Obesity is a central risk factor in the development of insulin resistance, which is characterized by an inability for insulin to inhibit glucose output from the liver and to increase glucose uptake into skeletal muscle 10,11
. Although the underlying mechanism is unclear, obesity has been found to disrupt insulin signaling in liver and adipose through chronic increases in endoplasmic reticulum stress 3
. Because hepatic glucose production is also increased in obesity, we investigated whether ER stress signals modulate the gluconeogenic program directly.
Previous studies showing an important role for the CREB coactivator CRTC2 in promoting hepatic gluconeogenesis 1,2
led us to examine effects of ER stress in this setting. Exposure of primary hepatocytes to the ER stress activators thapsigargin (THA) or tunicamycin (TUN) 6
stimulated CRTC2 dephosphorylation and nuclear entry (; sup. fig. 1); these effects were blocked when cells were pre-treated with cyclosporine, an inhibitor of the Ser/Thr phosphatase calcineurin/PP2B, which has been shown to mediate CRTC2 dephosphorylation 12,13
Figure 1 Nuclear translocation and association of CRTC2 with ATF6α in response to ER stress. A. Effects of ER stress activators (THA, TUN) and FSK on CRTC2 dephosphorylation (top) and Ad-CRE luc reporter activity (bottom) in primary hepatocytes. Pre-treatment (more ...)
We used an adenovirally encoded cAMP responsive (Ad-CRE luc) reporter to monitor CREB:CRTC2 activity in primary hepatocytes. Although they stimulated an ER stress-inducible reporter (X Box Binding Protein 1 (Xbp1)-luc) 7,14
, THA and TUN inhibited Ad-CRE luc activity, even when cells were co-stimulated with the cAMP activator forskolin (FSK; , bottom).
Having seen that ER stress promotes CRTC2 activation but not CREB-dependent transcription, we considered the potential involvement of a CRTC2 inhibitor in this process. In proteomic studies to identify cellular proteins that associate with CRTC2, we recovered the basic leucine zipper (bZIP) transcription factor ATF6α from immunoprecipitates of endogenous CRTC2 (sup. fig. 2a). We confirmed the CRTC2: ATF6α interaction in co-immunoprecipitation studies of primary hepatocytes using endogenous and epitope-tagged proteins (; sup. fig. 2a).
Localized to the ER under basal conditions, ATF6α undergoes intramembrane proteolysis and nuclear shuttling in response to ER stress, when it promotes cell viability by stimulating ER quality control gene expression 4–9
. CRTC2 was found to interact with the transcriptionally active cytoplasmic N-terminal (ATF6 N; aa 1–381) domain but not with the ER luminal C-terminal domain of ATF6α (ATF6 C; aa. 382–670) (). Conversely, ATF6α associated with an N-terminal CRTC2 polypeptide (aa. 1–120) that also mediates an interaction with CREB (sup. fig. 2b) 15,16
We examined effects of ER stress on the recruitment of CRTC2 to ATF6α-regulated genes. Under basal conditions, about one-third of cellular CRTC2 was localized to the cytoplasmic surface of the ER (sup. fig. 3). Following exposure of primary hepatocytes to THA or TUN, ATF6α and CRTC2 shuttled to the nucleus where they occupied the Xbp1 promoter 7
(, top). ATF6α over-expression augmented CRTC2 occupancy, while RNAi-mediated knockdown of ATF6α blocked it (, bottom). And CRTC2 over-expression increased Ad-Xbp1 luc reporter activity whereas RNAi-mediated depletion of CRTC2 reduced it (). Consistent with a requirement for ATF6α, CRTC2 did not up-regulate Ad-Xbp1 luc activity when cells were depleted of ATF6α.
Figure 2 CRTC2 stimulates the expression of ER quality control genes through an association with ATF6α. A. Top, chromatin immunoprecipitation (ChIP) assay showing occupancy of CRTC2 and ATF6α over the Xbp1 promoter in primary hepatocytes exposed (more ...)
During ad libitum
feeding, CRTC2 activity is silenced through phosphorylation at Ser171 by Salt Inducible Ser/Thr Kinase 2 (SIK2); these effects are reversed during fasting, when glucagon inhibits SIK2 activity via protein kinase A (PKA)-mediated phosphorylation 1
. Pointing to a role for this kinase in the ER stress response, SIK2 over-expression disrupted Ad-Xbp1 luc reporter activity in ER-stressed hepatocytes, while RNAi-mediated knockdown augmented it (sup. fig. 4a, b). Conversely, over-expression of phosphorylation-defective, active S171A mutant CRTC2 increased Ad-Xbp1 luc reporter activity constitutively in cells co-expressing active ATF6N (sup. fig. 4c).
