Evidence for functional redundancy of class IA PI3K isoforms in insulin signalling.

Recent genetic knock-in and pharmacological approaches have suggested that, of class IA PI3Ks (phosphatidylinositol 3-kinases), it is the p110alpha isoform (PIK3CA) that plays the predominant role in insulin signalling. We have used isoform-selective inhibitors of class IA PI3K to dissect further the roles of individual p110 isoforms in insulin signalling. These include a p110alpha-specific inhibitor (PIK-75), a p110alpha-selective inhibitor (PI-103), a p110beta-specific inhibitor (TGX-221) and a p110delta-specific inhibitor (IC87114). Although we find that p110alpha is necessary for insulin-stimulated phosphorylation of PKB (protein kinase B) in several cell lines, we find that this is not the case in HepG2 hepatoma cells. Inhibition of p110beta or p110delta alone was also not sufficient to block insulin signalling to PKB in these cells, but, when added in combination with p110alpha inhibitors, they are able to significantly attenuate insulin signalling. Surprisingly, in J774.2 macrophage cells, insulin signalling to PKB was inhibited to a similar extent by inhibitors of p110alpha, p110beta or p110delta. These results provide evidence that p110beta and p110delta can play a role in insulin signalling and also provide the first evidence that there can be functional redundancy between p110 isoforms. Further, our results indicate that the degree of functional redundancy is linked to the relative levels of expression of each isoform in the target cells.

Reduced PDE4 expression and activity contributes to enhanced catecholamine-induced cAMP accumulation in adipocytes from FOXC2 transgenic mice.

Overexpression of forkhead transcription factor FOXC2 in white adipose tissue (WAT) leads to a lean phenotype resistant to diet-induced obesity. This is due, in part, to enhanced catecholamine-induced cAMP-PKA signaling in FOXC2 transgenic mice. Here we show that rolipram treatment of adipocytes from FOXC2 transgenic mice did not increase isoproterenol-induced cAMP accumulation to the same extent as in wild type cells. Accordingly, phosphodiesterase-4 (PDE4) activity was reduced by 75% and PDE4A5 protein expression reduced by 30-50% in FOXC2 transgenic WAT compared to wild type. Thus, reduced PDE4 activity in adipocytes from FOXC2 transgenic mice contributes to amplified beta-AR induced cAMP responses observed in these cells.

Insulin induces SOCS-6 expression and its binding to the p85 monomer of phosphoinositide 3-kinase, resulting in improvement in glucose metabolism.

The suppressors of cytokine signaling (SOCS) family is thought to act largely as a negative regulator of signaling by cytokines and some growth factors. Surprisingly, the SOCS-6 transgenics had no significant defects in the cytokine signaling and hematopoietic system but displayed significant improvements in glucose metabolism. Insulin stimulation of Akt/protein kinase B was also potentiated. Biochemical analysis showed that, after insulin stimulation, SOCS-6 interacted with the monomeric p85 subunit of class-Ia phosphoinositide (PI) 3-kinase but not with p85/p110 dimers. Furthermore, SOCS-6 expression is transiently increased by serum and insulin in normal fibroblasts. However, both the mRNA and protein of SOCS-6 were rapidly degraded after induction by insulin. The degradation of the SOCS-6 protein was partially inhibited by a proteasome inhibitor, suggesting a proteasome-mediated degradation mechanism. In contrast, SOCS-6-associated p85 was not degraded and could be recruited to the newly synthesized SOCS-6 molecules in the presence of insulin, suggesting that SOCS-6 expression and its interaction with p85, but not the degradation, is regulated by insulin. The phenotype of SOCS-6 transgenic mice bears a striking resemblance to p85 knock-out mouse models in which glucose metabolism stimulated by insulin is significantly improved despite reduced activation of PI 3-kinase. This suggests that monomeric p85 might play a physiologically important role in attenuating signaling through PI 3-kinase-dependent pathways in unstimulated cells. Therefore, our results indicate that SOCS-6 may provide a dynamically regulated mechanism by which insulin can transiently overcome the negative effects that p85 monomers have on signaling via PI 3-kinase-dependent signaling pathways.

A winged helix forkhead (FOXD2) tunes sensitivity to cAMP in T lymphocytes through regulation of cAMP-dependent protein kinase RIalpha.

Forkhead/winged helix (FOX) transcription factors are essential for control of the cell cycle and metabolism. Here, we show that spleens from Mf2-/- (FOXD2-/-) mice have reduced mRNA (50%) and protein (35%) levels of the RIalpha subunit of the cAMP-dependent protein kinase. In T cells from Mf2-/- mice, reduced levels of RIalpha translates functionally into approximately 2-fold less sensitivity to cAMP-mediated inhibition of proliferation triggered through the T cell receptor-CD3 complex. In Jurkat T cells, FOXD2 overexpression increased the endogenous levels of RIalpha through induction of the RIalpha1b promoter. FOXD2 overexpression also increased the sensitivity of the promoter to cAMP. Finally, co-expression experiments demonstrated that protein kinase Balpha/Akt1 work together with FOXD2 to induce the RIalpha1b promoter (10-fold) and increase endogenous RIalpha protein levels further. Taken together, our data indicate that FOXD2 is a physiological regulator of the RIalpha1b promoter in vivo working synergistically with protein kinase B to induce cAMP-dependent protein kinase RIalpha expression, which increases cAMP sensitivity and sets the threshold for cAMP-mediated negative modulation of T cell activation.

