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Keywords:

  • type 2 diabetes;
  • obesity;
  • saturated fatty acids;
  • unfolded protein response;
  • fluidity;
  • membrane curvature

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Impacts of long-chain SFAs on the UPR- and ER stress pathways
  5. Lipid-induced ER stress and ER function
  6. An integrated view – biophysical consequences of SFA accumulation
  7. Future perspectives
  8. References

Exposure to long-chain saturated fatty acids (SFAs; e.g. palmitate) induces apoptosis in pancreatic β cells, a process that may contribute to the development of type 2 diabetes. Under palmitate treatment, β cells undergo a so-called endoplasmic reticulum (ER) stress that can be counteracted by the unfolded protein response (UPR). The UPR is a coordinated response, which is primarily devoted to helping the ER to cope with the accumulation of misfolded proteins. Sustained SFA exposure may ultimately overwhelm the UPR, resulting in cell death. By contrast, unsaturated fatty acids (e.g. oleate) are much less harmful to the cells and can even alleviate palmitate toxicity. Surprisingly, recent evidences indicate that a simple unicellular eukaryote, the budding yeast Saccharomyces cerevisiae, which is not routinely exposed to high-fat diets, also undergoes ER stress under lipotoxic conditions. This suggests that the mechanisms of SFA toxicity are largely conserved throughout eukaryotes and are not specific of a given cell type. The present review discusses the mechanisms of SFA toxicity in yeast and β cells, with a main emphasis on their potential impacts on ER-membrane organization/function and ER-based processes.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Impacts of long-chain SFAs on the UPR- and ER stress pathways
  5. Lipid-induced ER stress and ER function
  6. An integrated view – biophysical consequences of SFA accumulation
  7. Future perspectives
  8. References

Deficiency in insulin secretion through the loss of pancreatic β-cell function is believed to be a key feature of type 2 diabetes. In obese individuals, the incidence of this pathology is generally associated with hyperlipidemia that is characterized by high plasma levels of nonesterified fatty acids (NEFA) (Sabin et al., 2007; Kusminski et al., 2009). Even if still a debated issue, it has been proposed that sustained plasmatic NEFA levels could contribute, at least in part, to a decline in β-cell mass, a process that is generally referred to as ‘lipotoxicity’ (Butler et al., 2003; Rahier et al., 2008). Interestingly, lipotoxicity has also been reported in the liver, adipose tissue, skeletal muscle and brain under nutritional conditions associated with obesity and type 2 diabetes (for a review, see Lee & Glimcher, 2009). However, due to space limitations, the present review will focus on the mechanisms of NEFA toxicity in β cells, which appear to be conserved among the various cell types.

The cellular events of NEFA-induced toxicity have been studied thoroughly in the last few years by direct exposure of β cells in vivo and, more recently, in a model unicellular eukaryote, the yeast Saccharomyces cerevisiae. Interestingly, in both cell types, which may seem largely unrelated in terms of function and physiology, the impacts of NEFA on cell viability appear to be very similar. This suggests that the mechanisms of NEFA lipotoxicity are largely conserved throughout eukaryotes, from yeast to human cells.

In both organisms, the deleterious effects of NEFA vary significantly depending on the length and the unsaturation level of their fatty acid chain. Overall, it appears that NEFA with long-saturated chains [such as palmitate (C16:0) and stearate (C18:0)] are highly detrimental to the cells, whereas shorter saturates (C14:0 and below) and long-chain fatty acids bearing at least one unsaturation [unsaturated fatty acids (UFAs), such as palmitoleate (C16:1), oleate (C18:1), linoleate (C18:2) and linolenate (C18:3)], are relatively harmless (Wei et al., 2006; Laybutt et al., 2007; Cunha et al., 2008; Diakogiannaki et al., 2008; Morgan et al., 2008; Katsoulieris et al., 2009; Pineau et al., 2009). Moreover, a significant number of studies have demonstrated that UFA can efficiently counteract saturated fatty acid (SFA)-induced cell death (Stein et al., 1997; Wei et al., 2006; Cunha et al., 2008; Diakogiannaki et al., 2008; Katsoulieris et al., 2009; Pineau et al., 2009).

