Introduction:: Insulin resistance defines the broad physiological state before the onset of type-2 Diabetes where the glucose absorbing insulin receptor signaling network of insulin sensitive tissues undergoes signal attenuation. On stimulation with insulin, the insulin receptor signaling network comprising of the core PI3Kinase/Akt/mTOR signaling network, mediates the absorption of glucose into the cell via the activation of the kinase Akt. During insulin resistance, signal attenuation as evidenced in the output of Insulin receptor/PI3K/Akt/mTOR network, is characterized by a lower Akt peak and total area under the Akt curve during insulin stimulation. The homeostasis in the insulin receptor signaling network is achieved by atleast three feedback loops which serve to reduce the gain or sensitivity of the network by feedbacking and reducing the signaling levels at the input. While the substrate of mTOR signaling S6K degrades IRS1 in a feedback loop, the translation modifier mTOR signaling substrate 4EBP1 increases the translation of the negative regulator PTEN thus acting in concert to limit the signaling caused by the input. Akt signaling also causes Foxo proteins to sequester outside the nucleus and thus decrease the translation of IR thereby exacerbating the IR decrease that happens due to ligand mediated receptor degradation in a third feedback loop. We show that chronic increased insulin receptor signaling leads to increased negative feedback which then results in– decreased IRS1, decreased IR and increased PTEN. This new status of the feedback loops results in attenuated Akt signaling thus providing a mechanistic explanation for the emergence of insulin resistance.
Materials and Methods:: Insulin was purchased from Calbiochem and dissolved in DMSO. Antibodies against PTEN, P-AKT (S473), P-AKT (T308), AKT, P-HER3 (Y1197), P-HER2 (Y1221), P-EGFR (Y1068), EGFR, P-IGF1Rβ (Y1131)/IR (Y1146), IR, IGF1R, P-ERK (T202/T204), P-FoxO1 (T24)/FoxO3a (T32)/FoxO4 (T28), P-S6 (S235/236), S6, PRAS40, p-4E-BP1 (S65), p-4E-BP1 (T37/46), 4E-BP1, PTEN, Actin, Myc, p-eIF4E S209, eIF4E, p-eIF4B, eIF4B, p-eIF4G, eIF4G were from Cell Signaling Technology. Antibodies against HER3, CyclinD1 were from Santa Cruz Biotechnology. Antibodies against HER2 were from Millipore. Antibody against P-PRAS40 (T246) was from Invitrogen. Cells in culture were washed thrice in cold PBS and lysed with Cell Lysis Buffer (Cell Signaling #9803) supplemented with Halt protease and phosphatase inhibitors (Pierce Chemical). Lysates were briefly sonicated before centrifugation at 16,000×g for 15 minutes at 4°C. The supernatant was collected, and protein concentration was determined using the BCA kit (Pierce) per manufacturer’s instructions. Protein samples were diluted in 4X LDS sample Buffer with 10X Sample Reducing Agent (both from Invitrogen).
A detailed computational model of the Insulin Receptor Signaling network was built using a series of ordinary differential equations (ODEs). The model includes the phosphorylation of the insulin receptor (IR) in response to insulin binding, the recruitment of the Insulin receptor substrate-1 (IRS1) to the activated IR and then the subsequent activation of the PI3Kinase followed by the activation of Akt. Akt activates mTOR which then feedback regulates IRS-1 through S6Kinase and PTEN through negative feedback from 4EBP1. Akt also inhibits Foxo which regulates the transcription of IR, IGF1R. The model is implemented in Matlab.
Results, Conclusions, and Discussions:: NIH 3T3L1 pre-adipocyte cells were stimulated with saturating amounts of Insulin for up to 72 hours (Fig 1) and the signaling in the nodes of the PI3K/Akt/mTOR pathway were analyzed to find oscillations in the Akt-S6K-IRS1 feedback loop. The oscillations have a fundamental period of 6 hours and correspond to physiological process like periodicity in protein translation following insulin stimulation and oscillations in peripheral glucose levels in human adults following a large meal. A single period of these oscillations are driven by the bi-phasic control of IRS1 by the mTOR substrate pS6K in a negative feedback loop. The increase in signaling and IRS1 is driven by pS6K mediated protein translation while the subsequent decrease in signaling and IRS1 levels is also mediated by pS6K mediated degradation (Fig 2). The sustained oscillations are caused by the transcriptional control of IRS1 by Foxo3A in phase opposition to the signaling and thereby completing the core oscillatory IRS1-pAkt-pS6K axis (Fig3). While transient oscillations due to the IRS1-pAkt-pS6K are a characteristic, long term adaptation in the insulin signaling network is driven by insulin receptor degradation and decreased insulin receptor mRNA transcription. Prolonged activation of Akt via phosphorylation of FOXO1/3 results in decreased IR transcription and consequently decreased IR levels (Fig 4). Also, long term insulin stimulation results in increased phosphorylation of the EIF4E binding protein 4EBP1 which is a substrate of mTOR signaling and controls cap dependent translation. Recently we have shown that the negative regulator PTEN is translationally controlled by the 4EBP1, so increased insulin signaling results in increased PTEN as a consequence of the feedback loop (Fig 4). Given that there are multiple feedback loops that determine the signaling response, we show that the response of the insulin signaling network to long term insulin stimulation is a function of the status of its feedback loops i.e. decreased levels of IR, decreased levels of IRS1 and increased levels of PTEN in response to long term stimulation. These changes in the negative feedback loops render the system heavily damped with much lower Akt activation thus mimicking attenuated signaling in insulin resistance and prediabetes (Fig 5)
