Preventing Beta-Cell Loss and Diabetes With CCBs
Preventing Beta-Cell Loss and Diabetes With CCBs
INS-1 cells were grown in RPMI 1640 (Invitrogen) with 11.1 mmol/L glucose, 10% FBS, 1% penicillin/streptomycin, 1 mmol/L sodium pyruvate, 2 mmol/L l-glutamine, 10 mmol/L HEPES, and 0.05 mmol/L 2-mercaptoethanol and incubated with verapamil, diltiazem, NiCl2, EGTA, or cyclosporine A (CyA; Sigma). Mouse pancreatic islets were isolated by collagenase digestion and used for RNA or protein extraction or were embedded for immunohistochemistry and transferase-mediated dUTP nick-end labeling (TUNEL) analysis.
Human islets were obtained from the Islet Resource Facility of the University of Alabama at Birmingham Comprehensive Diabetes Center. After overnight incubation at 5 mmol/L glucose, human islets were incubated at 11.1 mmol/L glucose in the presence or absence of 50, 100, or 150 μmol/L verapamil for 24 h. Islets from the same donor were used as control.
All mouse studies were approved by the University of Alabama at Birmingham Animal Care and Use Committee.
Wild-type, 8-week-old, male C57BL/6 mice (The Jackson Laboratory) were rendered diabetic by multiple low-dose injection of streptozotocin (STZ; 40 mg/kg body wt per day for 5 days, prepared freshly in 0.1 mmol/L sodium citrate at pH 4.5).
As a model of type 2 diabetes, leptin-deficient, obese, insulin-resistant, and diabetic BTBRlep (ob/ob) mice (The Jackson Laboratory) were used. BTBRlep mice develop severe diabetes at 4–5 weeks of age and suffer from β-cell apoptosis.
Mice received verapamil in their drinking water (1 mg/mL), resulting in an average dose of 100 mg/kg/day, which is the commonly used dose for mouse studies of this kind. (A subset of mice received a lower dose of 0.5 mg/mL in their drinking water, resulting in an average dose of 50 mg/kg/day.) Control mice were housed under identical conditions without verapamil. Blood glucose was measured with a FreeStyle Lite glucometer; serum insulin was assessed using a mouse insulin ELISA kit (Alpco Diagnostics, Salem, NH).
Insulin tolerance tests (ITTs) were performed after a 4-h fast using insulin 1 IU/kg i.p.. At killing, mouse pancreata were collected for immunohistochemistry or for islet isolation.
Construction of the TXNIP promoter reporter and control LacZ plasmids were described previously, as was the carbohydrate response element–binding protein (ChREBP) overexpression plasmid.
To determine the TXNIP promoter region responsible for the verapamil effect, INS-1 cells were grown in 12-well plates and transfected with different TXNIP promoter deletion reporter plasmids (0.4 μg/well) together with pRL-TK control plasmid (20 ng/well; Promega) using DharmaFECT Duo transfection reagent (1 μL/well; Dharmacon/Thermo Scientific, Chicago, IL). Verapamil was added to the medium at a final concentration of 100 μmol/L. At 24 h after transfection, cells were harvested and luciferase activity was determined by Dual Luciferase assay kit (Promega).
To determine the role of ChREBP in the verapamil effects, INS-1 cells were grown in 6-well plates and transfected with ChREBP or control LacZ plasmids (2 μg/well) using DharmaFECT Duo transfection reagent (3 μL/well). At 24 h after transfection, verapamil was added to the medium with a final concentration of 50 μmol/L; 48 h after transfection, cells were harvested for RNA extraction.
Total RNA was extracted using RNeasy kit (QIAGEN) according to the manufacturer's instructions. RNA (1 μg) was reverse transcribed to cDNA using the first strand cDNA synthesis kit (Roche).
Quantitative real-time PCR was performed on a Prism 7000 Sequence Detection System using SYBR Green (Applied Biosystems, Foster City, CA). Rat TXNIP primers have been described previously. All samples were corrected for the 18S ribosomal subunit (Applied Biosystems) run as an internal standard.
Whole cell and nuclear protein extracts from INS-1 cells and mouse islets were prepared as described previously. The following antibodies were used: mouse anti-TXNIP IgG (1:400; JY2; MBL International), rabbit anti-ChREBP IgG (1:200; sc-33764), goat anti-mouse IgG (1:5,000; sc-2005), and goat anti-rabbit IgG (1:5,000; sc-2004) (Santa Cruz Biotechnology). Bands were visualized by ECL Plus (Amersham GE Health) and quantified by ImageQuant.
Insulin staining and TUNEL were performed as previously described.
Chromatin immunoprecipitation (ChIP) assays were performed as detailed previously.
Student t tests were used to calculate the significance of a difference between two groups. For datasets of more than two groups and to analyze changes overtime, we performed one-way ANOVA calculations.
