Reduced endoplasmic reticulum (ER)-to-Golgi protein trafficking contributes to ER stress in lipotoxic mouse beta cells by promoting protein overload
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- Preston, A.M., Gurisik, E., Bartley, C. et al. Diabetologia (2009) 52: 2369. doi:10.1007/s00125-009-1506-5
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.
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.
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.
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 protein-folding capacity of the ER.
KeywordsApoptosis Endoplasmic reticulum stress Lipotoxicity Pancreatic beta cell Protein trafficking Type 2 diabetes Vesicular stomatitis virus G protein
Green fluorescent protein
Vesicular stomatitis virus G protein
Apoptosis contributes to the beta cell failure that is essential for the development of type 2 diabetes, and can be recapitulated by chronic exposure of beta cells to saturated fatty acids (FAs). Recent work has implicated endoplasmic reticulum (ER) stress as a mechanism underlying both lipoapoptosis in vitro [1, 2] and the loss of beta cell mass observed in human type 2 diabetes and animal models of the disease . ER stress results when the load of client proteins entering the ER exceeds their capacity to be correctly folded. Cells can re-adjust the balance between protein production and folding by activating the unfolded-protein response, but if this fails to resolve the ER stress, apoptosis ensues .
Although the enhanced secretory demand that accompanies insulin resistance most probably contributes, ER stress is also triggered by direct actions of saturated FAs on the beta cell. The molecular mechanisms underlying these direct effects are currently unclear. One hypothesis invokes depletion of Ca2+ from the ER lumen, which thereby compromises the function of protein-folding chaperones. But Ca2+ depletion correlates rather poorly with the capacity of FAs to promote ER stress [3–5]. Here our aim was to investigate an alternative mechanism whereby FAs might induce ER stress by protein overload, secondary to reductions in ER-to-Golgi protein trafficking.
Cell culture and standard treatments
Cells of the mouse insulinoma cell line MIN6 were routinely cultured as previously described . After seeding (24 h) chronic treatments were commenced using Dulbecco’s modified Eagle’s medium containing 6 mmol/l glucose containing either BSA, or BSA coupled to FA (final 0.4 mmol/l FA:0.92% [wt/vol.] BSA) . Apoptosis ELISA assays and immunoblotting of ER stress markers were performed as detailed previously .
Trafficking and folding assays
MIN6 cells (3 × 106 per cuvette) were transfected (70% efficiency) with 3 μg DNA encoding the mutated vesicular stomatitis virus G protein VSV-Gt045 (VSVG) tagged with green fluorescent protein (GFP), or GFP control, by nucleofection (AMAXA Biosystems, Cologne, Germany). Cells were seeded onto coverslips and cultured overnight prior to chronic treatment as above. After 36 h, cells were switched to 40°C for 12 h. Trafficking assays were initiated by transfer to the permissive temperature of 32°C and addition of 20 μmol/l cyclohexamide for 0–30 min . After fixation, slides were exposed to primary antibodies for Golgi marker GOLGA2 (also known as GM130) (BD Transduction Labs, Franklin Lakes, NJ, USA) or calnexin (Stressgen, Victoria, BC, Canada). Slides were examined using an SP2 ABOS laser scanning confocal microscope (Leica Microscopes, Wetzler, Germany) with appropriate excitation wavelengths for individual fluorochromes, and channels scanned in series. Cells moderately overexpressing VSVG were scored for co-localisation with either GOLGA2 or calnexin by two independent observers, with inter-observer variation <5%. For folding assays, 1 μg/ml brefeldin A (BFA) was added as the cells were transferred to 32°C. After 6 h, cells were fixed and exposed to the I14 antibody for detection of native VSVG .
ER stress is potentially modulated by the rate of ER-to-Golgi trafficking but this segment of the secretory pathway is technically very challenging to assess using endogenous cargo protein. Therefore a reporter protein, the temperature-sensitive VSVG mutant, has been developed as the method of choice for this analysis, at least in readily transfectable cells where the relatively poor expression of the large transgene is negated. This protein is retained in the ER at 40°C but readily exits via the secretory pathway after switching to the permissive temperature of 32°C . Provided its ongoing synthesis is blocked, net transfer from ER to Golgi of the GFP-tagged VSVG can therefore be visualised. We therefore applied the VSVG technique to MIN6 cells that we, and others, have extensively characterised as undergoing a mild ER stress and lipoapoptosis [2, 6, 9] resembling that of primary mouse and human beta cells [4, 9]. As shown in Electronic supplementary material (ESM) Fig. 1, VSVG remains distributed with the ER marker calnexin immediately following the switch to the permissive temperature; after 10 min some co-localises with the Golgi marker GOLGA2, whereas transfer to the Golgi is virtually complete by 20 min (ESM Fig. 1). Hence, the 20 min time point was employed in subsequent analyses.
