Among Japanese Americans, increased baseline leptin levels are associated with increased risk of developing diabetes in men but not in women.

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.

The composition of the beta-cell exocytic machinery is very similar to that of neuronal synapses, and the developmental pathway of beta-cells and neurons substantially overlap. beta-Cells secrete gamma-aminobutyric acid and express proteins that, in the brain, are specific markers of inhibitory synapses. Recently, neuronal coculture experiments have identified three families of synaptic cell-surface molecules (neurexins, neuroligins, and SynCAM) that drive synapse formation in vitro and that control the differentiation of nascent synapses into either excitatory or inhibitory fully mature nerve terminals. The inhibitory synapse-like character of the beta-cells led us to hypothesize that members of these families of synapse-inducing adhesion molecules would be expressed in beta-cells and that the pattern of expression would resemble that associated with neuronal inhibitory synaptogenesis. Here, we describe beta-cell expression of the neuroligins, neurexins, and SynCAM, and show that neuroligin expression affects insulin secretion in INS-1 beta-cells and rat islet cells. Our findings demonstrate that neuroligins and neurexins are expressed outside the central nervous system and help confer an inhibitory synaptic-like phenotype onto the beta-cell surface. Analogous to their role in synaptic neurotransmission, neurexin-neuroligin interactions may play a role in the formation of the submembrane insulin secretory apparatus.

Background:The cell surface, transmembrane protein neurexin may play a role in insulin secretion. Results: Knockdown and knock-out of neurexin-1␣, the predominant ␤-cell isoform, increased insulin secretion and impaired secretory granule docking. Conclusion: Neurexin-1␣ helps mediate insulin granule docking and thereby constrains secretion. Significance: Neurexin-1␣ could couple localization and functioning of the insulin docking and secretory machinery to extracellular protein interactions.

␥-Aminobutyric acid (GABA) is stored in microvesicles in pancreatic islet cells. Because GAD65 and GAD67, which catalyze the formation of GABA, are cytoplasmic, the existence of an islet vesicular GABA transporter has been postulated. Here, we test the hypothesis that the putative transporter is the vesicular inhibitory amino acid transporter (VIAAT), a neuronal transmembrane transporter of GABA and glycine. We sequenced the human VIAAT gene and determined that the human and rat proteins share over 98% sequence identity. In vitro expression of VIAAT and immunoblotting of brain and islet lysates revealed two forms of the protein: an ϳ52-kDa and an ϳ57-kDa form. By immunoblotting and immunohistochemistry, we detected VIAAT in rat but not human islets. Immunohistochemical staining showed that in rat islets, the distribution of VIAAT expression parallels that of GAD67, with increased expression in the mantle. GABA, too, was found to be present in islet non-␤-cells. We conclude that VIAAT is expressed in rat islets and is more abundant in the mantle and that expression in human islets is very low or nil. The rat islet mantle differs from rat and human ␤-cells in that it contains only GAD67 and relatively increased levels of VIAAT. Cells that express only GAD67 may require higher levels of VIAAT expression. Diabetes 51:1763-1771, 2002 E xpression of GAD is a characteristic of the islets of Langerhans. GAD is produced in a number of different tissues but is most abundant in brain and pancreatic islets (1). There are two major isoforms of the enzyme: GAD65 and GAD67. For unknown reasons, islet expression of the two isoforms differs markedly in different animals. Rat islets express both GAD65 and GAD67. In contrast, human islets contain only GAD65 (2); GAD67 protein is not detectable, and GAD25, a nonenzymatically active GAD67 splice variant (3), is present in a relatively sparse subset of cells (S.D.C., W.T.S., unpublished observations) (2,4).GAD65 and GAD67 catalyze the formation of the neurotransmitter ␥-aminobutyric acid (GABA) from glutamate. The role of GABA in the islet is unclear; there is evidence that points to roles as a paracrine regulator of glucagon and somatostatin release, as a metabolic intermediary, and as an inhibitor of first-phase insulin release (4 -6). GAD65 is a major autoantigen in type 1 diabetes (2). GAD65 is primarily membrane-associated and targeted to the cytoplasmic surface of the synaptic-like microvesicle (SLMV), a secretory organelle found in endocrine cells that is the counterpart of the neuronal synaptic vesicle (7). GAD67 associates to a lesser extent with the SLMV; it is mostly distributed homogeneously throughout the cytosol (8).GABA accumulates within the SLMV, from which it is probably secreted in its role as a signaling molecule (6,9). Because neither GAD65 nor GAD67 is a transmembrane protein, another protein likely mediates GABA entry into the SLMV. Experiments using isolated microvesicles from a mouse ␤-cell line have provided evidence of such a SLMV GABA transporter. The micr...

Background:The synaptic protein neuroligin-2 is present on the pancreatic ␤ cell surface and affects insulin secretion. Results: Insulin secretion increases when ␤ cells contact adjacent cells expressing neuroligin; soluble neuroligin inhibits secretion. Conclusion: Clustered neuroligin-2 enhances insulin secretion in a transcellular manner, likely by promoting secretory microdomain formation. Significance: Transcellular protein-protein interactions paralleling the transsynaptic interactions that drive synapse formation may guide ␤ cell functional maturation.

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.

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 ␥-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.