In this study we examined the significance of insulin signaling on mitochondrial function in pancreatic β-cells. We report that disruption of insulin signaling in β-cells alters the stoichiometry of a mitochondrial BAD/GK complex, reduces GK activity and promotes mitochondrial dysfunction. Re-expression of the insulin receptor in β-cells that lack insulin receptors largely restores the complex and improves mitochondrial function, highlighting a significant role for insulin signaling in the maintenance of the BAD/GK complex and in the regulation of mitochondrial function.
The identification of a BAD/GK complex in both mouse and human β-cells in this study indicates conservation across species and supports the concept of a potential functional link between apoptosis and glycolysis at the mitochondria, which is similar to observations in hepatocytes 
. Indeed, the WAVE protein in the complex has been suggested to anchor GK and BAD to the mitochondria to efficiently co-ordinate spatial and temporal aspects of glucose metabolism 
. The alterations in the expression of different components in the complex in mitochondria from β-cells from βIRKO provide potential insights into the altered function of β-cells and may explain some of the phenotypes observed in βIRKO mice 
. Similar to effects observed in BAD-deficient hepatocytes, decreased BADS
protein in the βIRKO cells likely contributes to mitochondrial dysfunction and β-cell dysfunction, which may contribute to the glucose intolerance in both of these mutants 
. The BAD/GK complex was also disrupted in islets from humans with type 2 diabetes and showed an interesting similarity with islets from bIRKO mice. Thus it is possible that reduced BADS
in the mitochondria may be linked to the blunted glucose response and altered mitochondrial ultra structure observed in islets isolated from patients with type 2 diabetes 
Our study provides evidence that altered insulin signaling is associated with mitochondrial function in the β-cell, as has recently been reported in cardiomyocytes 
. The mechanism for mitochondrial dysfunction in βIRKO cells is multifactorial and includes reduced glucose-mediated augmentation in mitochondrial membrane potential and reduced ATP levels that could be due either to decreased synthesis or to increased consumption in order to maintain the membrane potential. Of interest, mitochondrial dysfunction occurs despite increased oxygen consumption. These observations suggest that despite reduced glucokinase activity and potentially reduced glucose delivery to βIRKO mitochondria they are also uncoupled. One potential mechanism for mitochondrial uncoupling is increased UCP2 expression, which may be driven by increased expression of PGC-1α and β 
. Moreover, increased UCP2 expression has been implicated in limiting stimulus-secretion coupling in β-cells in some animal models of diabetes but not in others 
. However, it is possible that this increased UCP2 expression may not contribute directly to mitochondrial dysfunction in βIRKO cells and it could be the result of ROS overproduction in response to glucose stimulation since it was found that deletion of insulin receptors in heart increases their propensity to enhance ROS when exposed to fatty acid substrates 
. Also, DNP experiments suggest that the capacity of the OXPHOS machinery may not be intrinsically impaired in βIRKO cells; the improvement in oxygen consumption that we see in βIRKO and the rescued cells might be due to altered mitochondrial mass (Fig. S3B
); that might increase total mitochondrial respiration. Together, the mitochondrial defects in βIRKO cells could be results of combined effects from reduced GK activity, ROS production and altered UCP2 expression. Finally, we also measured mitochondrial membrane potential response to glucose stimulation, and βIRKO cells that re-expressed the insulin receptors showed a trend towards improved membrane potential response in response to glucose (Fig. S3A
), which is also consistent with the increased respiration. Although our experiments have not definitively identified all mechanisms that could account for mitochondrial dysfunction, our study clearly shows that impaired insulin signaling in β-cells fundamentally alters mitochondrial bioenergetics and function, which may ultimately impair insulin secretion.