On the other hand, in both yeast and β cells, SFA toxicity is associated with the induction of a stress response in the endoplasmic reticulum (ER), also known as the unfolded protein response (UPR) (Wei et al., 2006; Guo et al., 2007; Laybutt et al., 2007; Cunha et al., 2008; Diakogiannaki & Morgan, 2008; Pineau et al., 2009). The ER is devoted to several crucial cellular processes including lipid synthesis, regulation of calcium homeostasis and biosynthesis of proteins destined for either intracellular organelles or the cell surface. This organelle is also the site where secretory proteins are primarily assembled and folded. ER stress results from an imbalance between ER client protein load and folding capacity. The UPR provides multiple strategies to avoid ER stress and to maintain ER integrity and function by helping this organelle to cope with the accumulation of misfolded proteins (Zhang & Kaufman, 2006; Kincaid & Cooper, 2007). Ultimately, failure to handle ER stress can result in apoptosis, a process that is believed to contribute to SFA-induced cell death.

Impacts of long-chain SFAs on the UPR- and ER stress pathways

  1. Top of page
  2. Abstract
  3. Introduction
  4. Impacts of long-chain SFAs on the UPR- and ER stress pathways
  5. Lipid-induced ER stress and ER function
  6. An integrated view – biophysical consequences of SFA accumulation
  7. Future perspectives
  8. References

In higher eukaryotes, the UPR is comprised of three distinct pathways controlled by integral proteins that act as sensors, namely inositol-requiring enzyme 1 (IRE1), protein-kinase-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6) (for reviews, see Kincaid & Cooper, 2007 and Zhang & Kaufman, 2006; Fig. 1). Induction of the UPR aims at limiting protein overload in the ER via a general decrease in translation initiation, an increase in the translation of specific mRNAs (such as ATF4; Fig. 1) and a coordinated upregulation of selective genes encoding a subset of enzymes involved in protein-folding and ER-associated degradation (ERAD). Interestingly, all these pathways are somehow activated when β cells are exposed to long-chain SFAs, such as palmitate (Table 1).

image

Figure 1.  The UPR pathway in higher eukaryotes. See text for details.

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Table 1.   Relative impacts of palmitate and oleate on selected actors of the UPR/ER stress pathways in β cells
 PalmitateOleatePalmitate+oleate
  1. [UPWARDS ARROW], upregulation; [DOWNWARDS ARROW], downregulation; 0, unchanged compared with nontreated cells; NT, not tested; (?), under debate.

IRE1 pathway
 Ire1α-P[UPWARDS ARROW]0N.T.
 XBP1 mRNA levels[UPWARDS ARROW]0 or [UPWARDS ARROW](?)[UPWARDS ARROW] (?)
 XBP1 mRNA splicing[UPWARDS ARROW]00
PERK pathway
 PERK-P[UPWARDS ARROW]00
 ATF4[UPWARDS ARROW]00
 elF2α-P[UPWARDS ARROW]00
ATF6 pathway
 ATF6Redistribution or[UPWARDS ARROW]0 or[UPWARDS ARROW](?)[UPWARDS ARROW] (?)
Apoptosis
 BCL2[DOWNWARDS ARROW][UPWARDS ARROW]0
 CHOP[UPWARDS ARROW]00
 Caspase-3[UPWARDS ARROW]0NT
 DNA laddering[UPWARDS ARROW]00
Chaperones
 Grp78/BiP/Kar2pRedistribution or [UPWARDS ARROW]0 or [UPWARDS ARROW](?)0 or [UPWARDS ARROW](?)
 Grp940 or [UPWARDS ARROW](?)0NT
 PDI0 or [UPWARDS ARROW](?)0NT
 C-calnexin[UPWARDS ARROW]NTNT

The Ire1p cascade

The Ire1p cascade is the only UPR pathway that is conserved throughout eukaryotes from yeast to humans (Fig. 1 and Table 2). In the yeast S. cerevisiae, the accumulation of unfolded proteins results in the dimerization of Ire1p, a process that activates its cytosolic endoribonuclease function. Kar2p, an ER-resident member of the HSP70 family, is an important regulator of the UPR in both yeast and mammalian cells [the mammalian ortholog of Kar2p is known as immunoglobulin-binding protein (BiP)]. In nonstressed cells, Kar2p associates with Ire1p to repress its activation. In response to the accumulation of unfolded proteins in the ER, Kar2p dissociates from Ire1p, resulting in Ire1p dimerization and its subsequent autophosphorylation, which activates its endoribonuclease function (Zhang & Kaufman, 2006). The substrate for Ire1p endonuclease activity is the transcript of Hac1p (the yeast ortholog of mammalian XBP1), a transcription factor that binds to promoter unfolded protein response elements (UPREs) and regulates the transcription of >380 yeast genes, i.e. approximately 5% of all yeast genes (Travers et al., 2000).

Table 2.   Comparative effects of palmitate treatment on yeast and β cells
 UPR pathwayApoptosis/growth arrestAccumulation of misfolded proteinsDepletion of ER calcium storesAltered ER morphology
Ire1PERKATF6
  1. NT, not tested; (?), under debate.

