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GRP94 KD/KO causes proinsulin misprocessing and increased cellular turnover...
Published: 22 January 2019
Figure 5 GRP94 KD/KO causes proinsulin misprocessing and increased cellular turnover. A: Control (Ctrl) and GRP94 KD INS-1E cells were cultured for 3 h in 2 mmol/L glucose containing Krebs-Ringer bicarbonate HEPES (KRBH) buffer and lysed. In order to preserve intramolecular disulfide bonds and visualize proinsulin folding intermediates, cell lysates were analyzed by nonreducing SDS-PAGE (Nu-Page 4–12% Bis-Tris Protein Gel), and proinsulin and insulin were visualized through WB with antiproinsulin and anti-insulin antibody (L6B10; Cell Signaling); n = 3. B: INS-1E Ctrl or GRP94 KO cells were pulse labeled with 35S-Met/Cys for 1 h (C0) and chased for 1 h (C1 and M1) in 11 mmol/L glucose. Cell lysates and culture supernatants were immunoprecipitated with anti-insulin antibody, and newly synthesized proinsulin was analyzed by nonreducing Tris-tricine-urea SDS-PAGE and phosphorimaging. Numbers 1–4 identify proinsulin 1 and 2 conversion intermediates. C, cells; M, media. n = 3. C: INS-1E Ctrl and GRP94 KD cells were cultured for 3 h in 2 mmol/L glucose–containing KRBH buffer, and subsequently 100 μmol/L of the protein synthesis inhibitor cycloheximide was added and cells were lysed at the indicated time points; n = 3. D: Ctrl and GRP94 KO cells were treated similarly, but 200 nmol/L of the inhibitor of exocytosis brefeldin A was added to the culture media from the start of the experiment. Cell lysates were analyzed via reducing SDS-PAGE, and proinsulin and insulin were visualized through WB with antiproinsulin antibody; n = 3. Quantification of proinsulin bands was performed with ImageJ and normalized to tubulin bands (C and D, right panels). The quantification data are presented as means ± SEM analyzed by Bonferroni-corrected paired Student t test of treatments vs. Ctrl. AUC, area under the curve. Figure 5. GRP94 KD/KO causes proinsulin misprocessing and increased cellular turnover. A: Control (Ctrl) and GRP94 KD INS-1E cells were cultured for 3 h in 2 mmol/L glucose containing Krebs-Ringer bicarbonate HEPES (KRBH) buffer and lysed. In order to preserve intramolecular disulfide bonds and visualize proinsulin folding intermediates, cell lysates were analyzed by nonreducing SDS-PAGE (Nu-Page 4–12% Bis-Tris Protein Gel), and proinsulin and insulin were visualized through WB with antiproinsulin and anti-insulin antibody (L6B10; Cell Signaling); n = 3. B: INS-1E Ctrl or GRP94 KO cells were pulse labeled with 35S-Met/Cys for 1 h (C0) and chased for 1 h (C1 and M1) in 11 mmol/L glucose. Cell lysates and culture supernatants were immunoprecipitated with anti-insulin antibody, and newly synthesized proinsulin was analyzed by nonreducing Tris-tricine-urea SDS-PAGE and phosphorimaging. Numbers 1–4 identify proinsulin 1 and 2 conversion intermediates. C, cells; M, media. n = 3. C: INS-1E Ctrl and GRP94 KD cells were cultured for 3 h in 2 mmol/L glucose–containing KRBH buffer, and subsequently 100 μmol/L of the protein synthesis inhibitor cycloheximide was added and cells were lysed at the indicated time points; n = 3. D: Ctrl and GRP94 KO cells were treated similarly, but 200 nmol/L of the inhibitor of exocytosis brefeldin A was added to the culture media from the start of the experiment. Cell lysates were analyzed via reducing SDS-PAGE, and proinsulin and insulin were visualized through WB with antiproinsulin antibody; n = 3. Quantification of proinsulin bands was performed with ImageJ and normalized to tubulin bands (C and D, right panels). The quantification data are presented as means ± SEM analyzed by Bonferroni-corrected paired Student t test of treatments vs. Ctrl. AUC, area under the curve. More
Meeting Abstracts
Journal: Diabetes
Diabetes 2001;50(suppl_1):S125
Published: 01 February 2001
... Bio-Gel P2, and perifused at 200 µl/min at 37°C. -Cells were Pasteur pipette. The digestion was stopped by adding cold Krebs-Ringer bicar- first equilibrated for 20 min in KRBH 0.7% FFA-BSA containing basal glucose bonate-HEPES buffer (KRBH) containing 140 mmol/l NaCl, 3.6 mmol/l KCl, (2.8 mmol/l...
