Rapamycin reverses insulin resistance (IR) in high-glucose medium without causing IR in normoglycemic medium

Abstract

Mammalian target of rapamycin (mTOR) is involved in insulin resistance (IR) and diabetic retinopathy. In retinal pigment epithelial (RPE) cells, insulin activates the mTOR pathway, inducing hypoxia-inducible factor-1α (HIF-1α) and HIF-dependent transcription in serum-free minimum essential medium Eagle (MEM). Serendipitously, we found that insulin failed to induce the HIF-1α-dependent response, when RPE cells were cultured in Dulbecco's modification of Eagle's medium (DMEM). Whereas concentration of glucose in MEM corresponds to normal glucose levels in blood (5.5 mM), its concentration in DMEM corresponds to severe diabetic hyperglycemia (25 mM). Addition of glucose to MEM also caused IR. Glucose-mediated IR was characterized by basal activation of mTORC1 and its poor inducibility by insulin. Basal levels of phosphorylated S6 kinase (S6K), S6 and insulin receptor substrate 1 (IRS1) S635/639 were high, whereas their inducibilities were decreased. Insulin-induced Akt phosphorylation was decreased and restored by rapamycin and an inhibitor of S6K. IR was associated with de-phosphorylation of IRS1 at S1011, which was reversed by rapamycin. Both short (16-40 h) and chronic (2 weeks) treatment with rapamycin reversed IR. Furthermore, rapamycin did not impair Akt activation in RPE cells cultured in normoglycemic media. In contrast, Torin 1 blocked Akt activation by insulin. We conclude that by activating mTOR/S6K glucose causes feedback IR, preventable by rapamycin. Rapamycin does not cause IR in RPE cells regardless of the duration of treatment. We confirmed that rapamycin also did not impair phosphorylation of Akt at T308 and S473 in normal myoblast C2C12 cells. Our work provides insights in glucose-induced IR and suggests therapeutic approaches to treat patients with IR and severe hyperglycemia and to prevent diabetic complications such as retinopathy. Also our results prompt to reconsider physiological relevance of numerous data and paradigms on IR given that most cell lines are cultured with grossly super-physiological levels of glucose.

Figure 1

The insulin/mTOR/HIF-1 pathway: a simplified schema. Glucose activates mTOR, which in turn blocks insulin signaling (a feedback loop). The mTOR pathway increases translation of the HIF-1α RNA. However, accumulation of the HIF-1α protein is tightly limited by its degradation via a PHD-feedback loop under normoxia.^, Red lines, inhibitory feedback loops

Figure 2

Induction of HRE-Luc in MEM, DMEM and MEM+additional glucose. RPE cells was plated in either MEM or DMEM with FBS. After cell attachment, the medium was changed to serum-free MEM or DMEM, as indicated. Cells were transfected with HRE-Luc (HIF-responsive-luciferase construct). After 1 day, cells were treated with 1 μg/ml insulin and 10% FBS as indicated. After 16 h, cellular luciferase activity was measured. (a) Comparison of MEM and DMEM. (b) Increasing concentrations of glucose (g/l) were added to MEM medium

Figure 3

Effects of rapamycin on insulin resistance in DMEM. Immunoblot analysis. (a) RPE cells were maintained in either DMEM (25 mM glucose) or MEM (5.5 mM glucose). Cells were incubated in serum-free medium for 42–44 h and then were stimulated with 1 μg/ml insulin for 15 min and lysed. If indicated Rap, stimulation in the presence of rapamycin. Rap, 10 nM rapamycin; o/n, rapamycin overnight before stimulation with insulin; chronic, rapamycin for 2 weeks before stimulation; IR, cells were treated with 1 μg/ml insulin overnight before stimulation; (b) RPE cell whole-cell lysates as in (a) were rerun in part and immunoblotting was performed with the indicated antibodies

Figure 4

Effects of S6K inhibitor and dose-response to rapamycin. Immunoblot analysis. RPE cells were maintained in either DMEM (25 mM glucose) or MEM (5.5 mM glucose) complete medium. Cells were incubated in serum-free respective medium in the absence or presence of rapamycin as indicated (in nM) or 10 μM PF4708671 (inhibitor of S6K1 kinase) for 42–44 h and then were stimulated with 1 μg/ml insulin for 20 min and lysed. Immunoblotting of membranes from two separate gels

Figure 5

Comparison of rapamycin and Torin 1 in RPE cells. Immunoblot analysis. RPE cells were incubated in serum-free MEM medium (42–44 h) and pre-treated as indicated with either 10 nM rapamycin (a) or 300 nM torin 1 for 42–44 h (b) and then were stimulated with 1 μg/ml insulin for 15 min and lysed. o/n, rapamycin overnight before stimulation; chronic, rapamycin for 2 weeks before stimulation. Immunoblotting of membranes from two separate gels

Figure 6

Comparison of rapamycin and Torin 1 in C2C12 cells. Immunoblot analysis. C2C12 cells were incubated in serum-free low-glucose (5.5 mM) DMEM for ∼46 h and then stimulated with 1 μg/ml insulin for 15 min and lysed. If indicated, cells were treated with 100 nM rapamycin or 300 nM Torin 1 for either 46 h or 4 h before stimulation with insulin. Blots were produced from immunoblotting of membranes from two separate gels

Figure 7

The insulin/mTOR pathway in RPE cells. (a) Insulin activates IRS and causes Akt phosphorylation, resulting in mTOR activation. As a negative feedback loop, mTOR and S6K both block insulin signaling. In high-glucose conditions, S6K is chronically active, causing insulin resistance. This resistance is characterized (in RPE cells) by phospho-S636/639 IRS1, p-S6 and impaired insulin-induced activation of Akt. (b) In the presence of rapamycin, the events downstream of mTORC1 are blocked and therefore there is no feedback inhibition of insulin signaling