Glucose-dependent regulation of cholesterol ester metabolism in macrophages by insulin and leptin.

Insulin resistance, obesity, and diabetes are characterized by hyperglycemia, hyperinsulinemia, and hyperleptinemia and are associated with increased risk of atherosclerosis. In an effort to understand how this occurs, we have investigated whether these factors cause disregulation of cholesterol ester metabolism in J774.2 macrophages. Raising glucose levels alone was sufficient to increase uptake of acetylated low density lipoprotein but did not stimulate synthesis of cholesterol esters. In the presence of high glucose, both insulin and leptin increased the rate of cholesterol ester synthesis, although they did not further increase uptake of acetylated low density lipoprotein. However, in the presence of high glucose both insulin and leptin caused a significant increase in the activity of acyl-CoA: cholesterol O-acyltransferase (ACAT) combined with a significant reduction in the level of hormone-sensitive lipase (HSL). Because ACAT is the main enzyme responsible for cholesterol ester synthesis and HSL contributes significantly to neutral cholesterol ester hydrolase activity, this suggests that glucose primes the J774.2 cells so that in the presence of high insulin or leptin they will store cholesterol esters. This contrasts with 3T3-L1 adipocytes, where HSL activity and expression are increased by insulin in high glucose conditions. These findings may provide an explanation for the observation that in conditions characterized by hyperglycemia, hyperleptinemia, and hyperinsulinemia, triglyceride lipolysis in adipocytes is increased while hydrolysis of cholesterol esters in macrophages is decreased, contributing to foam cell formation.

Mechanisms of FOXC2- and FOXD1-mediated regulation of the RI alpha subunit of cAMP-dependent protein kinase include release of transcriptional repression and activation by protein kinase B alpha and cAMP.

We have reported recently that mice overexpressing the forkhead/winged helix transcription factor FOXC2 are lean and show increased responsiveness to insulin due to sensitization of the beta-adrenergic cAMP-PKA(+) pathway and increased levels of the RI alpha subunit of cAMP-dependent protein kinase (PKA) (Cederberg, A., Grønning, L. M., Ahren, B., Taskén, K., Carlsson, P., and Enerbäck, S. (2001) Cell 106, 563-573). In this present study, we reveal that FOXC2 and a related factor, FOXD1, specifically activate the 1b promoter of the RI alpha gene in adipocytes and testicular Sertoli cells, respectively. By deletional mapping, we discovered two different mechanisms by which the Fox proteins activated expression from the RI alpha 1b promoter. In 3T3-L1 adipocytes, an upstream region represses promoter activity under basal conditions. Bandshift experiments indicate that overexpression of FOXC2 promotes the release of a potential repressor from this region. In Sertoli cells, sequences downstream of the transcription start sites mediate the activating effect of FOXD1, and protein kinase B alpha/Akt1 strongly induces this effect. Furthermore, we show that an inactive FOXD1 mutant lowers the cAMP-mediated induction of the RI alpha 1b reporter construct. In summary, winged helix transcription factors of the FOXC/FOXD families function as regulators of the RI alpha subunit of PKA and may integrate hormonal signals acting through protein kinase B and cAMP in a cell-specific manner.

Insulin and TNF alpha induce expression of the forkhead transcription factor gene Foxc2 in 3T3-L1 adipocytes via PI3K and ERK 1/2-dependent pathways.

We have recently identified the winged helix/forkhead gene Foxc2 as a key regulator of adipocyte metabolism that counteracts obesity and diet-induced insulin resistance. This study was performed to elucidate the hormonal regulation of Foxc2 in adipocytes. We find that TNF alpha and insulin induce Foxc2 mRNA in differentiated 3T3-L1 cells with the kinetics of an immediate early response (1-2 h with 100 ng/ml insulin or 5 ng/ml TNF alpha). This induction is, in both cases, attenuated by the PI3K inhibitor wortmannin as well as the MAPK kinase inhibitor PD98059. Furthermore, we show that stimulation of 3T3-L1 adipocytes with phorbol-12-myristate-13-acetate or 8-(4-chlorophenyl)thio-cAMP induces the expression of Foxc2. Interestingly, we find that the basal level of Foxc2 mRNA is down-regulated whereas hormonal responsiveness increases during differentiation of 3T3-L1 from preadipocytes to adipocytes. At the protein level, immunoblots with Foxc2 antibody demonstrated an induction of Foxc2 by insulin and TNF alpha in nuclear extracts of 3T3-L1 adipocytes. EMSA of nuclear proteins from phorbol-12-myristate-13-acetate- and TNF alpha-treated 3T3-L1 adipocytes using a forkhead consensus oligonucleotide revealed specific binding of a Foxc2/DNA complex. In conclusion, our data suggest that insulin and TNF alpha regulate the expression of Foxc2 via a PI3K- and ERK 1/2-dependent pathway in 3T3-L1 adipocytes. Also, signaling pathways downstream of PKA and PKC induce the expression of Foxc2 mRNA.