Chaperone treatment prevents beta-cell dysfunction under high glucose and palmitic acid treatment.

<p>hIAPP-INS1E cells were transduced with Ad-BiP, Ad-PDI or treated with TUDCA or PBA for 24 hours. After 24 hours, cells were treated with 25 mM of glucose and palmitic acid (HG+PA). Glucose-stimulated insulin secretion was performed at low (2.8 mM) and high (16 mM) glucose using INS1E cells as control as expressed by % of insulin content. Insulin levels were determined by ELISA. Results are expressed as mean ± S.E.M from three independent experiments. ^##<i>p</i><0.05 <i>vs</i> INS1E control, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 <i>vs</i>. hIAPP-INS1E cells treated with HG+PA. No statistical differences were found between Con and BiP, PDI, TUDCA and PBA.</p

Endogenous and chemical chaperones decrease ER stress markers in hIAPP-INS1E cells.

<p>A) BiP and tubulin levels were determined in hIAPP-INS1E cells previously transduced with Ad-BiP or Ad-GFP for 24 hours. Representative western blotting results showing protein expression levels of CHOP, ATF3, BiP, p-eIF2α and β-actin levels in hIAPP-INS1E cells cultured with B) 0.5 µM Thp for 8 hours or C) with 25 mM of glucose and 400 µM palmitic acid (HG+PA) for 24 hours. Representative results from 3 to 5 individual experiments are shown.</p

Thapsigargin induces ER stress and apoptosis in hIAPP-INS1E cells.

<p>A) INS1E control cells were cultured at 11 mM glucose and treated with 1 µM of thapsigargin for 8 and 24 hours. mRNA levels of ER stress markers ATF3, sXBP1 and CHOP were quantified by Real-Time PCR. *p<0.05, **p<0.01 and ***p<0.001 versus control cells at time 0. Results are expressed s mean ± S.E.M from five independent experiments. B) INS1E and hIAPP cells were exposed to 1 µM thapsigargin for 24 hours. Expression levels of CHOP, cleaved caspase 3, insulin, IAPP and β-actin were determined by Western blot. C) INS1E and hIAPP-INS1E control cells were exposed to 0.25, 0.5 and 1 µM of thapsigargin (Thp) for 8 and 24 hours. Protein levels of CHOP, cleaved caspase 3, IAPP and β-actin were determined by Western blot (n = 3). Representative Western blotting images are shown from 2 to 3 independent experiments.</p

Knockdown of CHOP protects hIAPP-INS1E cells from induced apoptosis.

<p>hIAPP-INS1E cells were transfected with 20 µM of siRNA CHOP or siRNA scramble (siRNA scr) as a control. Twenty-four hours after transfection, cells were treated with A) 0.5 µM Thp for 8 hours or B) high glucose (25 mM) and BSA-coupled palmitic acid (400 µM; HG+PA) for 24 hours, and protein expression levels for CHOP, ATF3 and tubulin were determined. Representative Western blotting images are represented from 3 to 5 individual experiments. Immunostaining (left panel) and quantification (right panel) of insulin positive beta-cells (red), cleaved caspase 3 (green) and nuclei (blue) of hIAPP-INS1E cells (Con) and hIAPP-INS1E cells transfected with siRNA scr or siRNA CHOP previously treated with C) Thp or D) HG+PA. Note the absence of cells containing insulin and cleaved caspsase 3 staining in cells transfected with siRNA CHOP as compared to Thp or HG+PA treated controls. Scale bar is 50 µm. Quantification is normalized to number of insulin + cells and expressed as mean ± S.E.M from three independent experiments. **<i>p</i><0.01 and ***<i>p</i><0.001 <i>vs</i>. controls. No statistical differences were found between controls and Thp siCHOP or HG+PA siCHOP.</p

Increased BiP and PDI expression after adenoviral transduction does not affect cell viability.

<p>A) hIAPP-INS1E cells were transduced with different doses (MOI) for 24 hours. Representative Western blotting shows BiP protein levels at indicated doses. B) GFP expression after adenoviral (Ad-PDI/GFP) transduction in hIAPP-INS1E cells for 24 hours. Note an increase in GFP expression that correlates with an increase in MOI. C) Live/death viability assay showing no cell death in hIAPP-INS1E cells transduced for 24 hours with Ad-BiP. Western blotting and images in B and C are representative from 3 independent experiments.</p

Thapsigargin and high glucose and palmitic acid potentiate ER stress gene expression in hIAPP-INS1E cells.

<p>ER stress expression markers CHOP, ATF3 and sXBP1 were determined by real-time PCR from hIAPP-INS1E, rIAPP-INS1E and INS1E control cells cultured at A) 11 mM glucose B) 11 mM glucose exposed to 0.5 µM of thapsigargin for 8 hours or C) 25 mM of glucose (HG) with 400 µM palmitic acid (PA) for 24 hours. Results are normalized to untreated INS1E, rIAPP-INS1E or hIAPP-INS1E cells (dashed line) and expressed as mean ± S.E.M from five independent experiments. *<i>p</i><0.05 **<i>p</i><0.01 and ***<i>p</i><0.001 <i>vs</i>. rIAPP and <i>^##p</i><0.01, <i>^###p</i><0.001 <i>vs</i>. INS1E control. n.s., not significant.</p

List of primer sequences used in qRT-PCR experiments.

<p>List of primer sequences used in qRT-PCR experiments.</p

Impaired endothelium-dependent relaxation in insulin resistant rat model and systemic amylin concentration.

<p>Panel A shows serum concentrations of amylin (pM) in over-night fasted control (CR) and insulin resistant rats (IRR). Data are expressed as mean±SE. N indicates the number of animals used for determinations. Samples were assessed in duplicate. p = 0.0004 versus CR by unpaired <i>t</i>-test. Panel B shows the relaxant response to acetylcholine (ACh; 1 nM to 10 μM) in mesenteric arterial segments derived from CR and IRR. Data are expressed as mean±SE of the remaining contraction induced by norepinephrine (NE). (n) is the number of vascular segments used for each curve. *** p < 0.0001 versus CR by two-factors ANOVA.</p

Body weight, fasting glucose and insulin of animals.

<p>Data are expressed as mean±SE. (N) is the number of rats used.</p><p>* p< 0.05</p><p>*** p< 0.0001 vs control rats by two-tailed unpaired Student′s <i>t</i>-test.</p><p>Body weight, fasting glucose and insulin of animals.</p

Acute treatment with amylin impairs endothelium-dependent vasodilation only in mesenteric arteries from control rats.

<p>Effects of preincubation with r-amylin (40 pM) on endothelium-dependent vasodilation elicited by acetylcholine (ACh; 1 nM to 10 μM) in isolated mesenteric arteries from control rats (CR) (A), and from insulin resistant rats (IRR) (B). Data are expressed as mean±SE of the remaining contraction induced by norepinephrine (NE). n indicates the number of vascular segments used for determinations. *** Indicates p < 0.0001 versus CR by two-factors ANOVA.</p