The requirement of Ser307 for insulin sensitivity in knock-in mice runs counter to prevailing notions about the function of this phosphorylation site. To date, however, almost all of the evidence linking Ser307 phosphorylation to insulin resistance in whole animals has been formally correlative in nature. In one exceptional study, it was recently shown that transgenic mice expressing triply-mutated (Ser 302/307/612→Ala) Irs1 in their skeletal muscle are protected against development of high-fat diet-induced insulin resistance (Morino et al., 2008
); however, the incompletely reductive design of this study makes it impossible to infer the specific contribution of Ser307. In contrast, we observed significant insulin resistance in high-fat-fed Ser307Ala knock-in mice (A/A), suggesting that the enhanced sensitivity of Ser 302/307/612 →Ala transgenic mice may owe to mutation of Ser302, Ser612, or both sites together.
In established cells that overexpress the IR and Irs1, Ser307 was previously shown to be required for the modest inhibition of Irs1 Tyr phosphorylation caused by TNFα treatment (Aguirre et al., 2000
). However, another cell-based study found no effect of a phospho-mimetic Ser307Asp mutation on insulin-stimulated glucose disposal, suggesting that Ser307 phosphorylation does not contribute substantially to insulin resistance (Weigert et al., 2008
). Compatible with this result, we observed mild effects of the Ser307Ala mutation in chow-fed A/A and A/+ mice that nevertheless progressed to insulin resistance upon high fat feeding or deletion of hepatic Irs2. In further contrast with established cell lines (Aguirre et al., 2000
; Weigert et al., 2008
), both HFD-fed A/A mouse muscles and primary hepatocytes from A/lox::LKO2 mice displayed slightly reduced or unchanged tyrosyl phosphorylation of A307Irs1
following insulin stimulation. In both cases, this was accompanied by decreased insulin-stimulated binding of PI3K to Irs1, without a marked decrease in Akt phosphorylation. In muscles of HFD-fed A/A mice, this may have owed to Akt activation via the Irs2 pathway; however, it is also possible that we did not choose the appropriate time to monitor Akt phosphorylation, which is typically rapidly saturated and long-lasting. We conclude that Ser307 in whole animals promotes insulin sensitivity through a moderate, but physiologically significant, effect to maintain PI3 kinase binding to Irs1.
As in other animal-based studies (e.g.,Um et al., 2004
), we noted significant stimulation of Ser307 phosphorylation by insulin, even in tissues of HFD-fed mice. Further, in primary hepatocytes infected with WT Irs1 adenovirus, Ser307 was strongly phosphorylated by insulin treatment. While these data are compatible with a positive role of Ser307 in signaling by Irs1, in other experiments we found that only a fraction of the total Irs1 in muscles of chow-fed mice or WT hepatocytes is phosphorylated on Ser307 by insulin stimulation (K.C., M.W.). Thus, Ser307 phosphorylation is not a sine qua non
for signaling via Irs1, but may function to maintain signaling by the Ser307-phosphorylated fraction. Fluctuation in the size of this fraction—for example, as influenced by diet—might therefore have modulated the severity of physiologic and signaling defects observed in our studies. The phosphorylation of other Ser/Thr sites is also likely to have affected Irs1 function; however, we did not observe markedly different phosphorylation of WT and A307 Irs1 at eight other Ser/Thr sites in adenovirus-infected hepatocytes, suggesting that the insulin resistance of A/A mice owed primarily to the effect of Ser307 status. Thus, despite clear upregulation of Ser307 phosphorylation in animal models of insulin resistance, this modification may have an adaptive, rather than pathogenic function. Extension of the knock-in mouse approach used here to other Ser/Thr sites will help to determine how common, or rare, this function might be.