aav 9 sst flp  (Addgene inc)

Journal: Diabetes

Article Title: Control of Insulin Secretion by Cholinergic Signaling in the Human Pancreatic Islet

doi: 10.2337/db13-1371

Figure Lengend Snippet: Human β-cells express functional M3 muscarinic receptors. M3 muscarinic receptors were present in human islets as detected by confocal microscopy of immunostained human pancreatic sections ( A ), Western blotting of lysates from five human islet preparations ( B ), and RT-PCR in human islets (I; n = 5) and brain (B) as a control ( C ). Molecular weight markers were run in parallel (shown is the 82-kDa marker). Scale bar, 20 μm. Confocal images of human pancreatic sections showing islets immunostained for M3 receptor ( D–F , green), glucagon ( D , red), somatostatin (Soma; E , red), or insulin ( G , red). Scale bars, 10 μm (in D applies to E and in F to G and H ). I : Traces of [Ca 2+ ] i responses in β-cells showing that responses to acetylcholine (ACh; 10 μmol/L) were inhibited in the presence of atropine (10 μmol/L). J : Quantification of results as in I shows that peak responses to ACh (Δ 340/380) were inhibited by atropine and the M3 receptor–specific antagonist J104129 (50 nmol/L), but not by the M1 receptor–specific antagonist MT7 (20 nmol/L) ( n = 4 islet preparations; Student t test, P < 0.05). K : Perifusion assay of insulin secretion showing that increases in insulin secretion induced by ACh were inhibited by J104129 (50 nmol/L). Quantification of [Ca 2+ ] i responses to ACh in the presence of thapsigargin ( L ) and in nominal 0 [Ca 2+ ] ( M ) ( n = 16 cells from three preparations; Student t test; P < 0.05). Au, arbitrary unit; RQ, relative quantification. Asterisks denote significance.

Article Snippet: Human whole islets were also transfected with an adenoviral construct driving expression of DsRed2 under the control of the somatostatin promoter (pAAV-fSST-RFP). pAAV-fSST-RFP was a gift from Edward Callaway (Addgene plasmid #22913).

Techniques: Functional Assay, Confocal Microscopy, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control, Molecular Weight, Marker, Quantitative Proteomics

Journal: Diabetes

Article Title: Control of Insulin Secretion by Cholinergic Signaling in the Human Pancreatic Islet

doi: 10.2337/db13-1371

Figure Lengend Snippet: Human β-cells express functional M5 muscarinic receptors. M5 muscarinic receptors were present in human islets as detected by confocal microscopy of immunostained human pancreatic sections ( A ), Western blotting of lysates from five human islet preparations ( B ), and RT-PCR in human islets (I; n = 5) and brain (B) as a control ( C ). Molecular weight markers were run in parallel (shown is the 60-kDa marker). Scale bar, 20 μm. Confocal images of human pancreatic sections showing islets immunostained for M5 receptor ( D–F , green), glucagon ( D , red), somatostatin (Soma; E , red), or insulin ( G , red). Scale bars, 10 μm (in D applies to E and in F to G and H ). I : Traces of [Ca 2+ ] i responses in β-cells showing responses to acetylcholine (ACh) alone (10 μmol/L, left ), in the presence of J104129 (50 nmol/L, middle ), and in the presence of J104129 and the M5 receptor modulator VU 0238429 (10 μmol/L, right ). J : Quantification of results as in I shows that peak responses to ACh (Δ 340/380) in the presence of J104129 were amplified by VU 0238429 ( n = 9 cells from three preparations; Student t test, P < 0.05). K : Perifusion assay of insulin secretion showing that increases in insulin secretion induced by ACh were amplified by VU 0365114, an allosteric modulator of M5 receptors (10 μmol/L). L : Quantification of results as in K shows that VU 0365114 increased insulin secretion stimulated by ACh ( n = 4 preparations; Student t test, P < 0.05). M : Quantification of perifusion assays of somatostatin secretion shows that VU 0365114 did not alter somatostatin secretion stimulated by ACh ( n = 3 preparations). Au/a.u., arbitrary unit; AUC, area under the curve; RQ, relative quantification. Asterisks denote significance.

Article Snippet: Human whole islets were also transfected with an adenoviral construct driving expression of DsRed2 under the control of the somatostatin promoter (pAAV-fSST-RFP). pAAV-fSST-RFP was a gift from Edward Callaway (Addgene plasmid #22913).

Techniques: Functional Assay, Confocal Microscopy, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control, Molecular Weight, Marker, Amplification, Quantitative Proteomics

Journal: Diabetes

Article Title: Control of Insulin Secretion by Cholinergic Signaling in the Human Pancreatic Islet

doi: 10.2337/db13-1371

Figure Lengend Snippet: Human δ-cells express functional M1 muscarinic receptors. M1 muscarinic receptors were present in human islets as detected by confocal microscopy of immunostained human pancreatic sections ( A ), Western blotting of lysates from five human islet preparations ( B ), and RT-PCR in human islets (I; n = 5) and brain (B) as a control ( C ). Molecular weight markers were run in parallel (shown is the 64-kDa marker). Scale bar, 20 μm. Confocal images of human pancreatic sections showing islets immunostained for M1 receptor ( D–F , green), insulin (Ins; D , red), glucagon ( E , red), or somatostatin (Soma; G , red). Scale bars, 10 μm (in D applies to E and in F to G and H ). Traces of [Ca 2+ ] i responses in δ-cells showing that responses to acetylcholine (ACh; 10 μmol/L) were inhibited in the presence of atropine (10 μmol/L; I ) and the M1 receptor–specific antagonist MT7 (20 nmol/L; J ). Quantification of results as in I and J shows that peak responses to ACh (Δ 340/380) were inhibited by atropine ( K ) and MT7 ( L ) ( n > 12 cells from three preparations; Student t test, P < 0.05). M : Perifusion assay of somatostatin secretion showing that basal somatostatin secretion at 3 mmol/L glucose concentration was inhibited by MT7 (20 nmol/L; n = 3 preparations). Au, arbitrary unit; RQ, relative quantification. Asterisks denote significance.

Article Snippet: Human whole islets were also transfected with an adenoviral construct driving expression of DsRed2 under the control of the somatostatin promoter (pAAV-fSST-RFP). pAAV-fSST-RFP was a gift from Edward Callaway (Addgene plasmid #22913).

Techniques: Functional Assay, Confocal Microscopy, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control, Molecular Weight, Marker, Concentration Assay, Quantitative Proteomics

Journal: Diabetes

Article Title: Control of Insulin Secretion by Cholinergic Signaling in the Human Pancreatic Islet

doi: 10.2337/db13-1371

Figure Lengend Snippet: Proposed model for paracrine cholinergic signaling in the human islet. Acetylcholine is released from α-cells and activates M1 receptors on δ-cells and M3 and M5 receptors on β-cells. Acetylcholine stimulates insulin secretion directly, but at the same time provides inhibition via somatostatin secretion from δ-cells. The net effect of acetylcholine on insulin secretion likely depends on the proximity of the different cells, the pharmacological properties of the different receptors, and glucose concentration. Not to be neglected is additional input from cholinergic innervation. PKC, protein kinase C.

Article Snippet: Human whole islets were also transfected with an adenoviral construct driving expression of DsRed2 under the control of the somatostatin promoter (pAAV-fSST-RFP). pAAV-fSST-RFP was a gift from Edward Callaway (Addgene plasmid #22913).

Techniques: Inhibition, Concentration Assay