Cell-sized lipid vesicles for cell-cell synaptic therapies.

Cell-sized lipid vesicles (CLVs) have shown great promise for therapeutic and artificial cell applications, but their fragility and short shelf life has hindered widespread adoption and commercial viability. We present a method to circumvent the storage limitations of CLVs such as giant unilamellar vesicles (GUVs) and single-compartment multisomes (SCMs) by storing them in a double emulsion precursor form. The double emulsions can be stored for at least 8 months and readily converted into either GUVs or SCMs at any time. In this study, we investigate the interfacial parameters responsible for this morphological change, and we also demonstrate the therapeutic potential of CLVs by utilizing them to present a transmembrane protein, neuroligin-2, to pancreatic ß-cells, forming cell-cell synapses that stimulate insulin secretion and cellular growth.

Identification and characterization of a novel isoform of the vesicular gamma-aminobutyric acid transporter with glucose-regulated expression in rat islets.

Pancreatic islets are unique outside the nervous system in that they contain high levels of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), synthesized by the enzyme glutamic acid decarboxylase (GAD). Since the role that GABA plays in the islet and the mechanisms whereby the two major GAD isoforms (GAD65 and GAD67) function as diabetes-associated autoantigens are unknown, continued characterization of the islet GAD-GABA system is important. We previously demonstrated that the GABA and glycine transporter vesicular inhibitory amino acid transporter (VIAAT also known as VGAT) is present in rat islets. Here we identify a novel 52 kDa variant of VIAAT in rat islets: VIAAT-52 (V52). V52 is an amino-terminally truncated form of VIAAT (V57) that likely results from utilization of a downstream start site of translation. V57 and V52 display different patterns of post-translational modification and cellular expression. Our results have indicated that islet content of V52, but not V57, is responsive to changes in glucose concentration and other extracellular conditions. VIAAT is expressed in the islet alpha cells, but there have been conflicting findings regarding the presence of VIAAT in the beta cells. Here we have also provided additional evidence for the presence of VIAAT in islet beta cells and show that the beta cell line INS-1 expresses V57. V52 may be better adapted than V57 to the unique rat alpha cell GAD-GABA system, which lacks GAD65 and in which VIAAT traffics to secretory granules rather than just to synaptic microvesicles.

Alternative splicing of GAD67 results in the synthesis of a third form of glutamic-acid decarboxylase in human islets and other non-neural tissues.

Two forms of glutamic-acid decarboxylase (GAD) have been identified in mammalian tissues: a 65-kDa form (GAD65) and a 67-kDa form (GAD67). Alternate splicing produces one or two smaller variants of GAD67 in the brain of embryonic mice and rats. Additionally, a short, heretofore unidentified transcript homologous to GAD67 has been detected in human testis RNA. Because GAD, the enzyme responsible for gamma-aminobutyric acid production and a key autoantigen in type I diabetes, has unclear function in non-neural tissue, it is important to understand its pattern of expression. Unlike GAD65, GAD67 is not produced in human pancreatic islets. Here, we describe a novel splice variant of GAD67 that is produced in human islets, testis, adrenal cortex, and perhaps other endocrine tissues, but not in brain. This transcript directs the synthesis of a protein without GAD enzymatic activity: GAD25. A unique peptide sequence at the carboxyl terminus of GAD25 is highly conserved between mice, rats, and humans. We conclude that humans produce a third form of GAD in non-neural tissues and that human islets, although they do not synthesize full-length GAD67, do express this shortened variant.

Association between baseline plasma leptin levels and subsequent development of diabetes in Japanese Americans.

OBJECTIVE: Plasma leptin levels correlate strongly with increased total adipose tissue, a known risk factor for type 2 diabetes, yet the role of leptin in the etiology of diabetes remains unclear. We sought to determine whether leptin is a risk factor for development of diabetes in Japanese Americans. RESEARCH DESIGN AND METHODS: We compared baseline leptin levels in 370 nondiabetic Japanese Americans who remained nondiabetic for 5-6 years of follow-up with those of 40 nondiabetic Japanese Americans who developed diabetes during follow-up. All participants had computed tomography measurements of baseline subcutaneous chest, abdomen, thigh, and intra-abdominal fat, with total fat defined as the sum of all these measurements. RESULTS: The mean age was 51.7 +/- 11.7 years for men and 51.9 +/- 12.0 years for women. The 23 men who developed diabetes had significantly higher leptin levels than the 212 men who remained nondiabetic (P < 0.01). Among men, baseline leptin levels predicted diabetes risk independent of baseline total fat, insulin, insulin resistance, glucose, or age in separate multiple logistic regression models (relative risk adjusted for baseline total fat = 1.80 per SD increase [2.7 ng/ml], 95% CI 1.02-3.17). This association was particularly strong among men in the top decile for intra-abdominal fat. In contrast, the 17 women who developed diabetes had leptin levels similar to those of the 158 women who remained nondiabetic (P = 0.31). CONCLUSIONS: Among Japanese Americans, increased baseline leptin levels are associated with increased risk of developing diabetes in men but not in women.