Consistent with the growth factor-induced phosphorylation and translocation of BADS 
we observed significant alterations in mitochondria isolated from β-cells lacking the insulin receptor. The high level of basal phosphorylation of Ser112-BAD and Ser136-BAD in the βIRKOs likely reflects the effects of signaling via the IGF-1 receptors. It is possible that the relative levels of activation of insulin versus IGF-1 receptors determine the overall phosphorylation state of BAD at the mitochondria, with Ser112 phosphorylation being specifically mediated by Akt-independent pathways, and Ser136 phosphorylation being activated by both Akt and non-Akt pathways 
Intact IGF-I signaling in βIRKO cells could mediate the activation of RSK and MSK1 to phosphorylate BAD at Ser-112 
. The phosphorylation state of BADS
has also been reported to impact its binding with other proteins such as Bcl-XL 
. Further, the silencing of all serine sites in the BAD3SA/3SA
mouse has been reported to reduce GK activity in hepatocytes while disruption of Ser-155 down-regulates GK activity in islets 
. These studies suggest that phosphorylation of BADS
can modulate GK activity and impact apoptosis, likely by disruption of protein interactions in the complex. Our data suggest that the level of BADS
phosphorylation correlates with function. This is reflected by the finding that βIRKO cells exhibit extremely high Ser112 phosphorylation in mitochondria ( and Fig. S2B
) and phosphorylation can potentially interrupt the interaction between BADS
and other proteins, including GK. Furthermore, re-expression of insulin receptors reversed the increase in Ser112 phosphorylation of BADS
and increased the content of total BADS
in the complex. Together, these data suggest the potential relevance of interactions between Ser112 phosphorylation and function of the complex. Based on the report by Danial et al 
, restoration of BADS
in the complex would augment glucokinase function and mitochondrial metabolism. Additional studies such as mutagenesis of Ser112 of BADS
will be necessary to prove a direct role of this phosphorylation event. In βIRKO mitochondria it is possible that phosphorylation at Ser-112 disrupts the residence of BADS
in the mitochondrial complex and alters glucokinase activity and mitochondrial metabolism without affecting Ser-155-BAD (data not shown). These observations are consistent with the altered glucose sensing and altered apoptosis of β-cells in βIRKO mice 
. The ability to reverse basal Ser-112 phoshorylation and restore BADS
content to control levels by re-expressing the insulin receptor in βIRKO cells further underscores the role of insulin signaling in regulating the mitochondrial complex.
Another signaling pathway that could potentially influence the stoichiometry of the BAD/GK complex is the elevated expression of PTEN secondary to enhanced IGF-I signaling in βIKRO cells 
. Increased PIP2 could decrease the binding of PKA in the VCA domain of the WAVE protein, which is known to anchor GK and BAD to the mitochondria to efficiently coordinate spatial and temporal aspects of glucose metabolism 
and may contribute to the partial normalization of PKA content upon re-expressing the insulin receptor in βIRKO cells.
In the cytosol, the elevated p70S6K activation in βIRKO cells could potentially serine-phosphorylate IRS1 and further dampen insulin-stimulation of Akt in βIRKO cells 
(), and potentiate IGF-I mediated pathways in the mutant cells. Indeed, activation of Akt/PKB has been reported to inhibit IGF-I activation of MAPK and mTOR pathways via Raf1 kinase, while adenoviral knockdown of Akt/PKB in INS-1 cells leads to enhanced ERK1/ERK2 signaling that is independent of IGF-I signaling 
Schematic showing the impact of insulin/IGF-I signaling on β-cell mitochondrial metabolism and function.
We observed high levels of JNK1 activation in mitochondria but not in cytosolic fractions in βIRKO β-cells, which is consistent with the report that JNK1 activation is linked to dysfunctional mitochondria 
. The activation of JNK1 by IGF-I, but not by insulin, in both cytosolic and mitochondrial fractions in control β-cells, implicates a role for IGF-I in the stress signaling pathway and is consistent with the potentiation of cytokine-mediated JNK following long-term IGF-I treatment 
. We suggest that JNK1 could potentially contribute to Ser-112 phosphorylation on BADS
to affect mitochondrial metabolism and function (). Our observations are consistent with the notion that down-regulation of Akt does not influence basal mitochondrial respiration 
. It will be useful to dissect further, how insulin and IGF-I signaling pathways differentially modulate MAPK and mTOR target proteins, such as p70S6K and JNK1, to regulate mitochondrial function in β-cells. Further work is also necessary to explore whether insulin/IGF-I signaling directly modulates PGC-1α to affect mitochondrial biogenesis and function 
In summary, this study provides novel insights into the role of insulin signaling in the regulation of the BAD/GK complex, glycolytic enzyme activity and mitochondrial metabolism in pancreatic β-cells. Ser112-BADS and its upstream kinases may be potential targets for the maintenance of the BAD/GK complex that is necessary for normal mitochondrial function and the regulation of β-cell survival.