β cells+++ (?)++++
Yeast cells+NoneNone++NT+

Palmitate-treated cells, including rat INS-1, mouse MIN6 and human islets, display most of the trademarks of IRE1-pathway induction, with elevated XBP1 mRNA or protein levels (Karaskov et al., 2006; Laybutt et al., 2007; Choi et al., 2008; Cunha et al., 2008) and increased XBP1 mRNA splicing (Kharroubi et al., 2004; Wei et al., 2006; Cunha et al., 2008). Moreover, increased levels of IRE1 phosphorylation have also been found in H4IIE liver cells (Wei et al., 2006). In contrast, this pathway is poorly sensitive to the monounsaturated fatty acid oleate (Karaskov et al., 2006; Wei et al., 2006; Laybutt et al., 2007; Cunha et al., 2008) and oleate can efficiently counteract palmitate-induced XBP1 mRNA splicing (Cunha et al., 2008).

In yeast, activation of the Ire1p pathway in response to SFA was assayed using a reporter gene (LACZ), encoding β-galactosidase, under the control of four UPREs (Cox & Walter, 1996; Pineau et al., 2009). In this context, the induction of β-galactosidase activity reflects the cellular amounts of functional Hac1p/XBP1. As reported for mammalian cells, the accumulation of SFA in yeast, originating from both endogenous (by preclusion of fatty acid desaturation) or exogenous sources (by addition of palmitate to the culture medium), resulted in full-fledged activation of the Ire1p pathway. Moreover, this induction was also abolished by oleate addition (Pineau et al., 2009).

PERK pathway

The second UPR sensor/transducer is PERK, a type I transmembrane ER protein (Fig. 1). Similar to IRE1, PERK is maintained in an inactive state by binding to BiP (Bertolotti et al., 2000). The accumulation of misfolded proteins leads to the activation of PERK cytosolic kinase domain, which in turn autophosphorylates and catalyzes the phosphorylation of the translation-initiation factor eIF2α (elF2α-P). elF2α is a subunit of elF2 (with elF2β and elF2γ) that belongs to a ternary complex consisting of elF2, GTP and the methionyl-initiator tRNA (Met-tRNAiMet) (for a review, see Holcik & Sonenberg, 2005). This ternary complex delivers Met-tRNAiMet to the ribosome to initiate translation. As GTP is hydrolyzed during translation initiation, elF2 must be recharged via a GDP/GTP exchange process catalyzed by elF2b. Phosphorylation of elF2α inhibits the GDP/GTP exchange reaction by reducing the dissociation rate of eIF2B. Ultimately, this results in an attenuation of general translation (Shi et al., 1998). A main aim of this pathway of the UPR is therefore to limit the protein load in the ER under stress conditions. Paradoxically, elF2α-P also increases the translation of a subset of genes, including the transcription factor ATF4. In turn, ATF4, in conjunction with XBP-1, upregulates the expression of selective UPR-activated genes, among which are the ER chaperone BiP (also known as GRP78) and the proapoptotic transcription factor CHOP (C/EBP homologous protein) (Harding et al., 2000a; Scheuner et al., 2001).

The induction of the PERK pathway in various β-cell lines in response to palmitate treatment has been studied in considerable detail. Palmitate, but not oleate, induces the respective phosphorylations of PERK (Karaskov et al., 2006; Cunha et al., 2008) and eIF2α (Karaskov et al., 2006; Laybutt et al., 2007; Cunha et al., 2008; Diakogiannaki et al., 2008). As a corollary, downstream in the PERK-eIF2α pathway, the amounts of ATF4 mRNA and protein levels are increased under palmitate treatment (Kharroubi et al., 2004; Karaskov et al., 2006; Laybutt et al., 2007; Diakogiannaki et al., 2008). Moreover, palmitate was also demonstrated to induce a general attenuation of protein synthesis (Cnop et al., 2007). As for the Ire1p branch of the UPR pathway, oleate, but also palmitoleate (C16:1), have been shown to efficiently counteract the inductive effects of palmitate (Cunha et al., 2008; Diakogiannaki et al., 2008).

Although yeasts lack a direct equivalent of PERK in the UPR pathway, a kinase, named Gcn2p, can phosphorylate eIF2α under ER stress, inhibiting general protein translation and specifically enhancing the synthesis of the ATF4 homolog Gcn4p. This transcription factor activates the transcription of at least 539 genes (Dever et al., 1992; Hinnebusch, 1997; Natarajan et al., 2001).

