LKB1 has been implicated in a diverse array of roles, many of which impact on human health and disease. In addition to functioning as a tumor suppressor and the causative agent of Peutz-Jeghers syndrome (21
), for example, LKB1 has been shown to control glucose homeostasis in the liver (41
). Since cancer and diabetes constitute the second and sixth leading causes of death among Americans (Health, United States, 2005; http://www.cdc.gov/nchs/hus.htm
), continuing studies of the function of LKB1 are likely to provide novel insights into diseases that affect the health of many worldwide. In this study, we show that LKB1 plays a prominent role in the negative regulation of glucose homeostasis in skeletal muscle. Specifically, MLKB1KO mice displayed improved whole-body glucose homeostasis, enhanced insulin sensitivity in skeletal muscle, and increased insulin-stimulated Akt signaling in skeletal muscle. Interestingly, our preliminary studies show that the improved glucose tolerance is maintained in MLKB1KO mice following 5 weeks on a high-fat diet (N. Fujii, H. J. Koh, and L. J. Goodyear, unpublished observations).
Given the putative links between AMPK activation and improvements in insulin sensitivity (5
), as well as the role of LKB1 as a positive regulator of glucose homeostasis in the liver (41
), our finding of improved insulin sensitivity and glucose homeostasis in MLKB1KO mice is surprising. The improved whole-body glucose homeostasis is associated with increased skeletal muscle insulin-stimulated glucose uptake in vivo, and enhanced Akt phosphorylation could be an important mechanism for this effect (7
). Given the role of Akt in cell proliferation and tumor genesis, increased signaling through Akt in the absence of LKB1 is consistent with the well-established role of LKB1 as a tumor suppressor.
We believe that the enhancements in Akt signaling and ultimately in whole-body glucose homeostasis in the MLBK1KO mice are due, at least in part, to downregulation of TRB3 expression in skeletal muscle. Consistent with this hypothesis is our finding that heterozygous mice, which do not have enhanced glucose tolerance or decreases in fasting blood glucose or insulin concentrations, also do not have decreases in muscle TRB3 expression. In recent studies, TRB3 has been found to inhibit Akt activation in liver by physically binding to Akt and masking the Thr308
activation site (10
). In this study, we show that TRB3 binds Akt in skeletal muscle and that increases in TRB3 expression in C2C12 cells result in decreased insulin-stimulated Akt phosphorylation. TRB3 thus appears to downregulate Akt signaling in skeletal muscle, as it does in liver. Since reduced TRB3 protein levels in both liver (10
) and skeletal muscle (current study) are associated with enhanced glucose tolerance in vivo, the development of TRB3 inhibitors could be extremely valuable in the treatment of type 2 diabetes.
An important implication of the current study is that LKB1 regulates TRB3 expression, providing a novel link between this tumor suppressor protein and a key signaling molecule mediating cell metabolism. In the liver, PGC1α and PPARα have been shown to regulate TRB3 expression (25
). Here, our data suggest that LKB1 regulation of TRB3 expression occurs through the PGC1α/PPARα pathway in skeletal muscle. Previous reports on skeletal muscle have shown that increases in LKB1 protein with chronic exercise training (45
) and activation of AMPK via AICAR (24
) correlate with increases in PGC1α expression in skeletal muscle. Given that AMPKα2 is required for the AICAR effect in skeletal muscle (24
), it seems plausible that the reductions in AMPKα2 activity in MLKB1KO muscle may partially mediate the decreases in PGC1α and PPARα expression. However, compared to MLKB1KO mice, transgenic mice expressing dominant-negative AMPKα2 (13
) do not have decreases in PGC1α or PPARα and have only slight decreases in TRB3 (N. Fujii, H. J. Koh, and L. J. Goodyear, unpublished observations), suggesting that other LKB1-regulated kinases play a role in the regulation of these proteins in muscle.
While we believe that increased Akt signaling mediated by downregulation of TRB3 is likely the primary cause of the enhanced glucose tolerance and insulin signaling in the MLKB1KO mice, we cannot entirely rule out other possibilities that may contribute to the phenotype of these mice. One alternative potential mechanism is that the lack of AMPKα2 activity and marked decrease in ACC phosphorylation result in a severe reduction in muscle fatty acid oxidation. This hypothesis is supported by the increased intramuscular triglycerides that we observed in the MLKB1KO mice. In response to a reduction in fatty acid oxidation, MLKB1KO mice may preferentially utilize glucose over fatty acids for ATP generation, leading to the reductions in muscle glycogen content that we observed. Lower muscle glycogen content and increased glycogen synthase activity are a combination that is well known to increase insulin-stimulated glucose uptake in skeletal muscle (22
). However, this hypothesis is not fully supported by previous data for whole-body AMPK knockout mice. Similarly to MLKB1KO mice, AMPKα2 knockout mice have decreased ACC phosphorylation and lower muscle glycogen content, yet the AMPKα2 knockout mice have impaired glucose tolerance, which could be due to nonmuscle effects (23
). In addition, there is no significant phenotype in the AMPKα1 knockout mice (46
). Thus, the available data suggest that the enhanced glucose homeostasis in the MLKB1KO mice is due to upregulation of Akt signaling, stemming from decreased TRB3 expression caused by the loss of LKB1 activity.
