Arthur Suckow, Ph.D.

GPR40 (FFAR1) and GPR120 (FFAR4) are G-protein coupled receptors (GPCRs) that are activated by long chain fatty acids (LCFAs). GPR40 is expressed at high levels in islets and mediates the ability of LCFAs to potentiate glucose-stimulated insulin secretion (GSIS). GPR120 is expressed at high levels in colon, adipose and pituitary, and at more modest levels in pancreatic islets. The role of GPR120 in islets has not been explored extensively. Here, we confirm that saturated (e.g. palmitic acid)… Show more

GPR40 (FFAR1) and GPR120 (FFAR4) are G-protein coupled receptors (GPCRs) that are activated by long chain fatty acids (LCFAs). GPR40 is expressed at high levels in islets and mediates the ability of LCFAs to potentiate glucose-stimulated insulin secretion (GSIS). GPR120 is expressed at high levels in colon, adipose and pituitary, and at more modest levels in pancreatic islets. The role of GPR120 in islets has not been explored extensively. Here, we confirm that saturated (e.g. palmitic acid) and unsaturated (e.g. docosahexaenoic acid (DHA)) LCFAs engage GPR120 and demonstrate that palmitate- and DHA-potentiated glucagon secretion are greatly reduced in isolated GPR120 KO islets. Remarkably, LCFA potentiated glucagon secretion is similarly reduced in GPR40 KO islets. Compensatory changes in mRNA expression of GPR120 in GPR40 KO islets, and vice versa, do not explain that LCFA potentiated glucagon secretion seemingly involves both receptors. LCFA-potentiated GSIS remains intact in GPR120 KO islets. Consistent with previous reports, GPR120 KO mice are hyperglycemic and glucose intolerant; however, our KO mice display evidence of a hyperactive counter-regulatory response rather than insulin resistance during insulin tolerance tests. An arginine stimulation test and a glucagon challenge confirmed both increases in glucagon secretion and liver glucagon sensitivity in GPR120 KO mice relative to WT mice. Our findings demonstrate that GPR120 is a nutrient sensor that is activated endogenously by both saturated and unsaturated long chain fatty acids and that an altered glucagon axis likely contributes to the impaired glucose homeostasis observed in GPR120 KO mice. Show less

Pancreatic islet beta-cells contain synaptic-like microvesicles (SLMVs). The origin, trafficking, and role of these SLMVs are poorly understood. In neurons, synaptic vesicle (SV) biogenesis is mediated by two different cytosolic adaptor protein complexes, a ubiquitous AP-2 complex and the neuron-specific AP-3B complex. Mice lacking AP-3B subunits exhibit impaired GABAergic (inhibitory) neurotransmission and reduced neuronal vesicular GABA transporter (VGAT) content. Since beta-cell maturation… Show more

Pancreatic islet beta-cells contain synaptic-like microvesicles (SLMVs). The origin, trafficking, and role of these SLMVs are poorly understood. In neurons, synaptic vesicle (SV) biogenesis is mediated by two different cytosolic adaptor protein complexes, a ubiquitous AP-2 complex and the neuron-specific AP-3B complex. Mice lacking AP-3B subunits exhibit impaired GABAergic (inhibitory) neurotransmission and reduced neuronal vesicular GABA transporter (VGAT) content. Since beta-cell maturation and exocytotic function seem to parallel that of the inhibitory synapse, we predicted that AP-3B-associated vesicles would be present in beta-cells. Here, we test the hypothesis that AP-3B is expressed in islets and mediates beta-cell SLMV biogenesis. A secondary aim was to test whether the sedimentation properties of INS-1 beta-cell microvesicles are identical to those of bona fide SLMVs isolated from PC12 cells. Our results show that the two neuron-specific AP-3 subunits beta3B and mu3B are expressed in beta-cells, the first time these proteins have been found to be expressed outside the nervous system. We found that beta-cell SLMVs share the same sedimentation properties as PC12 SLMVs and contain SV proteins that sort specifically to AP-3B-associated vesicles in the brain. Brefeldin A, a drug that interferes with AP-3-mediated SV biogenesis, inhibits the delivery of AP-3 cargoes to beta-cell SLMVs. Consistent with a role for AP-3 in the biogenesis of GABAergic SLMV in beta-cells, INS-1 cell VGAT content decreases upon inhibition of AP-3 delta-subunit expression. Our findings suggest that beta-cells and neurons share molecules and mechanisms important for mediating the neuron-specific membrane trafficking pathways that underlie synaptic vesicle formation.