Although the effects of SFA on protein translation via Gcn2p have not been directly addressed in yeast, a general attenuation of translation is unlikely in this organism under SFA-accumulating conditions. Indeed, it has been shown that the amounts of several client proteins, namely the uracil permease Fur4p, the proton-ATPase Pma1p and the secreted enzyme invertase, are not significantly modified under SFA accumulation (Pineau et al., 2008). However, the possible involvement of Gcn2p and Gcn4p in SFA-induced ER stress will require more in-depth investigations.

The ATF6 pathway

The third sensor/transducer of the UPR in higher eukaryotes, ATF6, is a type II membrane protein that displays a luminal sensor domain and a bZIP transcription-factor domain oriented toward the cytoplasmic face of the ER (Yoshida et al., 1998; Haze et al., 1999). Under ER stress, ATF6 moves from the ER to the Golgi, where it is cleaved by the S1P and S2P proteases (Ye et al., 2000). ATF6 retention within the ER is likely due to its interaction with BiP. Under conditions of misfolded protein overload, BiP would dissociate from ATF6, unmasking two Golgi-targeting sequence signals (Shen et al., 2002).

After proteolytic cleavage of the ATF6 cytosolic fragment, this transcription factor is relocalized to the nucleus, where it activates the transcription of selective UPR genes, in cooperation with the general transcription factor NF-Y (Haze et al., 1999; Yoshida et al., 2000).

The studies regarding the impacts of palmitate on the ATF6 pathway in β cells are more controversial than for the other two branches of the UPR cascade (Tables 1 and 2). Karaskov et al. (2006) have demonstrated, by transiently transfecting an HA-tagged version of ATF6 in INS-1 cells, that palmitate altered the distribution of the tagged protein that failed to form the typical reticular staining, which is observed in control and oleate-treated cells. Instead, HA-ATF6 distributes around the nucleus and at the periphery of the cell. However, the authors did not address the nature of the intracellular compartment of ATF6 redistribution. In MIN6 cells, cleaved (activated) ATF6 levels are transiently increased under palmitate treatment (Laybutt et al., 2007). However, in INS-1E cells transfected with a UPR-luciferase reporter construct, which is responsive to active ATF6 and XBP1, all fatty acids tested (i.e. palmitate, oleate or a mix between both fatty acids) were shown to activate the reporter (Cunha et al., 2008).

Downstream targets of the UPR

ER chaperones

ATF6 and XBP1 regulate a subset of genes encoding ER-resident chaperones, folding enzymes and various actors of the ERAD pathway (Okada et al., 2002; Lee et al., 2003). Several studies suggest that XBP1 and ATF6 would act in parallel and may interact with each other upon ER stress (Yoshida et al., 2000, 2001; Lee et al., 2002, 2003).

The BiP protein is crucial to the UPR pathway because it serves both as a chaperone and as a sensor of protein misfolding. BiP is upregulated during ER stress and, even if controversy still remains, could be regulated by ATF6 during this process (Okada et al., 2002; Lee et al., 2003). Interestingly, the role of BiP under palmitate-induced ER stress is also under debate. Depending on the cell type considered, BiP has been alternatively shown to be upregulated by palmitate, but not oleate (Wei et al., 2006; Laybutt et al., 2007), relatively insensitive to palmitate (Laybutt et al., 2007; Diakogiannaki et al., 2008) or induced by both fatty acids (Kharroubi et al., 2004; Cunha et al., 2008). In mouse MIN6 cells, BiP overexpression has been demonstrated to attenuate ER stress and to protect against SFA-induced apoptosis (Laybutt et al., 2007). However, high expression levels of BiP may be required to ensure such a protection, because overexpressing BiP by approximately twofold in MIN6 cells is not sufficient to reduce palmitate-induced apoptosis significantly (Lai et al., 2008). Accordingly, a threefold overexpression of BiP in INS-1 cells failed to protect against palmitate-induced activation of ER stress pathways and palmitate-induced cell death (Lai et al., 2008). These discrepancies could be explained, at least in part, by differential basal levels of BiP in the various cell lines and by their different propensities to desaturate exogenously supplied SFA (Lai et al., 2008). Indeed, MIN6 and human islets, which are intrinsically more resistant to palmitate-related apoptosis, express significantly higher steady-state levels of stearoylcoenzyme A desaturase-1 (SCD1) mRNA than INS-1 cells (Lai et al., 2008). Such variations in SCD1 amounts have also been evidenced among various MIN6 cell lines, and high SCD1 amounts correlate with increased resistance to lipoapoptosis (Busch et al., 2005). Therefore, for a direct comparison between various studies, a precise evaluation of the SFA/UFA ratio within the total cellular lipids would be required.