Increased plasma leptin levels are associated with fat accumulation in Japanese Americans.

Although the hormone leptin seems to play a role in ensuring the maintenance of adequate energy stores and thereby protects against starvation, its role in the regulation of body weight and adiposity under normal circumstances is unclear. Overweight individuals have markedly elevated circulating leptin levels, suggesting that leptin's effect on food intake and thermogenesis is diminished or absent in obesity. Recent evidence, though, indicates that weight gain in Pima Indians is associated with relatively decreased levels of the hormone. Because it is important to understand whether a deficiency in circulating leptin contributes to the development of obesity, we sought to determine whether there is a relationship between leptin levels and subsequent changes in adiposity in a more typical population. We compared baseline plasma leptin concentrations to changes over 5 years in body weight, BMI, and computed tomography-determined total fat in 492 second- and third-generation Japanese Americans. Subjects were of 100% Japanese ancestry; male subjects had a mean BMI at baseline of 25.4 kg/m2 and a mean age of 54 years; female subjects had a mean BMI of 23.1 kg/m2 and a mean age of 53 years. Changes in weight (men: r = 0.17, P < 0.05; women: r = 0.20, P < 0.05), BMI (men: r = 0.17, P < 0.05; women: r = 0.18, P < 0.05), and total fat (men: r = 0.19, P < 0.05; women: r = 0.20, P < 0.01) were positively correlated with baseline leptin levels adjusted for baseline adiposity, fasting insulin, and age. In Japanese Americans, then, relatively increased leptin levels are associated with greater subsequent gains in weight and adiposity. We concluded that in this population, fat accumulation is associated not with leptin deficiency but possibly with leptin resistance and is preceded by increased leptin levels.

Endoplasmic reticulum-mediated quality control of type I collagen production by cells from osteogenesis imperfecta patients with mutations in the pro alpha 1 (I) chain carboxyl-terminal propeptide which impair subunit assembly.

A heterozygous single base change in exon 49 of COL1A1, which converted the codon for pro alpha 1(I) carboxyl-terminal propeptide residue 94 from tryptophan (TGG) to cysteine (TGT) was identified in a baby with lethal osteogenesis imperfecta (OI64). The C-propeptide mutations in OI64 and in another lethal osteogenesis imperfecta cell strain (OI26), which has a frameshift mutation altering the sequence of the carboxyl-terminal half of the propeptide (Bateman, J. F., Lamande, S. R., Dahl, H.-H. M., Chan, D., Mascara, T. and Cole, W. G. (1989) J. Biol. Chem. 264, 10960-10964), disturbed procollagen folding and retarded the formation of disulfide-linked trimers. Although assembly was delayed, the presence of slowly migrating, overmodified alpha 1(I) and alpha 2(I) chains indicated that mutant pro alpha 1(I) could associate with normal pro alpha 1(I) and pro alpha 2(I) to form pepsin-resistant triple-helical molecules, a proportion of which were secreted. Further evidence of the aberrant folding of mutant procollagen in OI64 and OI26 was provided by experiments demonstrating that the endoplasmic reticulum resident molecular chaperone BiP, which binds to malfolded proteins, was specifically bound to type I procollagen and was coimmunoprecipitated in the osteogenesis imperfecta cells but not control cells. Experiments with brefeldin A, which inhibits protein export from the endoplasmic reticulum, demonstrated that unassembled mutant pro alpha 1(I) chains were selectively degraded within the endoplasmic reticulum resulting in reduced collagen production by the osteogenesis imperfecta cells. This biosynthetic deficiency was reflected in the inability of OI64 and OI26 cells to produce a substantial in vitro collagenous matrix when grown in the continuous presence of ascorbic acid to allow collagen matrix formation. Both these carboxyl-terminal propeptide mutants showed a marked reduction in collagen accumulation to 20% (or less) of control cultures, comparable to the reduced collagen content of tissues from OI26.