Journal Articles
Journal: Diabetes
Diabetes 2004;53(suppl_3):S79–S83
Published: 01 December 2004
... then washed twice and preincubated at 37°C for 1 h in 1 ml glucose-free Krebs-Ringer bicarbonate HEPES buffer (KRBH) containing (in mmol/l) 135 NaCl, 3.6 KCl, 5 NaHCO3, 0.5 Na H2PO4, 0.5 MgCl2, 1.5 CaCl2, 10 HEPES, and 0.1% BSA 0.1% (glucose free and FFA...
Journal Articles
Journal: Diabetes
Diabetes 2001;50(10):2296–2300
Published: 01 October 2001
... at 37°C in a Krebs-Ringer bicarbonate HEPES buffer (KRBH), pH 7.4, containing 10 mmol/l NaHCO3, 30 mmol/l HEPES, 1% (w/v) bovine serum albumin (BSA) (fraction V; Intergen, Purchase, NY) and 200 nmol/l adenosine as previously described ( 14 ). The cells were then washed three times with KRBH...
Journal Articles
Journal: Diabetes
Diabetes 2006;55(4):1029–1033
Published: 01 April 2006
... in rat pancreatic islets. Am J Physiol   287 : E828 –E833, 2004 DIABETES 2006 DMA, dimethylamiloride HBSS, Hanks’ balanced salt solution KRBH, Krebs-Ringer bicarbonate HEPES buffer nNOS, neuronal nitric oxide synthase pHi, intracellular pH TDI, time-dependent inhibition TDP...
Journal Articles
Journal: Diabetes
Diabetes 1997;46(9):1414–1418
Published: 01 September 1997
... stored at-20°C Kakizaki; KRBH, Krebs-Ringer bicarbonate-HEPES buffer; MODY, maturity- onset diabetes of the young; PFK, phosphofructokinase; RIA, radioim- munoassay; Tg, transgenic. 1414 DIABETES, VOL. 46, SEPTEMBER 1997 H. KNOBLER AND ASSOCIATES until used for immunoreactive insulin measurements...
Journal Articles
Journal: Diabetes
Diabetes 2001;50(9):1970–1975
Published: 01 September 2001
... and preincubated for 30 min in Krebs-Ringer bicarbonate buffer with 10 mmol/l HEPES (KRBH; pH 7.4) containing 0.07% bovine serum albumin (BSA; Fraction V RIA grade; Sigma, St. Louis, MO) at 3 mmol/l glucose. Groups of 10 islets (6 replicas per genotype) were distributed in 12-well plates and submitted to three...
Journal Articles
Journal: Diabetes
Diabetes 2004;53(12):3152–3158
Published: 01 December 2004
... metabolism ( 12 ), effects on phosphofructokinase and hexokinase ( 13 ), and inhibition of proinsulin biosynthesis ( 14 ). DAG, diacylglycerol α-KIC, α-ketoisocaproic acid KRBH, Krebs-Ringer bicarbonate HEPES buffer PKC, protein kinase C PLD, phospholipase D TDP, time-dependent potentiation...
Journal Articles
Journal: Diabetes
Diabetes 1994;43(3):375–383
Published: 01 March 1994
... factor II; STZ, streptozocin; BSA, bovine serum phorylation of the cytosolic portion of the p-subunit on albumin; PMSF, phenylmethylsulfonyl fluoride; WGA, wheat germ agglutinin; tyrosine residues (15-18). The tyrosine-autophosphory- tV2, half-time; KRBH, Krebs-Ringer bicarbonate-HEPES buffer; KRH, KRBH...
Journal Articles
Journal: Diabetes
Diabetes 2001;50(2):233–240
Published: 01 February 2001
..., both 3T3-L1 adipocytes and C2C12 myotubes were serum starved for 3 h in Krebs-Ringer bicarbonate buffer solution with 25 mmol/l Krebs-Ringer bicarbonate HEPES buffer (KRBH)(pH 7.4) containing 100 μmol/l bovine serum albumin (BSA) (Sigma, St. Louis, MO) essentially fatty acid free, and 5 mmol/l glucose...
Journal Articles
Journal: Diabetes
Diabetes 2002;51(1):105–113
Published: 01 January 2002
..., IN). Pancreatic islets were isolated according to the collagenase digestion method described by Lacy and Kostianovsky ( 21 ), with slight modifications. The animals were fed ad libitum until they were killed. Krebs-Ringer bicarbonate (KRB) solution of pH 7.4 buffered with 10 mmol/l HEPES (KRBH) was used...