The induction of several other chaperones has also been investigated in response to palmitate, such as Grp94 (Karaskov et al., 2006), members of the protein disulfide isomerase family (Karaskov et al., 2006; Laybutt et al., 2007; Choi et al., 2008) or C-calnexin (Choi et al., 2008). Overall, such as for BiP, it appears that the downstream chaperone targets of the UPR pathway are not as clearly and uniformly induced in response to palmitate than the upstream components. This observation even led some authors to postulate that discrepancies may exist between the ‘regular’ UPR pathway and lipid-induced ER stress, at least in the latest step of the process (Lai et al., 2008).

Apoptosis

Two main proapoptotic pathways emanating from the UPR are directly mediated by the PERK and IRE1 transducers (Fig. 1).

As already mentioned, the activation of the UPR transducer PERK leads to the phosphorylation of eIF2α, in a rapid and reversible manner. Sustained phosphorylation of eIF2α is proapoptotic because it induces the synthesis of transcription factor ATF4, which subsequently activates the transcription of CHOP. CHOP potentiates apoptosis, possibly by repressing the expression of the apoptotic repressor BCL2 and the consecutive cleavage/activation of caspase-3 (Harding et al., 2000b; DeGracia et al., 2002; Ma et al., 2002; Zhang & Kaufman, 2006). As expected, palmitate exposure could be correlated to CHOP increased expression levels (Laybutt et al., 2007; Diakogiannaki et al., 2008), BCL2 downregulation (Maedler et al., 2003) and induction of caspase-3 activity (Wei et al., 2006). Oleate, but also palmitoleate, can efficiently counteract palmitate induction of this proapoptotic cascade and, as a consequence, significantly protect β cells from palmitate-induced apoptosis (Maedler et al., 2003; Cunha et al., 2008; Diakogiannaki et al., 2008).

Interestingly, CHOP depletion in β cells results in protection (at least transiently) against palmitate-induced cell death (Akerfeldt et al., 2008; Cunha et al., 2008). This effect could be correlated to reduced cleaved (activated) caspase-3 levels in response to palmitate (Akerfeldt et al., 2008). Moreover, the presence of a general caspase inhibitor prevents palmitate-induced caspase-3 activation and consequent apoptosis-associated DNA laddering in H4IIE liver cells (Wei et al., 2006). Altogether, these results point to the PERK/CHOP pathway as being central in palmitate-induced apoptosis.

Under sustained ER stress, activated IRE1 can bind c-Jun-N-terminal inhibitory kinase and recruits cytosolic adapter TRAF2 to the ER membrane (Urano et al., 2000). TRAF2 activates the apoptosis-signaling kinase 1 that, in turn, leads to the activation (phosphorylation) of the JNK protein kinase (Urano et al., 2000; Nishitoh et al., 2002). Increased phosphorylation of JNK could also be observed in response to palmitate treatment in INS-1 β cells (Akerfeldt et al., 2008).

Although the mechanisms of SFA-induced cell death have not been studied thoroughly in yeast, palmitate and, to a lower extent, shorter saturates, result in growth arrest, a process that can be fully suppressed by oleate addition (Pineau et al., 2008, 2009; Table 2).

Lipid-induced ER stress and ER function

  1. Top of page
  2. Abstract
  3. Introduction
  4. Impacts of long-chain SFAs on the UPR- and ER stress pathways
  5. Lipid-induced ER stress and ER function
  6. An integrated view – biophysical consequences of SFA accumulation
  7. Future perspectives
  8. References

ER calcium stores

Because the ER stress response can be activated under the depletion of ER calcium stores (Paschen, 2003), several efforts have been made to evaluate the impacts of palmitate on this process in β cells. Despite some discrepancies between several studies (Karaskov et al., 2006; Cunha et al., 2008; Gwiazda et al., 2009), it appears that palmitate may indeed promote the depletion of ER calcium stores (Cunha et al., 2008; Gwiazda et al., 2009). It has been suggested that this phenomenon could be related to the direct effects of palmitate on sarcoplasmic–endoplasmic reticulum calcium ATPase-2b (SERCA) pump activity (Cunha et al., 2008). This interesting hypothesis ought to be related to in vitro studies, which demonstrated that SERCA activity is inhibited when reconstituted into microsomes containing phosphoslipids with saturated fatty acyl chains (Li et al., 2004). One may therefore postulate that palmitate incorporation within ER phospholipid could be the direct cause of depleted calcium stores, by modifying ER-membrane fluidity or order.