Mutations in the carboxyl-terminal propeptide of the pro alpha 1(I) chain of type I collagen result in defective chain association and produce lethal osteogenesis imperfecta.

Fibroblast cell strains from three infants with perinatal lethal osteogenesis imperfecta (OI) carry unique mutations in COL1A1 (the gene encoding the pro alpha 1(I) chain of type I procollagen) that impair chain association. The three mutations are: substitution of arginine for leucine at position 170 in the carboxyl-terminal propeptide, substitution of histidine for aspartic acid at position 59 of the propeptide, and a 6-base pair deletion that results in loss of 2 amino acid residues, glutamic acid and tyrosine, at positions 119 and 120 of the propeptide. In each cell strain the rate of association of the abnormal chain into molecules is slowed and formation of interchain disulfide bonds is impaired. The substitution at residue 59 alters the most highly conserved region of the propeptide and has a much greater effect on the incorporation of pro alpha 2(I) chains into disulfide-bonded trimers than the other two mutations. The molecules that contain the abnormal chains are extensively overmodified by post-translational modifying enzymes, yet have a normal thermal stability. These findings extend the range of lethal mutations in the type I collagen genes and help to identify regions of the carboxyl-terminal propeptide that may be important for chain-chain recognition and molecular assembly.

BiP binds type I procollagen pro alpha chains with mutations in the carboxyl-terminal propeptide synthesized by cells from patients with osteogenesis imperfecta.

Of 20 fibroblast cell strains from patients with osteogenesis imperfecta (OI), a disease caused by mutations in the genes encoding type I procollagen, three had increased synthesis of BiP (GRP78), an hsp70-related, endoplasmic reticulum-resident protein. All three strains carry unique mutations in pro alpha 1(I) chains which impair type I procollagen chain association. Immunoprecipitation and pulse-chase experiments show that BiP (immunoglobulin heavy chain-binding protein) stably binds pro alpha 1(I) chains in these three cell strains after a brief lag. Ascorbate, which increases procollagen synthesis, increases BiP synthesis and content in these three strains and not in the others. In one of these three strains, BiP content is constitutively elevated prior to ascorbate treatment, and BiP is less inducible. This strain also has relatively high levels of synthesis and content of GRP94, another endoplasmic reticulum-resident stress protein. Pretreating each of the three cell strains to increase their BiP content reduces subsequent ascorbate-mediated BiP induction. BiP synthesis in the 17 other OI strains examined, which had a variety of type I procollagen mutations, was normal. These results suggest that BiP is induced by and binds procollagen with specific types of mutations: ones in the carboxyl-terminal propeptide that interfere with chain association. The recognition by BiP of such procollagen in OI cell strains shows that BiP plays a role in the physiological response to the production of some disease-producing abnormal proteins.

Defective folding and stable association with protein disulfide isomerase/prolyl hydroxylase of type I procollagen with a deletion in the pro alpha 2(I) chain that preserves the Gly-X-Y repeat pattern.

We have studied the folding, processing, and association with two endoplasmic reticulum (ER) resident proteins of the abnormal type I procollagen molecules produced by a strain of fibroblasts harboring a 4.5 kilobase deletion in an allele of COL1A2 (Willing, M. C., Cohn, D.H., Starman, B. Holbrook, K.A., Greenberg, C.R., and Byers, P.H. (1988) J. Biol. Chem. 263, 8398-8404). By sequencing cDNA, we found that the mutant allele encodes pro alpha 2(I) chains that are shortened by 180 amino acids but retain the Gly-X-Y repeat pattern crucial for collagen triple helix formation. The type I procollagen molecules that incorporated the shortened chain were retained intracellularly and were stable. The triple helical domain in these molecules did not attain a normal conformation and remained accessible to posttranslational modifying enzymes amino-terminal to the deletion site for a prolonged period. The abnormal molecules folded into a triple helical conformation more slowly than the normal molecules, and the amino-terminal ends of the pro alpha 1(I) chains failed to become protease-resistant. While the abnormal procollagen molecules were not bound by the ER-resident protein BiP, they stably associated with protein disulfide isomerase, the beta-subunit of prolyl-4-hydroxylase. These results indicate that some mutations in type I collagen genes both transiently delay folding and permanently disrupt the structure of the triple helix and suggest that binding to prolyl-4-hydroxylase helps to retain certain abnormal procollagen molecules within the ER.