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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… Show more

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.

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The metastatic potential of cancer cells is directly attributed to their ability to invade through the extracellular matrix. The mechanisms regulating this cellular invasiveness are poorly understood. Here, we show that junctional adhesion molecule A (JAM-A), a tight junction protein, is a key negative regulator of cell migration and invasion. JAM-A is robustly expressed in normal human mammary epithelium, and its expression is down-regulated in metastatic breast cancer tumors. In breast cancer… Show more

The metastatic potential of cancer cells is directly attributed to their ability to invade through the extracellular matrix. The mechanisms regulating this cellular invasiveness are poorly understood. Here, we show that junctional adhesion molecule A (JAM-A), a tight junction protein, is a key negative regulator of cell migration and invasion. JAM-A is robustly expressed in normal human mammary epithelium, and its expression is down-regulated in metastatic breast cancer tumors. In breast cancer cell lines, an inverse relationship between JAM-A expression and the ability of these cells to migrate on a collagen matrix was observed, which correlates with the known ability of these cells to metastasize. The T47D and MCF-7 cells, which migrate least, are found to express high levels of JAM-A, whereas the more migratory MDA-MB-468 cells have lower levels of JAM-A on the cell surface. MDA-MB-231 cells, which are highly migratory, express the least amount of JAM-A. Overexpression of JAM-A in MDA-MB-231 cells inhibited both migration and invasion through collagen gels. Furthermore, knockdown of JAM-A using short interfering RNAs enhanced the invasiveness of MDA-MB-231 cells as well as T47D cells. The ability of JAM-A to attenuate cell invasion correlated with the formation of increased numbers of focal adhesions and the formation of functional tight junctions. These results show for the first time that an immunoglobulin superfamily cell adhesion protein expressed at tight junctions could serve as a key negative regulator of breast cancer cell invasion and possibly metastasis. Furthermore, loss of JAM-A could be used as a biomarker for aggressive breast cancer.

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The enzyme glutamate decarboxylase-65 (GAD65) is a major autoantigen in autoimmune diabetes. The mechanism whereby autoreactivity to GAD65, an intracellular protein, is triggered is unknown, and it is possible that immunoreactive GAD65 is released by injured pancreatic islet beta-cells. There is a great need for methods by which to detect and monitor ongoing islet injury. If GAD65 were released and, furthermore, were able to reach the circulation, it could function as a marker of beta-cell… Show more

The enzyme glutamate decarboxylase-65 (GAD65) is a major autoantigen in autoimmune diabetes. The mechanism whereby autoreactivity to GAD65, an intracellular protein, is triggered is unknown, and it is possible that immunoreactive GAD65 is released by injured pancreatic islet beta-cells. There is a great need for methods by which to detect and monitor ongoing islet injury. If GAD65 were released and, furthermore, were able to reach the circulation, it could function as a marker of beta-cell injury. Here, a novel GAD65 plasma immunoassay is used to test the hypotheses that beta-cell injury induces GAD65 discharge in vivo and that discharged GAD65 reaches the bloodstream. Plasma GAD65 levels were determined in rats treated with alloxan, and with diabetogenic and low, subdiabetogenic doses of streptozotocin. beta-Cell injury resulted in GAD65 release into the circulation in a dose-dependent manner, and low-dose streptozotocin resulted in a more gradual increase in plasma GAD65 levels than did diabetogenic doses. Plasma GAD65 levels were reduced in rats that had undergone partial pancreatectomy and remained undetectable in mice. Together, these data demonstrate that GAD65 can be released into the circulation by injured beta-cells. Autoantigen shedding may contribute to the pathogenesis of islet autoimmunity in the multiple low-dose streptozocin model and perhaps, more generally, in other forms of autoimmune diabetes. These results demonstrate that, as is true with other tissues, islet injury, at least in some circumstances, can be monitored by use of discharged, circulating proteins. GAD65 is the first such confirmed protein marker of islet injury.

