Luminal H ₂ O ₂ promotes ER Ca ²⁺ dysregulation and toxicity of palmitate in insulin‐secreting INS‐1E cells

Abstract

The endoplasmic reticulum (ER) lumen is not only the major site for the assembly and folding of newly synthesized proteins but also the main intracellular Ca2+ store. Ca2+ ions are involved in versatile biochemical processes, including posttranslational processing and folding of nascent proteins. Disruption of ER Ca2+ homeostasis is usually accompanied by an ER stress response that can ultimately lead to apoptosis if unresolved. Abnormal ER Ca2+ depletion has been linked to pancreatic β‐cell dysfunction and death under lipotoxic conditions. However, the underlying mechanisms how the β‐cell toxic saturated free fatty acid palmitate perturbs ER Ca2+ homeostasis and its interplay with other organelles are not fully understood. In the present study, we demonstrate that treatment of insulin‐secreting INS‐1E cells with palmitate diminished ER Ca2+ levels, elevated cytosolic/mitochondrial Ca2+ content, lowered the mitochondrial membrane potential, and ATP content. In addition, palmitate‐pretreated β‐cells contained significantly less luminal Ca2+, revealed a severely impaired ER Ca2+ reuptake rate, and substantially lower insulin content. Importantly, detoxification of luminal H2O2 by expression of the ER‐resident glutathione peroxidase 8 (GPx8) abrogated the lipotoxic effects of palmitate. Moreover, GPx8 supported oxidative protein folding and preserved insulin content under lipotoxic conditions. A direct involvement of luminal H2O2 in palmitate‐mediated ER Ca2+ depletion could be corroborated by the ectopic expression of an ER‐luminal active catalase. Our data point to the critical role of luminal H2O2 in palmitate‐mediated depletion of ER Ca2+ through redox‐dependent impairment of Ca2+ ATPase pump activity upstream of mitochondrial dysfunction in insulin‐secreting INS‐1E cells.

Spore Oil-Functionalized Selenium Nanoparticles Protect Pancreatic Beta Cells from Palmitic Acid-Induced Apoptosis via Inhibition of Oxidative Stress-Mediated Apoptotic Pathways

“…However, the long-term increase of free fatty acids represented by PA leads to the apoptosis of pancreatic β-cells. As a result, the function of insulin secretion is reduced [ 44 , 45 , 46 ]. Therefore, pancreatic β-cell retention may be an effective strategy to prevent diabetes.…”

Non-esterified fatty acid palmitate facilitates oxidative endoplasmic reticulum stress and apoptosis of β-cells by upregulating ERO-1α expression

“…The ratio between PA and added BSA was 0.5 mM: 1 % (w/v) for INS-1E and 0.5 mM: 0.67 % (w/v) for EndoC cells (equivalent to a PA/BSA molar ratio of 3.3 or 5, respectively, representing the FFA/BSA ratios under pathological conditions [ 40 ]). Considering the stepwise equilibrium model and the respective binding affinities of PA for BSA [ 41 , 42 ], the calculated free, unbound PA fraction lies at approximately 27 or 41 nM, respectively [ 5 , 43 , 44 ], which represent approximately three- to five-times the concentration of unbound FFAs measured in the plasma of healthy lean individuals [ 45 ]. These calculated concentrations of free, unbound PA may correlate with the three-fold elevated concentrations of FFAs observed in individuals diagnosed with T2DM [ 46 ].…”

“…Western blot analysis was performed as described earlier [ 5 ]. In brief, cell lysates were sonified in phosphate-buffered saline (PBS; supplemented with complete protease inhibitor mix (Roche Diagnostics, Manheim, Germany).…”

“…Many studies have linked increased plasma concentrations of free fatty acids (FFAs), particularly palmitate (PA; C16:0), to the prevalence of T2DM. Accumulating evidence suggests that high circulating concentrations of PA lead to β-cell apoptosis [ [3] , [4] , [5] , [6] , [7] ] via the induction of oxidative and endoplasmic reticulum (ER) stress, two mechanisms that are intimately interrelated [ [8] , [9] , [10] , [11] ]. The ER lumen provides a unique environment for the correct formation of disulphide bonds within nascent polypeptides, which are vital for maintaining protein structure and function [ 10 , 12 ].…”

“…Especially, within the ER, with its limited luminal H 2 O 2 efflux [ 21 , 22 ], substantial amounts of H 2 O 2 can accumulate, thus affecting ER proteostasis. Indeed, emerging evidence suggests that accumulation of H 2 O 2 in the ER lumen is required for PA-mediated dysregulation of ER Ca 2+ homeostasis and ER stress induction in pancreatic β-cells [ 5 , 6 ]. Specific detoxification of luminal H 2 O 2 effectively prevented PA-mediated induction of fulminant ER stress and β-cell death [ 6 ].…”