Research Design and Methods
Cell Culture
INS-1 cells were grown in RPMI 1640 (Invitrogen) with 11.1 mmol/L glucose, 10% FBS, 1% penicillin/streptomycin, 1 mmol/L sodium pyruvate, 2 mmol/L l-glutamine, 10 mmol/L HEPES, and 0.05 mmol/L 2-mercaptoethanol and incubated with verapamil, diltiazem, NiCl2, EGTA, or cyclosporine A (CyA; Sigma). Mouse pancreatic islets were isolated by collagenase digestion and used for RNA or protein extraction or were embedded for immunohistochemistry and transferase-mediated dUTP nick-end labeling (TUNEL) analysis.
Human islets were obtained from the Islet Resource Facility of the University of Alabama at Birmingham Comprehensive Diabetes Center. After overnight incubation at 5 mmol/L glucose, human islets were incubated at 11.1 mmol/L glucose in the presence or absence of 50, 100, or 150 μmol/L verapamil for 24 h. Islets from the same donor were used as control.
Animal Studies
All mouse studies were approved by the University of Alabama at Birmingham Animal Care and Use Committee.
Wild-type, 8-week-old, male C57BL/6 mice (The Jackson Laboratory) were rendered diabetic by multiple low-dose injection of streptozotocin (STZ; 40 mg/kg body wt per day for 5 days, prepared freshly in 0.1 mmol/L sodium citrate at pH 4.5).
As a model of type 2 diabetes, leptin-deficient, obese, insulin-resistant, and diabetic BTBRlep (ob/ob) mice (The Jackson Laboratory) were used. BTBRlep mice develop severe diabetes at 4–5 weeks of age and suffer from β-cell apoptosis.
Mice received verapamil in their drinking water (1 mg/mL), resulting in an average dose of 100 mg/kg/day, which is the commonly used dose for mouse studies of this kind. (A subset of mice received a lower dose of 0.5 mg/mL in their drinking water, resulting in an average dose of 50 mg/kg/day.) Control mice were housed under identical conditions without verapamil. Blood glucose was measured with a FreeStyle Lite glucometer; serum insulin was assessed using a mouse insulin ELISA kit (Alpco Diagnostics, Salem, NH).
Insulin tolerance tests (ITTs) were performed after a 4-h fast using insulin 1 IU/kg i.p.. At killing, mouse pancreata were collected for immunohistochemistry or for islet isolation.
Plasmid Construction and Transient Transfection Assays
Construction of the TXNIP promoter reporter and control LacZ plasmids were described previously, as was the carbohydrate response element–binding protein (ChREBP) overexpression plasmid.
To determine the TXNIP promoter region responsible for the verapamil effect, INS-1 cells were grown in 12-well plates and transfected with different TXNIP promoter deletion reporter plasmids (0.4 μg/well) together with pRL-TK control plasmid (20 ng/well; Promega) using DharmaFECT Duo transfection reagent (1 μL/well; Dharmacon/Thermo Scientific, Chicago, IL). Verapamil was added to the medium at a final concentration of 100 μmol/L. At 24 h after transfection, cells were harvested and luciferase activity was determined by Dual Luciferase assay kit (Promega).
To determine the role of ChREBP in the verapamil effects, INS-1 cells were grown in 6-well plates and transfected with ChREBP or control LacZ plasmids (2 μg/well) using DharmaFECT Duo transfection reagent (3 μL/well). At 24 h after transfection, verapamil was added to the medium with a final concentration of 50 μmol/L; 48 h after transfection, cells were harvested for RNA extraction.
Quantitative Real-time RT-PCR
Total RNA was extracted using RNeasy kit (QIAGEN) according to the manufacturer's instructions. RNA (1 μg) was reverse transcribed to cDNA using the first strand cDNA synthesis kit (Roche).
Quantitative real-time PCR was performed on a Prism 7000 Sequence Detection System using SYBR Green (Applied Biosystems, Foster City, CA). Rat TXNIP primers have been described previously. All samples were corrected for the 18S ribosomal subunit (Applied Biosystems) run as an internal standard.
Western Blotting
Whole cell and nuclear protein extracts from INS-1 cells and mouse islets were prepared as described previously. The following antibodies were used: mouse anti-TXNIP IgG (1:400; JY2; MBL International), rabbit anti-ChREBP IgG (1:200; sc-33764), goat anti-mouse IgG (1:5,000; sc-2005), and goat anti-rabbit IgG (1:5,000; sc-2004) (Santa Cruz Biotechnology). Bands were visualized by ECL Plus (Amersham GE Health) and quantified by ImageQuant.
Immunohistochemistry and TUNEL Assay
Insulin staining and TUNEL were performed as previously described.
Chromatin Immunoprecipitation
Chromatin immunoprecipitation (ChIP) assays were performed as detailed previously.
Statistical Analysis
Student t tests were used to calculate the significance of a difference between two groups. For datasets of more than two groups and to analyze changes overtime, we performed one-way ANOVA calculations.
