By focusing on one branch of the UPR - the eIF2(αP)-mediated ISR - and by restricting the genetic manipulation to a single tissue - the liver - this study clarifies earlier work in mice with pervasive alterations in the ER stress response that were effected by germline mutations in UPR pathway genes. Both the hepatic ISR-defective Alb::GC
mice described here and the globally ISR-defective homozygous Eif2aS51A
mutant mice (Scheuner et al., 2001
), share a tendency towards fasting hypoglycemia and reduced hepatic glycogen content. This argues that at least part of the metabolic consequences of a defective ISR are autonomous to the hepatocyte.
The aforementioned hepatic defect is well explained by lower levels of C/EBPα and β protein in the liver of the Alb::GC
mice, as both C/EBPα and β are known to positively regulate genes involved in glycogen synthesis and hepatic glucose production (Wang et al., 1995
; Liu et al., 1999
). Our findings argue that ISR mediated activation of C/EBPα and β can proceed by a translational mechanism that is mobilized within minutes of induction eIF2α phosphorylation and functions independently of new mRNA synthesis. Translational activation is aided by previously-recognized transcriptional upregulation, which likely reinforces further C/EBP expression (Chen et al., 2004
). In these respects the C/EBPs resemble the well-validated translational target of the mammalian ISR, ATF4, and its yeast counterpart, GCN4. However, the arrangement of conserved upstream open reading frames in the C/EBPα and β genes suggests important differences between the molecular mechanism by which eIF2(αP) promotes their translation and that of ATF4/GCN4. Two conserved upstream open reading frames specify regulated translational re-initiation at the ATF4 (and GCN4) coding sequence when levels of phosphorylated eIF2α are high (Hinnebusch and Natarajan, 2002
; Lu et al., 2004a
; Vattem and Wek, 2004
). In the case of C/EBPα & β, our observations and those of Calkhoven and colleagues (Calkhoven et al., 2000
) are more readily explained by a model whereby eIF2(αP) disfavors initiation at the single inhibitory short open reading frame conserved in their mRNAs and favors initiation at downstream AUG(s). The mechanism linking eIF2(αP) to regulated initiation at two consecutive open reading frames, remains to be resolved, however the phenomena is not restricted to C/EBPα and β, as it is observed on the ATF4 mRNA when the 5’-most of the 2 upstream open reading frames is deleted (Lu et al., 2004a
Our study also implicates the ISR in regulating lipid metabolism in the liver, as the Alb::GC
mice accumulated less neutral lipid in their liver when placed on a high fat diet. This alteration, too, appears to be mediated by changes in gene expression, as levels of enzymes involved in fatty acid synthesis were lower in the ISR-defective transgenic mice, compared with the wildtype. Lower levels of PPARγ might explain part of this defect, as a requirement for hepatic PPARγ in the development of hepatic steatosis has been noted recently (Gavrilova et al., 2003
). C/EBP proteins positively regulate PPARγ expression (Millward et al., 2007
; Rahman et al., 2007
), which is consistent with a linear pathway from eIF2(αP) to the C/EBP proteins and from there to PPARγ (). Furthermore, the ISR defective Alb::GC
transgenic mice gain significantly less weight, when placed on high fat diet. While a detailed understanding of the physiological mechanisms awaits further studies, the pervasive role of eIF2(αP) in regulating genes involved in glycogenesis and lipid synthesis is consistent with the idea that impaired conversion of ingested nutrients to their storage forms limits weight gain in these ISR-defective animals.
In cultured cells transient activation of the ISR promotes survival and adaptation, whereas unremitting signaling promotes cell death (Rutkowski et al., 2006
). The observations made here provide an interesting parallel in terms of intermediary metabolism in the liver: Low level signaling in the ISR (as observed under physiological circumstances) promotes expression of genes involved in glycogen synthesis, gluconeogenesis and fatty acid synthesis, whereas higher levels of signaling repress the expression of the same genes. These findings might be explained by differential responsiveness of the ISR’s effectors to signaling at different intensities, as proposed (Rutkowski et al., 2006
). CHOP, ATF3 and C/EBPβ mRNA levels increase monophasically with signal strength, but expression of downstream C/EBP target genes decline at high levels of ISR activity (). CHOP-mediated inhibition of C/EBP proteins (Ron and Habener, 1992
) and direct repression of PEPCK by ATF3 (Allen-Jennings et al., 2002
) could contribute to the declining limb of the bi-phasic relationship between strength of ISR signal and downstream target gene expression.
Bi-phasic regulation of enzymes involved in fatty acid biosynthesis may also explain an apparent discrepancy between this study, in which signaling in the hepatic ISR is shown to promote steatosis in mice fed a high fat diet, and the observation that global Gcn2
deletion predisposes mice fed a leucine deficient diet to steatosis (Guo and Cavener, 2007
). Perhaps leucine deficiency is associated with levels of ISR signaling that repress lipid synthesis in wildtype mice but fail to achieve this level in the Gcn2−/−
mice. Alternatively, the lower levels of amino acid transporters noted in ISR defective cells (Harding et al., 2003
) may further reduce hepatic uptake of leucine and sensitize amino-acid deprived Gcn2−/−
mice to fatty liver by further reducing the building blocks for lipoprotein synthesis and thereby lipid export.
It has previously been reported that a partial compromise in downstream signaling in the IRE1 branch of the UPR (effected by haploid insufficiency for XBP-1) accentuates insulin resistance and promotes glucose intolerance in obese mice (Ozcan et al., 2004
) and that protein and chemical chaperones that reduce ER stress in insulin target tissues ameliorate that phenotype (Ozawa et al., 2005
; Ozcan et al., 2006
). Our study suggests a parallel process operating in hepatocytes, whereby heightened activity of the ISR and its downstream target genes contributes to the link between (physiological) ER stress and the metabolic syndrome of obesity and diabetes. In regard to intermediary metabolism, signaling by IRE1 and the ISR proceed in parallel and neither seems to dominate the metabolic phenotype. This is exemplified by the observations that insulin signaling to its proximal targets is not obviously affected by the ISR perturbation (data not shown); whereas such a defect is predicted in a system dominated by IRE1 signaling. Though loss of translational control in the ISR(−) state leads to more IRE1 signaling, which impairs insulin signaling, our study suggests that the net effect of a compromised ISR is to ameliorate the metabolic phenotype in mice exposed to nutrient excess.
It is interesting to speculate on the evolutionary origins of the link between the ISR and intermediary metabolism. Gene knockout experiments show that in most mammalian tissues the ER stress inducible kinase PERK dominates ISR activity (Harding et al., 2001
). However, the GCN2-posessing ancestor in which PERK first evolved already had in place a gene expression program responsive to eIF2(αP) that modulated intermediary metabolism (Hinnebusch and Natarajan, 2002
). We propose that this pre-existing link was co-opted by PERK to coordinate intermediary metabolism with nutritionally entrained variation in load of unfolded client proteins that enter the ER of insulin responsive tissues. This model is supported by the observation that yeasts, that lack PERK, also use ER activity as a proxy for nutrient availability, but effect this by IRE1 signaling (Schroder et al., 2000
; Patil et al., 2004
). Presumably, under conditions of limited nutrient availability this fine tuning of intermediary metabolism by physiological levels of ER stress (activity) is adaptive, however in the presence of excess nutrients the ISR’s contribution to lipid synthesis and hepatic glucose production are counterproductive and promote glucose intolerance and liver steatosis. Time will tell if this example of failure of homeostasis can be exploited therapeutically, by targeting distinct facets of the ISR with inhibitors.