The data in this report provide evidence for calcium-mediated regulation of HGP as part of a pathway that can be summarized as follows: glucagon/fasting → cAMP/PKA → IP3R1 → Ca2+i
→ nuclear FoxO1 → HGP. CaMKII also mediates elevated HGP in obese mice (), and although more work is needed in this area, it is possible that driving force here is also glucagon (Sorensen et al., 2006
; Unger and Cherrington, 2012
; Saltiel, 2001
). As such, the present findings have implications for three fundamental areas related to HGP: the molecular mechanisms whereby glucagon and fasting, as well as obesity/insulin resistance, stimulate HGP; the molecular links between intracellular calcium and HGP; and the regulation of FoxO1 nuclear transport. The latter issue is of particular interest, because while there have been many reports on how insulin/AKT-mediated phosphorylation of FoxO1, as well as FoxO1 acetylation, promote nuclear exclusion of FoxO1 (Lin and Accili, 2011
; van der Horst and Burgering, 2007
), there has been little emphasis on the regulation of FoxO1 nuclear entry
that occurs in the absence of insulin or in the setting of insulin resistance.
The CaMKII pathway is downstream of cAMP/PKA, and so it would naturally complement other glucagon-PKA pathways that stimulate HGP. Thus far, our data suggest that these other pathways occur in parallel with the CaMKII pathway rather than also being downstream of CaMKII. For example, glucagon-PKA directly phosphorylates cAMP response element binding (CREB) protein, which transcriptionally induces the FoxO1 transcriptional co-factor PGC1α (Herzig et al., 2001
), but there was no difference in nuclear CREB in livers from adeno-LacZ vs. KD-CaMKII mice (Figure S3B
). This was an important finding, because there are in vitro
data in neurons and in RANKL-treated RAW264.7 cells that CaMKII can activate/phosphorylate CREB in certain settings (Dash et al., 1991
; Sheng et al., 1991
; Ang et al., 2007
). We also found that CaMKII deficiency did not affect nuclear Crtc2 (Figure S3C
), which is another transcriptional activator involved in HGP. These data indicate that CaMKII in liver works in parallel with these other pathways, which together effect the nuclear localization of the proper array of transcriptional factors to mediate HGP. The case with Crtc2 is particularly interesting, because glucagon/PKA-mediated IP3R activation and ER calcium release promotes Crtc2 nuclear localization through another calcium-sensing enzyme, calcineurin (Y. Wang, G. Li, J. Goode, J. C. Paz, R. Screaton, W. H. Fischer, I. Tabas, and M. Montminy, manuscript submitted for publication). Indeed, we found that inhibition of CaMKII and calcineurin are additive in terms of suppressing forskolin-induced G6pc
mRNA (unpublished data). Thus, a common proximal signaling pathway leads to the coordinated nuclear entry of two key HGP transcription factors, Crtc2 and FoxO1, by different distal mechanisms. In this regard, it is interesting to note a previous study showing that drugs that promote calcium entry through the plasma membrane actually decrease Pck1
mRNA in HCs (Valera et al., 1993
), which may suggest that the route of calcium entry into the cytoplasm is a factor in determining downstream events.
FoxO1 is phosphorylated at Thr24, Ser253, and Ser316 (murine sequence numbers) by insulin/growth factors via Akt to promote its nuclear exclusion. It would be counterintuitive to propose that CaMKII phosphorylates these sites, because CaMKII promotes FoxO1 nuclear localization, but CaMKII could theoretically activate a phosphatase that de-phosphorylates these sites. However, CaMKIIγ deficiency did not affect the phosphorylation of these three residues, and it also did not affect FoxO1 acetylation (Figure S4A–B
). Instead, we found evidence that CaMKII mediates the phosphorylation of other Ser residues on FoxO1, and our Ser-Ala FoxO1 mutant experiments suggest that this action plays a role in CaMKII-mediated FoxO1 nuclear localization.
The link between CaMKII and FoxO1 phosphorylation may be direct or indirect. An indirect mechanism, i.e.
, whereby CaMKII activates another kinase, could be linked to previous findings that other kinases can phosphorylate FoxO on non-Akt sites in a manner that promotes their nuclear retention (Essers et al., 2004
; Chiacchiera and Simone, 2010
). Based on the p38 inhibitor and gene-targeting data herein and the study of Asada et al.
(Asada et al., 2007
), we suggest that p38 MAPK may also be able to carry out this function and, indeed, may be the mediator of CaMKII-induced FoxO1 nuclear localization. In support of this hypothesis are reports of links between CaMKII and p38 and between p38 and HGP (Cao et al., 2005
; Blanquet, 2000
). While there is no direct evidence yet that p38 phosphorylates and thereby activates FoxO1, the ability of glucocorticoids to promote FoxO1 nuclear localization in rat cardiomyocytes correlated with activation/phosphorylation of nuclear p38, and immunofluorescence microscopy and IP/immunoblot data suggested that p-P38 and FoxO1 may interact with each other (Puthanveetil et al., 2010
). Interestingly, there is evidence that FoxO1 may be able to activate p38 in HCs (Naimi et al., 2007
), and so it is possible that a FoxO1-p38 feed-forward pathway might amplify the effect the CaMKII-p38 pathway suggested here on FoxO1 nuclear localization. However, more work is needed to establish the role of p38 and to further elucidate the mechanisms whereby CaMKII promotes FoxO1 nuclear localization.
The discovery of the role of calcium-CaMKII in HGP not only provides insight into the physiologic defense against fasting hypoglycemia but may also reveal therapeutic targets for the disturbed glucose metabolism that occurs in the setting of insulin resistance, as suggested by the data in . Indeed, in type 2 diabetes, disproportionate HGP and an imbalance of glucagon vs. insulin signaling contributes to fasting hyperglycemia (Sorensen et al., 2006
; Saltiel, 2001
). Moreover, glucagon signaling has also been implicated in type 1 diabetes (Unger and Cherrington, 2012
). In this context, future studies will further address the pathophysiologic role(s) and mechanisms of hepatic CaMKIIγ in obesity, insulin resistance, and diabetes and thereby evaluate its potential as a therapeutic target in these disorders.