LKB1 has been termed a “master kinase” that can increase not only AMPK activity but also the activities of at least 13 AMPK-related kinases (20
). Of these additional 13 kinases, only MARK2/3, MARK4, QSK, and QIK/SIK2 are detectable in skeletal muscle, and none are regulated by AICAR, contraction, or phenformin (36
). In the current investigation we found that MARK4 but not MARK2/3 activities were reduced for MLKB1KO mice. Whether downregulation of skeletal muscle MARK4 is a mechanism for improved glucose homeostasis and alterations in TRB3 expression will be an important area of future investigation, and those studies are ongoing in our laboratory.
Recently, Sakamoto et al. reported generation of LKB1 knockout mice that have an approximately 90% reduction in LKB1 in all tissues and ablation of LKB1 activity in skeletal muscle (38
). While our model and theirs show similar effects in regard to AMPKα2 activity, there appear to be very significant differences in the metabolic phenotypes. While we show a pronounced improvement in glucose homeostasis and insulin sensitivity for MLKB1KO mice, Sakamoto et al. reported normal blood glucose concentrations up to 10 weeks of age and reported little additional physiological data. The major differences between the studies may be due to the 90% hypomorphic phenotype of their LKB1flox/flox
mice; our double-floxed mice did not have significant decreases in LKB1 protein or enzyme activity (Fig. ). The significance of this difference is highlighted by a recent report that hepatic LKB1 is essential for the regulation of gluconeogenesis and normal glucose tolerance (41
). Given that loss of hepatic LKB1 leads to glucose intolerance and fasting hyperglycemia and our data showing that loss of skeletal muscle LKB1 leads to improvements in glucose tolerance, it seems plausible that for the hypomorphic animals described by Sakamoto, the beneficial effects of a reduction in skeletal muscle LKB1 expression on glucose homeostasis are masked by the detrimental effects of a reduction in hepatic LKB1 expression. Interestingly, loss of LKB1 in liver leads to increases in PGC1α (41
), whereas we clearly show decreases in PGC1α in skeletal muscle (Fig. ), which likely explains the opposite effects of liver and muscle LKB1 deficiency on glucose homeostasis. Recently, a decrease in TORC2 phosphorylation has been proposed to be a mechanism by which LKB1 regulates PGC1α in liver. However, we found that TORC2 was barely detectable in mouse skeletal muscles compared to levels in liver, and we found no evidence for alterations in TORC2 phosphorylation in MLKB1KO mice (H. J. Koh and L. J. Goodyear, unpublished observation), implying that LKB1 regulates PGC1α and TRB3 through a distinct mechanism in skeletal muscle.
While LKB1 has been reported to regulate both AMPKα1 and α2 activities in vitro and in cell culture systems (15
), our results suggest that LKB1 is necessary for the activation of AMPKα2 but not AMPKα1. This finding is consistent with recent studies performed with cardiac muscle, in which LKB1 was found to be necessary for hypoxia-induced activation of AMPKα2 but not α1 (39
). We are unable to determine whether this is due to isoform-specific AMPKα2 activation by LKB1 or to the presence of an alternative AMPKα1 kinase. Another possible explanation for the divergence between AMPKα1 and AMPKα2 activities in the MLKB1KO mice is that the majority of AMPKα1 activity present in muscle tissue is not of skeletal muscle origin. AMPKα1 is highly expressed in vascular endothelial and smooth muscle cells, which are embedded in skeletal muscle tissue (35
). Therefore, it is possible that AMPKα1 is regulated by LKB1 but is expressed only at low levels in skeletal muscle and that most of the AMPKα1 activity detected may be from nonmuscle cells which would have normal LKB1 expression in MLKB1KO animals.
In summary, in addition to serving as the major upstream kinase for the regulation of AMPKα2 activity in skeletal muscle, skeletal muscle LKB1 is a negative regulator of glucose homeostasis and insulin sensitivity. Disruption of LKB1 in skeletal muscle results in enhanced glucose tolerance and upregulation of insulin sensitivity for glucose uptake and signaling in skeletal muscle; this affect appears to be mediated, at least in part, through PGC1α- and PPARα-mediated downregulation of TRB3 expression, resulting in enhanced insulin-stimulated Akt signaling. Given that whole-body AMPKα2 knockout mice have impaired glucose homeostasis (46
), it is possible that LKB1 itself or non-AMPK substrates, such as the MARK proteins, are responsible for the beneficial adaptations to muscle insulin sensitivity in MLKB1KO mice. The concept of enhanced insulin or growth factor signaling in the absence of LKB1, including upregulation of Akt, is consistent with the well-established role of LKB1 as a tumor suppressor and provides additional evidence for the dual function of LKB1 in both cell metabolism and cancer biology.