We examined whether CRTC2 modulates hepatic ER stress responses in vivo. Triggering of the ER stress pathway by intraperitoneal (IP) injection of TUN increased Ad-Xbp1 luc reporter activity and ER stress gene expression in both fasted and ad libitum fed mice (; Xbp1, GRP78, and CHOP; sup. figs. 4d, 5, 6); these effects were attenuated by RNAi-mediated depletion of hepatic ATF6α (sup. fig. 7). Similar to its effects in hepatocytes, Ad-CRTC2 expression also enhanced hepatic Ad-Xbp1 luc reporter activity and ER stress gene expression in TUN-injected mice, while RNAi-mediated depletion of CRTC2 down-regulated it (; sup. fig. 8). Taken together, these results indicate that CRTC2 promotes the expression of ER quality control genes in liver via an association with ATF6α.
We considered that ATF6α could interfere with induction of the gluconeogenic program through the CREB:CRTC2 pathway should cellular levels of CRTC2 be limiting. Supporting this idea, exposure of primary hepatocytes to THA or TUN increased the binding of ATF6α (p50) to CRTC2 and reciprocally reduced amounts of CRTC2 associated with CREB (, top). Over-expression of nuclear, active ATF6N also decreased the CREB:CRTC2 interaction, and it blocked recruitment of CRTC2 to the gluconeogenic G6Pase promoter in FSK-treated cells (; sup. fig. 9). ATF6N expression also decreased Ad-CRE luc reporter activity, gluconeogenic gene expression, and glucose output from primary hepatocytes, while RNAi-mediated depletion of ATF6α increased it (; sup. fig. 9). Confirming the importance of the ATF6α:CRTC2 interaction, a mutant (Arg337Ala) ATF6α polypeptide with lower affinity for CRTC2 was less potent in disrupting CREB activity relative to wild-type (sup. fig. 10).
Figure 3 CRTC2 mediates cross-talk between hepatic ER stress and fasting pathways. A. Top, immunoblot showing recovery of CREB from co-IPs of CRTC2 prepared from nuclear extracts of primary hepatocytes exposed to FSK, TUN, or THA. Bottom, effect of ATF6α (more ...)
Because ATF6α and CREB bind to the same domain in CRTC2, CREB may reciprocally down-regulate ATF6α activity. Indeed, Ad-CREB expression not only reduced Ad-Xbp1 luc reporter activity, but it also increased CRE-luc reporter activity and glucose production in hepatocytes expressing ATF6N (, sup. fig. 11). Taken together, these results indicate that CREB and ATF6α exert counter-regulatory effects on gluconeogenesis, in part by competing for CRTC2 .
We tested the role of ATF6α in modulating glucose balance in vivo. Modest hepatic over-expression of ATF6α lowered the fasting gluconeogenic profile - which consists of hepatic Ad-CRE luc activity, gluconeogenic gene expression, and circulating blood glucose concentrations - in control mice, and to a greater extent in mice injected IP with TUN (; sup. fig. 12a). By contrast, RNAi-mediated depletion of hepatic ATF6α increased the fasting gluconeogenic profile in both control and TUN-injected animals (; sup. fig. 12b).
Considering that ER stress is chronically upregulated in obesity 3
and that ATF6α undergoes proteasome-dependent degradation when ER stress is prolonged 8
, we tested whether hepatic ATF6α activity is altered in this setting. Relative to lean controls, obese (ob/ob
) mice exhibited lower Ad-Xbp1 luc reporter activities, and they had reduced hepatic ATF6α protein amounts (; sup. fig. 13). By contrast, Ad-CRE luc activity, gluconeogenic gene expression, and circulating blood glucose concentrations were all elevated in both ob/ob
Figure 4 Reciprocal down-regulation of ATF6α and up-regulation of CREB in obesity. A. Left, hepatic Ad-Xbp1 luc and Ad-CRE luc activities in obese db/db mice relative to lean controls. Right, immunoblot of hepatic extracts from wild-type and db/db mice (more ...)
Because RNAi-mediated depletion of ATF6α increases CRE-luc activity and hepatic glucose output, we wondered whether the obesity-related loss of hepatic ATF6α would have similar effects on CREB and CRTC2. In that case, ATF6α over-expression should improve glucose balance by lowering gluconeogenic gene expression. Supporting this notion, adenoviral expression of active ATF6N reduced fasting hepatic Ad-CRE luc activity as well as mRNA amounts for gluconeogenic genes in both db/db and high fat diet-fed (DIO) mice (; sup. fig. 14). Over-expression of ATF6N also lowered circulating blood glucose levels and enhanced glucose tolerance (; sup. fig. 15). Arguing against a significant effect of ATF6N on insulin signaling per se, hepatic amounts for inactive phospho (Ser307) Insulin Receptor Substrate 1 (IRS1) or active phospho-(Thr308) AKT appeared comparable between ATF6N-expressing and control mice (sup. fig. 15).
Taken together, these results indicate that CRTC2 functions as a dual sensor for fasting and ER stress signals (sup. fig. 16). The attendant cross-talk between these pathways appears to protect against excessive increases in hepatic gluconeogenesis that otherwise lead to chronic hyperglycemia in obesity. In addition to its effects on ATF6α, chronic ER stress has also been found to increase hepatic gluconeogenesis and lipogenesis by triggering other branches of the unfolded protein response 17
. Future studies should reveal the extent to which these pathways promote glucose intolerance through CREB or other bZIP transcription factors.