Finally we compared the effects of FAs, BFA and thapsigargin on apoptosis (Fig. 2b), along with phosphorylation of EIF2AK3 (also known as PERK) and levels of DDIT3 (also known as CHOP) as ER stress markers (Fig. 2c). The results demonstrate that ER stress and apoptosis in beta cells is not only triggered by disruption of protein-folding capacity with thapsigargin, but also by protein overload caused by even brief blockade of ER-to-Golgi trafficking with BFA. This establishes that the trafficking defect observed with palmitate could contribute to ER stress and hence lipoapoptosis.
Our results clearly demonstrate that saturated, but not unsaturated, FAs specifically slowed ER-to-Golgi protein trafficking. Moreover, disruption of trafficking at this site with BFA was sufficient to cause ER stress, whereas direct induction of strong ER stress with thapsigargin caused only a modest decrease in protein export out of the ER. This places defective trafficking upstream of ER stress. Collectively our data suggest that protein overload should now be considered as a mechanism contributing to ER stress in beta cells chronically exposed to saturated FAs. By slowing the transit of protein from the ER to Golgi, unfolded protein could accumulate under these conditions, even in the absence of changes in the rate of folding, simply because of the build-up of protein in the ER lumen (ESM Fig. 3).
Although the VSVG assay is the method of choice for assessing, directly and specifically, the trafficking of protein between ER and Golgi, it will be important in future studies to confirm our findings using independent techniques, and in primary beta cells. The latter caveat also holds for the other main mechanism previously advanced to explain how saturated FAs promote ER stress in beta cells, namely inhibition of the rate of protein folding in beta cells [4, 9]. Specifically it has been proposed that depletion of ER Ca2+ impacts negatively on protein-folding efficiency . However, the effects of different saturated FAs on luminal Ca2+ do not always correlate well with ER stress [3–5]. On the other hand, such studies are difficult to quantify accurately, so we certainly do not exclude this possibility. Indeed, it is probable that multiple mechanisms contribute to the overall induction of ER stress. This is especially likely in vivo, where the direct effects of saturated FAs on beta cells may be expected to exacerbate the secretory load that is already imposed in trying to overcome peripheral insulin resistance (ESM Fig. 3).
Palmitate pretreatment is also known to impair proinsulin processing . Although it is tempting to speculate that the trafficking defect described here may contribute to this impairment, specific alterations in post-Golgi processing have also been described [11, 12]. Evaluating the relative contribution of these alternative mechanisms would therefore require a better understanding of how palmitate actually reduces protein trafficking, since this would facilitate investigation of whether rescuing the trafficking defect could improve proinsulin conversion. Amongst several potential effects of palmitate, an impact on ER membrane dynamics is perhaps the most likely explanation for the defective trafficking, although alternative mechanisms may involve alterations in signalling or gene expression.
In conclusion, our findings provide further evidence that saturated FAs act directly on beta cells to induce ER stress. In establishing that protein trafficking is specifically disrupted under these conditions, we provide the important conceptual advance that protein overload potentially contributes as an underlying mechanism. A better understanding of these processes will facilitate the design of future experiments to quantify the relative contributions of protein overload vs protein misfolding to the induction of ER stress during beta cell failure.
This work was funded by a Project Grant, and Postgraduate Scholarships, from the National Health and Medical Research Council of Australia. We gratefully acknowledge J. Lippincott-Schwartz (NIH, Bethesda, MD, USA) and D. Lyles (Wake Forest University School of Medicine, Winston-Salem, NC, USA) for the generous gifts of the VSVG reporter construct and I14 antibody, respectively. We also thank C. Achard and J. Stoeckli (Garvan Institute of Medical Research, Sydney, NSW, Australia) for critical review of the manuscript.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.