Reduced endoplasmic reticulum (ER)-to-Golgi protein trafficking contributes to ER stress in lipotoxic mouse beta cells by promoting protein overload

Abstract

Aims/hypothesis Saturated fatty acids augment endoplasmic reticulum (ER) stress in pancreatic beta cells and this is implicated in the loss of beta cell mass that accompanies type 2 diabetes. However, the mechanisms underlying the induction of ER stress are unclear. Our aim was to establish whether saturated fatty acids cause defects in ER-to-Golgi protein trafficking, which may thereby contribute to ER stress via protein overload. Methods Cells of the mouse insulinoma cell line MIN6 were transfected with temperature-sensitive vesicular stomatitis virus G protein (VSVG) tagged with green fluorescent protein to quantify the rate of ER-to-Golgi protein trafficking. I14 antibody, which detects only correctly folded VSVG, was employed to probe the folding environment of the ER. ER stress markers were monitored by western blotting.Results Pretreatment with palmitate, but not oleate, significantly reduced the rate of ER-to-Golgi protein trafficking assessed using VSVG. This was not secondary to ER stress, since thapsigargin, which compromises chaperone function by depletion of ER calcium, markedly inhibited VSVG folding and promoted strong ER stress but only slightly reduced protein trafficking. Blockade of ER-to-Golgi protein trafficking with brefeldin A (BFA) was sufficient to trigger ER stress, but neither BFA nor palmitate compromised VSVG folding. Conclusions/interpretation Reductions in ER-to-Golgi protein trafficking potentially contribute to ER stress during lipoapoptosis. In this case ER stress would be triggered by protein overload, rather than a disruption of the proteinfolding capacity of the ER.

Hypoxia reduces ER-to-Golgi protein trafficking and increases cell death by inhibiting the adaptive unfolded protein response in mouse beta cells

“…The temperature-sensitive VSVG mutant protein is retained in the ER at 40°C (ESM Fig. 4) and translocates to the Golgi after the switch to the permissive temperature of 32°C [21]. Interestingly, in comparison to the normoxic condition, cells treated with hypoxia (1% O 2 ) retained substantial VSVG in the ER (Fig.…”

“…Trafficking assay ER-to-Golgi protein trafficking was assessed using the temperature-sensitive mutant vesicular stomatitis viral glycoprotein (VSVG) assay [21,22]. MIN6 cells seeded on coverslips were transfected with VSVG protein tagged with green fluorescent protein (GFP).…”

“…A recent study showed that impaired Golgi-to-ER retrograde transport can induce beta cell ER stress [44]. Moreover, blockage of ER-to-Golgi protein trafficking causes accumulation of proteins in the ER and thus ER stress in beta cells chronically exposed to saturated fatty acids [21,22,45].…”

Upregulation of the Rab27a-Dependent Trafficking and Secretory Mechanisms Improves Lysosomal Transport, Alleviates Endoplasmic Reticulum Stress, and Reduces Lysosome Overload in Cystinosis

“…Cystinotic cells have upregulated ER stress. Defects in vesicular transport mechanisms may lead to the development of ER stress (31,51,52), and ER stress is upregulated in some lysosomal storage disorders (34). To evaluate whether Ctns Ϫ/Ϫ cells have an upregulated unfolded protein response, we first analyzed the expression level of the established UPR target genes Grp78 (Bip) and Grp94, which are transcriptionally upregulated and play a central role during the response to conditions of ER stress (53,54).…”

Targeting surface voids to counter membrane disorders in lipointoxication-related diseases

“…For example, exogenously supplied saturated fatty acids (SFAs), which efficiently incorporate within phospholipids, have a major impact on the integrity of the organelles from the loose-lipid packing territory. Early in the secretory pathway, SFAs have been shown to alter vesicular budding in the ER, promote the accumulation of unfolded proteins within this compartment and induce a so-called ER stress, which can ultimately lead to cell death by apoptosis (Borradaile et al, 2006;Dhayal and Morgan, 2011;Guo et al, 2007;Gwiazda et al, 2009;Laybutt et al, 2007;Pineau et al, 2009;Preston et al, 2009). Later in the pathway, SFAs have also been demonstrated to affect vesicle formation at the Golgi in a process that could be directly connected to increased lipid packing (Payet et al, 2013).…”