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BACKGROUND:Glutamic acid decarboxylase-65 (GAD65) is a major autoantigen in autoimmune diabetes and is discharged from injured islet beta cells.GAD65 may also be released by transplanted islets undergoing immunological rejection.To test hypotheses regarding the utility of GAD65 as a biomarker for transplant rejection or diabetes-associated islet damage and also regarding the timing and instigators of GAD65 release in humans or animal models, a sensitive assay capable of measuring GAD65 in serum… Show more

BACKGROUND:Glutamic acid decarboxylase-65 (GAD65) is a major autoantigen in autoimmune diabetes and is discharged from injured islet beta cells.GAD65 may also be released by transplanted islets undergoing immunological rejection.To test hypotheses regarding the utility of GAD65 as a biomarker for transplant rejection or diabetes-associated islet damage and also regarding the timing and instigators of GAD65 release in humans or animal models, a sensitive assay capable of measuring GAD65 in serum or plasma will be necessary.Ideally, this assay would also be resistant to interference by anti-GAD65 autoantibodies.
METHODS:A novel,magnetic bead-based assay was developed based on GAD65 capture by a monoclonal antibody directed to the only region of the protein known not to be significantly targeted by autoantibodies.A subsequent denaturation step allows sensitive immunodetection to proceed using anti-GAD65 polyclonal antibodies that would otherwise potentially be blocked by bound autoantibodies.
RESULTS:The GAD65 assay worked equally well with serum and plasma as with a solution of bovine serum albumin(BSA).The limit of blank was 31 pg/mL and did not differ significantly in the BSA solution (27 pg/mL).Mean recovery of GAD65 from the plasma of control subjects and GAD65 autoantibody-positive and -negative subjects with type 1 diabetes was 101 +/- 4.6%, 88 +/- 7.8%, and 99 +/- 7.0% (+/- SEM), respectively. The assay was used to quantify both recombinant GAD65 and the GAD65 content of human and rodent islets and other tissue extracts that were added to human plasma samles.

CONCLUSIONS: A sensitive, autoantibody-resistant GAD65 assay has been developed that is compatible with detection in serum and plasma and therefore will likely also work with a variety of other biologic fluids. This assay may enable the use of circulating GAD65 as a biomarker of islet damage or transplant rejection and will facilitate in vivo studies of the pathogenesis of anti-GAD65 autoreactivity.
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Junctional adhesion molecule-A (JAM-A, JAM-1, F11R) is an Ig domain containing transmembrane protein that has been proposed to function in diverse processes including platelet activation and adhesion, leukocyte transmigration, angiogenesis, epithelial cell shape and endothelial cell migration although its function in vivo is less well established. In the mouse eye, JAM-A protein expression is first detected at 12.5 dpc in the blood vessels of the tunica vasculosa, while it is first detected in… Show more

Junctional adhesion molecule-A (JAM-A, JAM-1, F11R) is an Ig domain containing transmembrane protein that has been proposed to function in diverse processes including platelet activation and adhesion, leukocyte transmigration, angiogenesis, epithelial cell shape and endothelial cell migration although its function in vivo is less well established. In the mouse eye, JAM-A protein expression is first detected at 12.5 dpc in the blood vessels of the tunica vasculosa, while it is first detected in both the corneal epithelium and lens between 13.5 and 14.5 dpc. In the corneal epithelium, JAM-A levels remain appreciable throughout life, while JAM-A immunostaining becomes stronger in the lens as the animals age. Both the cornea and lens of mice lacking an intact JAM-A gene are transparent until at least a year of age, although the cells of the JAM-A null corneal epithelium are irregularly shaped. In wild-type mice, JAM-A protein is found at the leading edge of repairing corneal epithelial wounds, however, corneal epithelial wound repair was qualitatively normal in JAM-A null animals. In summary, JAM-A is expressed in the corneal epithelium where it appears to regulate cell shape.

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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… Show more

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.

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