmic free Ca(2+) concentration ([Ca(2+)]i) and apoptosis. Decreasing apoCIII in vivo results in improved glucose tolerance, and pancreatic apoCIII knockout islets transplanted into diabetic mice, with high systemic levels of the apolipoprotein, demonstrate a normal [Ca(2+)]i response pattern and no hallmarks of inflammation. Hence, under conditions of islet insulin resistance, locally produced apoCIII is an important diabetogenic factor involved in impairment of ß-cell function and may thus constitute a novel target for the treatment of type 2 diabetes mellitus.

A GLP-1 receptor agonist conjugated to an albumin-binding domain for extended half-life.

Glucagon-like peptide 1 (GLP-1) and related peptide agonists have been extensively investigated for glycaemic control in Type 2 diabetes, and may also have therapeutic applications for other diseases. Due to the short half-life (t1/2 < 2 min) of the endogenous peptide, caused by proteolytic degradation and renal clearance, different strategies for half-life extension and sustained release have been explored. In the present study, conjugates between a GLP-1 analogue and a 5 kDa albumin-binding domain (ABD) derived from streptococcal protein G have been chemically synthesized and evaluated. ABD binds with high affinity to human serum albumin, which is highly abundant in plasma and functions as a drug carrier in the circulation. Three different GLP-1-ABD conjugates, with the two peptides connected by linkers of two, four, and six PEG units, respectively, were synthesized and tested in mouse pancreatic islets at high (11 mM) and low (3 mM) glucose concentration. Insulin release upon stimulation was shown to be glucose-dependent, showing no significant difference between the three different GLP-1-ABD conjugates and unconjugated GLP-1 analogue. The biological activity, in combination with the high affinity binding to albumin, make the GLP-1-ABD conjugates promising GLP-1 receptor agonists expected to show extended in vivo half-life.

Lowering apolipoprotein CIII delays onset of type 1 diabetes.

Serum levels of apolipoprotein CIII (apoCIII) are increased in type 1 diabetic patients, and when ß cells are exposed to these diabetic sera, apoptosis occurs, an effect abolished by an antibody against apoCIII. We have investigated the BB rat, an animal model that develops a human-like type 1 diabetes, and found that apoCIII was also increased in sera from prediabetic rats. This increase in apoCIII promoted ß-cell death. The endogenous levels of apoCIII were reduced by treating prediabetic animals with an antisense against this apolipoprotein, resulting in a significantly delayed onset of diabetes. ApoCIII thus serves as a diabetogenic factor, and intervention with this apolipoprotein in the prediabetic state can arrest disease progression. These findings suggest apoCIII as a target for the treatment of type 1 diabetes.

Transthyretin binds to glucose-regulated proteins and is subjected to endocytosis by the pancreatic ß-cell.

Transthyretin (TTR) is a functional protein in the pancreatic ß-cell. It promotes insulin release and protects against ß-cell death. We now demonstrate by ligand blotting, adsorption to specific magnetic beads, and surface plasmon resonance that TTR binds to glucose-regulated proteins (Grps)78, 94, and 170, which are members of the endoplasmic reticulum chaperone family, but Grps78 and 94 have also been found at the plasma membrane. The effect of TTR on changes in cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) was abolished if the cells were treated with either dynasore, a specific inhibitor of dynamin GTPase that blocks clathrin-mediated endocytosis, or an antibody against Grp78, that prevents TTR from binding to Grp78. The conclusion is that TTR binds to Grp78 at the plasma membrane, is internalized into the ß-cell via a clathrin-dependent pathway, and that this internalization is necessary for the effects of TTR on ß-cell function.

The cytotoxic effects of the anti-bacterial peptides on leukocytes.

Antimicrobial peptides are small molecular weight proteins with a large antibacterial spectrum. They can reach high local concentrations in tissues with active inflammation, being largely produced by immunocompetent cells. However, their effect on eukaryotic cells is still unclear. We have, therefore, studied three structurally different antimicrobial peptides (cecropin P1, PR-39 and NK-lysin) for their cytotoxic effects on blood mononuclear cells. None of the antimicrobial peptides tested exhibited significant cytotoxic effect on resting lymphocytes isolated either from peripheral blood or from the spleen with the exception of high concentrations (ten times higher than IC100 for Escherichia coli) of NK-lysin. Activated lymphocytes were, however, more sensitive to the cytotoxic effect of the antimicrobial peptides. Both activated T-cells and B-cells were dose dependent sensitive to NK-lysin while only activated B-cells but not activated T-cells were sensitive to PR-39. Cecropin did not exhibit any cytotoxic effect on activated lymphocytes either. By using several cell lines (3B6, K562, U932 and EL-4) we were able to show that NK-lysin has a broad necrotic effect while PR-39 has a cell specific apoptotic effect dependent on the specifically cellular uptake. In conclusion we show here that antimicrobial peptides are not cytotoxic for the resting eukaryotic cells but can be cytotoxic on activated immune cells through distinct mechanisms of cell death.

SNAP-25b-deficiency increases insulin secretion and changes spatiotemporal profile of Ca2+oscillations in ß cell networks.