Journal Articles
Journal: Diabetes
Diabetes 2006;55(5):1413–1420
Published: 01 May 2006
..., horseradish peroxidase KRBH, Krebs-Ringer bicarbonate HEPES buffer The extracellular matrix plays a critical role in modulating epithelial cell morphology, growth, migration, and differentiation. The response of cells to extracellular matrix attachment is mediated primarily by the integrin family...
Journal Articles
Journal: Diabetes
Diabetes 1998;47(2):255–262
Published: 01 February 1998
... min at 37°C. The embryonic cells were resuspended in Krebs- cence, whereas the addition of alloxan or H2O2 to cells gave Ringer bicarbonate HEPES buffer (KRBH) containing 1 mmol/1 glucose and 100 a strongfluorescence(results not shown). Moreover, addition umol/1 DCFH-DA (Acros Organics, Pittsburgh, PA...
Journal Articles
Journal: Diabetes
Diabetes 1997;46(9):1424–1433
Published: 01 September 1997
... channel, ATP-sensitive K+ channel; KRBH, Krebs-Ringer bicarbonate-HEPES buffer; MAP, mitogen-activated protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5 tetra- zolium bromide; NGF, nerve growth factor; PMA, phorbol 12-myristate 13- acetate; PKA, protein kinase A; PKC, protein kinase C; VDCC, voltage...
Journal Articles
Journal: Diabetes
Diabetes 2004;53(12):3142–3151
Published: 01 December 2004
..., ATP-sensitive K+ channel KRBH, Krebs-Ringer bicarbonate HEPES buffer RIA, radioimmunoassay SPA, [d-Arg1, d-Phe5, d-Trp7,9, Leu11]-substance P TEA, tetraethylammonium Ghrelin, a novel acylated 28-amino acid...
Journal Articles
Journal: Diabetes
Diabetes 2006;55(10):2826–2834
Published: 01 October 2006
... chromatography KATP channel, ATP-sensitive K+ channel KRBH, Krebs-Ringer bicarbonate HEPES buffer PIP, phosphatidylinositol phosphate SulfLacCer, sulfated lactosyl ceramide TLC, thin-layer chromatography Sulfatide is a glycosphingolipid present in mammalian organisms ( 1...
Journal Articles
Journal: Diabetes
Diabetes 2005;54(6):1808–1815
Published: 01 June 2005
... 26 1 2005 DIABETES 2005 FACS, fluorescence-activated cell sorter GLP-1, glucagon-like peptide 1 KATP channel, ATP-sensitive K+ channel KRBH, Krebs-Ringer bicarbonate HEPES buffer SUR, sulfonylurea receptor Glucagon, a hormone secreted from α-cells in the pancreatic...
Meeting Abstracts
Journal: Diabetes
Diabetes 2000;49(6):981–991
Published: 01 June 2000
...- dotransferase; GlcN, glucosamine; GlcN-6-P, glucosamine-6-phosphate; HPLC, high-pressure liquid chromatography; HSP, hexosamine biosynthetic pathway; IRS, insulin receptor substrate; KRBH, Krebs-Ringer bicarbonate/HEPES buffer; LDM, low-density microsomal; PBS, phosphate-buffered saline; PCA, per- chloric acid...
Journal Articles
Journal: Diabetes
Diabetes 1997;46(11):1684–1690
Published: 01 November 1997
... antibody; a-PY, antiphosphotyrosine antibody; mmol/1 MgSO4. Krebs-Ringer bicarbonate HEPES (KRBH) buffer, pH 7.4, con- CytPTK, cytosolic protein tyrosine kinase; HDM, high-density microsomes; sisted of 117 mmol/1 NaCl, 10 mmol/1 NaHCO3,1 mmol/1 CaCl2,1 mmol/1 MgSO4, HPLC, high-performance liquid...
Journal Articles
Journal: Diabetes
Diabetes 1991;40(2):290–294
Published: 01 February 1991
... ± 1.6t 128 ± 13t 5.1 ± 0.7 20.0 ± 2.3 27.6 ± 1.9 0 100 5.5 ± 0.9 118 ± 7.9* 6.0 ± 0.9 19.5 ± 2.8 30.8 ± 1.3 25 100 6.1 ± 0.6f Values are means ± SE for 7 experiments. Islets were incubated in 0.25 ml Krebs-Ringer bicarbonate buffer, 2 mg/ml bovine serum albumin, and 10 mM HEPES (KRBH) medium with rlL-1...