Protein-folding capacity

Even if the relieving of ER stress by overexpression of protein chaperones remains under debate, the fact that the treatment of the cells with pharmacological chaperones, such as 4-phenyl butyric acid (4-PBA) or taurine-conjugated ursodeoxycholic acid, alleviates SFA-induced UPR suggests that misfolded protein overload in the ER could be a major factor in the process (Ozcan et al., 2006; Akerfeldt et al., 2008; Choi et al., 2008; Pineau et al., 2009). Such chemical chaperones are known to stabilize protein conformation, improve ER folding capacity and facilitate the trafficking of mutant proteins (Welch & Brown, 1996). More specifically, 4-PBA has been shown to prevent protein aggregation both in vitro and in vivo (Kubota et al., 2006; de Almeida et al., 2007). Interestingly, the protective effect of molecular chaperones could be observed both in yeast (Pineau et al., 2009) and in β cells (Akerfeldt et al., 2008; Choi et al., 2008).

In β cells, treatment with 4-PBA alleviates most of the trademarks of palmitate induction of the IRE1 and PERK UPR-signaling pathway, because it was shown to inhibit XBP1 activation by selective splicing of its mRNA (Akerfeldt et al., 2008) and PERK/eIF2α phosphorylations and the subsequent expression of the ATF4 and CHOP transcription factors (Akerfeldt et al., 2008; Choi et al., 2008). The same inhibitory effects of 4-PBA could be observed on the downstream targets of the UPR related to programmed cell death, such as caspase-3 activation/cleavage and JNK phosphorylation (Akerfeldt et al., 2008). As a corollary, 4-PBA could efficiently protect INS-1 cells from palmitate-induced apoptosis (Akerfeldt et al., 2008). This protective effect could also be obtained with another chemical chaperone, trimethylamine oxide (Akerfeldt et al., 2008).

Consistently, in yeast, 4-PBA was also shown to alleviate the Ire1p/UPR induction related to SFA accumulation, originating from both endogenous (preclusion of fatty acid desaturation) and exogenous sources.

These observations therefore point to misfolded protein overload as being the molecular signal of SFA-induced UPR induction.

Conversely, palmitoleate has been demonstrated to attenuate tunicamycin-induced ER stress (Diakogiannaki et al., 2008). This suggests that UFA could also act as ‘lipid chaperones,’ by inducing positive effects on protein folding and/or by preventing aggregate formation.

ER morphology

Electron microscopy of INS-1 β-cells also revealed that palmitate markedly alters ER morphology (Moffitt et al., 2005; Karaskov et al., 2006; Diakogiannaki et al., 2008). Indeed, in palmitate-treated cells, ER appears to be dilated, forming electron-lucent clefts that extend throughout the cytoplasm. If β cells treated with both palmitate and palmitoleate also displayed abnormal morphological features, the ER distension was less extensive than in cells exposed to palmitate alone (Diakogiannaki et al., 2008).

Similar dilated structures could also be detected in yeast cells treated with exogenously supplied palmitate (Pineau et al., 2009). Interestingly, such clefts do not form in yeast, when SFA accumulation results from the inhibition of Ole1p, the unique fatty acid desaturase in this organism (Pineau et al., 2009). These distinct behaviors are probably related to the fact that Ole1p inhibition results in the accumulation of SFA with shorter chains, such as lauric acid (C12:0) and myristic acid (C14:0). Compared with palmitate, these fatty acids are also associated with lower Ire1p/UPR induction and appear to be less toxic to the cells (Pineau et al., 2009). This is very consistent with the fact that β cells also tolerate the presence of elevated concentrations of octanoic (C8:0), lauric or myristic acids much better than they tolerate palmitate (Welters et al., 2004; Morgan et al., 2008). Altogether, these results suggest that alterations in the ER morphology correlate with a high UPR induction in response to long SFAs. A plausible explanation to this observation would be that long-chain SFA are more prone to impact protein folding in the ER than shorter chain SFA, and that elevated misfolded protein levels could result, in turn, in an altered ER structure. This hypothesis is reinforced by the fact that 4-PBA seems to restore normal ER morphology in palmite-treated β cells (Choi et al., 2008).