SNAP-25 is a protein of the core SNARE complex mediating stimulus-dependent release of insulin from pancreatic ß cells. The protein exists as two alternatively spliced isoforms, SNAP-25a and SNAP-25b, differing in 9 out of 206 amino acids, yet their specific roles in pancreatic ß cells remain unclear. We explored the effect of SNAP-25b-deficiency on glucose-stimulated insulin release in islets and found increased secretion both in vivo and in vitro. However, slow photo-release of caged Ca2+ in ß cells within pancreatic slices showed no significant differences in Ca2+-sensitivity, amplitude or rate of exocytosis between SNAP-25b-deficient and wild-type littermates. Therefore, we next investigated if Ca2+ handling was affected in glucose-stimulated ß cells using intracellular Ca2+-imaging and found premature activation and delayed termination of [Ca2+] i elevations. These findings were accompanied by less synchronized Ca2+-oscillations and hence more segregated functional ß cell networks in SNAP-25b-deficient mice. Islet gross morphology and architecture were maintained in mutant mice, although sex specific compensatory changes were observed. Thus, our study proposes that SNAP-25b in pancreatic ß cells, except for participating in the core SNARE complex, is necessary for accurate regulation of Ca2+-dynamics.

Conserved structure and function in the granulysin and NK-lysin peptide family.

Granulysin and NK-lysin are homologous bactericidal proteins with a moderate residue identity (35%), both of which have antimycobacterial activity. Short loop peptides derived from the antimycobacterial domains of granulysin, NK-lysin, and a putative chicken NK-lysin were examined and shown to have comparable antimycobacterial but variable Escherichia coli activities. The known structure of the NK-lysin loop peptide was used to predict the structure of the equivalent peptides of granulysin and chicken NK-lysin by homology modeling. The last two adopted a secondary structure almost identical to that of NK-lysin. All three peptides form very similar three-dimensional (3-D) architectures in which the important basic residues assume the same positions in space. The basic residues in granulysin are arginine, while those in NK-lysin and chicken NK-lysin are a mixture of arginine and lysine. We altered the ratio of arginine to lysine in the granulysin fragment to examine the importance of basic residues for antimycobacterial activity. The alteration of the amino acids reduced the activity against E. coli to a larger extent than that against Mycobacterium smegmatis. In granulysin, the arginines in the loop structure are not crucial for antimycobacterial activity but are important for cytotoxicity. We suggest that the antibacterial domains of the related proteins granulysin, NK-lysin, and chicken NK-lysin have conserved their 3-D structure and their function against mycobacteria.

Transthyretin constitutes a functional component in pancreatic beta-cell stimulus-secretion coupling.

Transthyretin (TTR) is a transport protein for thyroxine and, in association with retinol-binding protein, for retinol, mainly existing as a tetramer in vivo. We now demonstrate that TTR tetramer has a positive role in pancreatic beta-cell stimulus-secretion coupling. TTR promoted glucose-induced increases in cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) and insulin release. This resulted from a direct effect on glucose-induced electrical activity and voltage-gated Ca(2+) channels. TTR also protected against beta-cell apoptosis. The concentration of TTR tetramer was decreased, whereas that of a monomeric form was increased in sera from patients with type 1 diabetes. The monomer was without effect on glucose-induced insulin release and apoptosis. Thus, TTR tetramer constitutes a component in normal beta-cell function. Conversion of TTR tetramer to monomer may be involved in the development of beta-cell failure/destruction in type 1 diabetes.

Increased diversity of intestinal antimicrobial peptides by covalent dimer formation.

Antimicrobial peptides are essential effector molecules of the innate immune system. Here we describe the structure, function and diversity of cryptdin-related sequence (CRS) peptides, a large family of antimicrobial molecules. We identified the peptides as covalent dimers in mouse intestinal tissue in amounts comparable to those of Paneth cell-derived enteric alpha-defensins. CRS peptides caused rapid and potent killing of commensal and pathogenic bacteria. The CRS peptides formed homo- and heterodimers in vivo, thereby expanding the repertoire of antimicrobial peptides and increasing the peptide diversity of Paneth cell secretions. CRS peptides might therefore be important in the maintenance of the microbial homeostasis within the intestinal tract.

Apolipoprotein CIII promotes Ca2+-dependent beta cell death in type 1 diabetes.

In type 1 diabetes (T1D), there is a specific destruction of the insulin secreting pancreatic beta cell. Although the exact molecular mechanisms underlying beta cell destruction are not known, sera from T1D patients have been shown to promote Ca(2+)-induced apoptosis. We now demonstrate that apolipoprotein CIII (apoCIII) is increased in serum from T1D patients and that this serum factor both induces increased cytoplasmic free intracellular Ca(2+) concentration ([Ca(2+)](i)) and beta cell death. The apoCIII-induced increase in [Ca(2+)](i) reflects an activation of the voltage-gated L-type Ca(2+) channel. Both the effects of T1D sera and apoCIII on the beta cell are abolished in the presence of antibody against apoCIII. Increased serum levels of apoCIII can thus account for the increase in beta cell [Ca(2+)](i) and thereby beta cell apoptosis associated with T1D.