ER-to-Golgi trafficking

Finally, it has also been demonstrated that ER-to-Golgi protein trafficking is precluded in palmitate-treated β cells (Preston et al., 2009). Palmitate (but not oleate) exposure reduced the rate of ER-to-Golgi trafficking, as monitored using a temperature-sensitive vesicular stomatitis virus G protein (VSVG; Preston et al., 2009). However, whether ER-to-Golgi trafficking disruption is the cause of protein overload or, conversely, whether misfolded protein accumulation impacts ER integrity and function and therefore, as a second side effect, ER-secreting capacity, is still unclear. It has been shown in yeast that short-chain SFA, which induce UPR in a process that can be fully alleviated by 4-PBA treatment, do not affect the ER structure and do not significantly impair ER-to-Golgi protein trafficking (Pineau et al., 2009). In contrast, in β cells, ER-to-Golgi trafficking defect is unlikely to be secondary to the accumulation of misfolded proteins, because pharmacological depletion of ER calcium stores using thapsigargin markedly inhibited VSVG folding and promoted strong ER stress, but impacted VSVG delivery to the Golgi apparatus only moderately (Preston et al., 2009). In any event, defects in ER-to-Golgi trafficking are likely to contribute to misfolded protein overload in response to palmitate, and could account for the induction of full-fledged ER stress in both yeast and human cells.

An integrated view – biophysical consequences of SFA accumulation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Impacts of long-chain SFAs on the UPR- and ER stress pathways
  5. Lipid-induced ER stress and ER function
  6. An integrated view – biophysical consequences of SFA accumulation
  7. Future perspectives
  8. References

A fundamental question is how palmitate, and more generally long-chain SFA, can exert so many toxic effects on ER function, i.e. impact protein folding, calcium homeostasis, ER-to-Golgi secretion and ER morphology. A possible explanation would be that long-chain SFA may alter ER-membrane biophysical properties and therefore most (if not all) ER-membrane-related functions.

Exogenously supplied SFA are efficiently incorporated into phospholipids

The lipid composition of the ER is different from that of other organelles. First, it has the highest proportion of phosphatidylcholine (Zinser et al., 1991; Borradaile et al., 2006; Pineau et al., 2009), a phospholipid that is highly unsaturated under normal conditions in both yeast and human cells (Borradaile et al., 2006; Pineau et al., 2008). Second, cholesterol (or ergosterol in yeast) amounts are very low in this organelle (Zinser et al., 1993; Feng et al., 2003; Pineau et al., 2009). Collectively, these two features suggest that ER membranes have a relatively low lipid chain order, a parameter that may be essential for specialized ER-based processes, such as protein translocation.

A first important observation is that exogenously supplied SFA are efficiently incorporated into phospholipid species and particularly into phosphatidylcholine (Busch et al., 2005; Borradaile et al., 2006; Pineau et al., 2009). As a consequence, the unsaturation ratio of phosphatidylcholine acyl chains declines from 55% in yeast grown on oleate to 15% in cells exposed to palmitate (Pineau et al., 2009). More specifically, palmitate treatment results in a sevenfold increase of phosphatidylcholine species bearing two C16:0 acyl chains, in ER-enriched fractions (Pineau et al., 2009). Such modifications of the phospholipid acyl chain content can hardly be without consequences on the biophysical properties of the ER membrane. Similar effects have also been observed for other phospholipid species, such as phosphatidylethanolamine (Pineau et al., 2009).

The unsaturation level of phospholipids can regulate several properties of biological membranes including microdomain formation (such as rafts), fluidity/order and membrane curvature. However, at least in yeast, SFA accumulation has little impact on raft formation (Pineau et al., 2008) and, more surprisingly, ER-membrane fluidity (Pineau et al., 2009). However, it cannot be excluded that membrane order can be increased locally within specific ER subdomains, without significant impacts on the ER membrane as a whole.

Membrane curvature is regulated by the proportion of cylindrical vs. conical-shaped lipids (van den Brink-van der Laan et al., 2004; Boumann et al., 2006; de Kroon, 2007). Because the cross-sectional area of the lipid headgroup is similar to the cross-sectional area of the acyl chains (but this latest section may vary depending on the nature of the fatty acyl chains), some phospholipids such as phosphatidylcholine display an overall cylindrical shape and tend to organize themselves in bilayers. In contrast, other phospholipids with smaller headgroups such as phosphatidylethanolamine display conical shapes (type II lipids) and they tend to form nonlamellar phases with a negative curvature such as the hexagonal phase HII (van den Brink-van der Laan et al., 2004; de Kroon, 2007). When present in phospholipid bilayers, hexagonal phase-promoting lipids result in curvature stress, an overall property of the membrane that can influence the structure and activity of membrane-anchored proteins (for reviews, see Booth, 2003; van den Brink-van der Laan et al., 2004). It has been proposed that the presence of nonbilayer lipids may result in increased lateral pressure in the acyl chain region that could favor the packing of transmembrane helices (van den Brink-van der Laan et al., 2004). Curvature stress has also been shown to be a major driving force for vesicle budding (Huttner & Schmidt, 2002). The overall shape of a given phospholipid species can also be influenced by its fatty acyl chain composition (Boumann et al., 2006). As shown in Fig. 2, increasing the saturation level of phosphatidylcholine species would result in a decrease in the cross-sectional area of the acyl chains and, therefore, in a shift from a rather conical to a cylindrical shape. One may therefore speculate that increasing the amount of SFA in phosphatidylcholine species (and even most drastically in phosphatidylethanolamine) would decrease their contribution to membrane curvature/lateral stress and would also result in a higher membrane order.

image

Figure 2.  Schematic representation of the molecular shapes of phosphatidylcholine depending on its fatty acyl content. Space fill representations (obtained with rasmol 2.6) of phosphatidylcholine species bearing either two unsaturated (C16:1) (a) or two saturated (C16:0) (b) fatty acyl chains. Under palmitate treatment, the incorporation of long-saturated fatty acyl chains (C16:0) within PC would induce its shift from a rather conical to a cylindrical shape (c).

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Such a phenomenon could account for most of the effects attributed to palmitate toxicity both in yeast and in human cells. First, decreasing membrane curvature and/or increasing membrane order could impact protein folding in the ER, with a main effect on membrane-anchored protein, a process that may explain the observed misfolded protein overload associated with palmitate-induced ER stress (Lee, 2003, 2004). Second, this effect could also impair protein translocation to the ER lumen, because the translocation process has been shown to be highly sensitive to increased membrane order in vitro (Nilsson et al., 2001). Third, it could account for perturbations in ER–calcium homeostasis, by affecting, among others, the activity of SERCA (Li et al., 2004). Finally, it may also result in the disruption of ER-to-Golgi protein trafficking by direct impacts on vesicle formation in this organelle.

Suppressive effects of UFAs

By competing with SFA for phospholipid-synthesizing enzymes, oleate, and more generally UFA (such as myristoleic acid, C14:1), are efficiently incorporated into phospholipid, even under conditions of excess SFA. This is true when oleate originates from either exogenous (Pineau et al., 2008, 2009) or endogenous sources, i.e. when fatty acid desaturation is increased, due to elevated SCD1/Ole1p activity (Busch et al., 2005). As a consequence, under competition between SFA and UFA, the unsaturation level of fatty-acyl-containing lipids is, at least partly, restored (Busch et al., 2005; Pineau et al., 2009). The fact that high SCD1 levels can efficiently counteract SFA-associated toxicity in β cells (Busch et al., 2005; Lai et al., 2008) may be of physiological significance, because SCD1 is upregulated in obesity and can provide partial protection from obesity-induced insulin resistance (Pinnamaneni et al., 2006).

If the protective effects of UFA in β-cells likely result from a combination of factors, including fatty acid transport and/or trafficking, altered signal transduction and changes in transcriptional profiles, such a substrate-competition mechanism could account for part of these suppressive effects by reversing phospholipid conformation from a cylindrical to a conical shape (Fig. 2) and, consequently, by restoring low order/high lateral pressure within cellular membranes.

Future perspectives

  1. Top of page
  2. Abstract
  3. Introduction
  4. Impacts of long-chain SFAs on the UPR- and ER stress pathways
  5. Lipid-induced ER stress and ER function
  6. An integrated view – biophysical consequences of SFA accumulation
  7. Future perspectives
  8. References

Among the many perspectives in the field, elucidating the mechanisms that underlie SFA impacts on ER function will deserve special attention. In particular, studying the relative contribution of SFA to (still potential) preclusion of protein translocation, integral protein folding/activity and vesicle budding will be of particular interest. In this context, yeast genetics could undoubtedly contribute to address these questions.

Finally, a crucial point will be to demonstrate unambiguously the physiological and pathological significance of long-chain SFA toxicity, observed with β-cells in vitro, in the development of type 2 diabetes. More specifically, a clear-cut demonstration of the lipotoxic effects of SFA accumulation within cellular lipids in vivo (and particularly within phospholipid) under an SFA-enriched diet will have to be provided. Indeed, even if the phospholipid composition of selective tissues (including the liver and pancreas) has been shown to be responsive to SFA- and UFA-rich diets, their overall fatty acid content remains relatively constant over a wide range of dietary variations (Hulbert et al., 2005). Additional studies will therefore be required to determine whether such discrete variations in phospholipid fatty acyl content can indeed account for β-cell death in vivo.

References

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  2. Abstract
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  4. Impacts of long-chain SFAs on the UPR- and ER stress pathways
  5. Lipid-induced ER stress and ER function
  6. An integrated view – biophysical consequences of SFA accumulation
  7. Future perspectives
  8. References
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