p75 neurotrophin receptor regulates glucose homeostasis and insulin sensitivity

Abstract

Insulin resistance is a key factor in the etiology of type 2 diabetes. Insulin-stimulated glucose uptake is mediated by the glucose transporter 4 (GLUT4), which is expressed mainly in skeletal muscle and adipose tissue. Insulin-stimulated translocation of GLUT4 from its intracellular compartment to the plasma membrane is regulated by small guanosine triphosphate hydrolases (GTPases) and is essential for the maintenance of normal glucose homeostasis. Here we show that the p75 neurotrophin receptor (p75(NTR)) is a regulator of glucose uptake and insulin resistance. p75(NTR) knockout mice show increased insulin sensitivity on normal chow diet, independent of changes in body weight. Euglycemic-hyperinsulinemic clamp studies demonstrate that deletion of the p75(NTR) gene increases the insulin-stimulated glucose disposal rate and suppression of hepatic glucose production. Genetic depletion or shRNA knockdown of p75(NTR) in adipocytes or myoblasts increases insulin-stimulated glucose uptake and GLUT4 translocation. Conversely, overexpression of p75(NTR) in adipocytes decreases insulin-stimulated glucose transport. In adipocytes, p75(NTR) forms a complex with the Rab5 family GTPases Rab5 and Rab31 that regulate GLUT4 trafficking. Rab5 and Rab31 directly interact with p75(NTR) primarily via helix 4 of the p75(NTR) death domain. Adipocytes from p75(NTR) knockout mice show increased Rab5 and decreased Rab31 activities, and dominant negative Rab5 rescues the increase in glucose uptake seen in p75(NTR) knockout adipocytes. Our results identify p75(NTR) as a unique player in glucose metabolism and suggest that signaling from p75(NTR) to Rab5 family GTPases may represent a unique therapeutic target for insulin resistance and diabetes.

Fig. 1.

p75^NTR−/− mice exhibit improved insulin sensitivity on normal diet. (A) Body weight of WT and p75^NTR−/− mice on normal chow (n = 10; NS, not significant). (B) Glucose tolerance of fasted WT and p75^NTR−/− mice on normal chow (n = 10, *P < 0.05, **P < 0.01, ***P < 0.001). (C) Insulin tolerance of fasted WT and p75^NTR−/− mice on normal chow (n = 10, **P < 0.01, ***P < 0.001). (D) Percentage of initial blood glucose in insulin tolerance test (n = 10, *P < 0.05). (E) Glucose disposal rates (GDRs) from WT and p75^NTR−/− mice on normal chow (n = 10, **P < 0.01). (F) Insulin-stimulated glucose disposal rates (IS-GDRs) of WT and p75^NTR−/− mice on normal chow (n = 10, *P < 0.05). (G) Hepatic glucose production (HGP) from WT and p75^NTR−/− mice on normal chow (n = 10, *P < 0.05). (H) Suppression of HGP in WT and p75^NTR−/− mice on normal chow (n = 10, *P < 0.05). Data are shown as the means ± SEM. Statistical comparisons between means were made with one-way ANOVA (B–D) and Student's t test (A and E–H).

Fig. 2.

p75^NTR regulates glucose uptake in adipocytes and skeletal muscle cells. (A) Basal and insulin-stimulated glucose uptake in WT and p75^NTR−/− MEF-derived adipocytes (NS, not significant; *P < 0.05). (B) Basal and insulin-stimulated glucose uptake in WT and p75^NTR−/− primary isolated adipocytes (NS, not significant; *P < 0.05). (C) p75^NTR expression in 3T3L1 adipocytes infected with p75^NTR shRNA or scrambled shRNA lentivirus. Gapdh was used as a loading control (Upper). Basal and insulin-stimulated glucose uptake in 3T3L1 cells differentiated to adipocytes and infected with lentivirus containing p75^NTR shRNA or scrambled shRNA control (*P < 0.05; **P < 0.01) (Lower). (D) p75^NTR expression in L6 myocytes tranfected with p75^NTR siRNA or siRNA control. Gapdh was used as a loading control (Upper). Basal and insulin-stimulated glucose uptake in L6 myocytes cells transfected with p75^NTR siRNA or siRNA control (**P < 0.01; ***P < 0.001) (Lower). (E) Basal and insulin-stimulated glucose uptake in WT and p75^NTR−/− MEF-derived adipocytes electroporated with p75FL, p75ICD, or GFP control expression vectors. After 72 h, cells were serum starved for 3 h and then stimulated with 100 nM insulin (*P < 0.05). Data are shown as the means ± SEM of a minimum of three different experiments performed in triplicate. Statistical comparisons between means were made with one-way ANOVA (A, B, and E) and Student's t test (C and D).

Fig. 3.

p75^NTR regulates GLUT4 trafficking in adipocytes. (A) Immunocytochemistry of WT and p75^NTR−/− MEF-derived adipocytes transfected with GFP-tagged GLUT4 and stimulated with 100 nM of insulin for 30 min. (Scale bar, 75 μm.) Representative images of two independent experiments are shown (Left). GLUT4 plasma membrane (PM) translocation was quantified by determining the percentage of cells with GFP rim staining. At least 50 cells per condition were counted (*P < 0.05 by Student's t test). (B) Western blot for GLUT4 in the plasma membrane fraction of epididymal WAT from WT and p75^NTR−/− mice on normal chow injected with (1 g/kg) of dextrose after 6 h fasting. Ponceau red was used as a loading control.

Fig. 4.

p75^NTR regulates Rab5 and Rab31 GTPase activity in adipocytes. (A) WT and p75^NTR−/− MEF-derived adipocytes were stimulated with insulin for 5 and 20 min. The activation state of Rab31 and Rab5 was determined using GST-EEA1/NT. Rab31-GTP and Rab5-GTP levels normalized to Rab31 and Rab5, respectively, were quantified by densitometry (Δ represents mean value). Representative blot of three independent experiments is shown. (B) Basal and insulin-stimulated glucose uptake in WT and p75^NTR−/− MEF-derived adipocytes electroporated with Rab31Q69L, Rab5S34N, or GFP control expression vector. After 72 h, cells were serum starved for 3 h and then stimulated with insulin (*P < 0.05 vs. WT basal; ^#P < 0.05 vs. p75^NTR−/− basal). Data are shown as the means ± SEM of a minimum of three different experiments performed in triplicate. Statistical comparisons between means were made with one-way ANOVA.

Fig. 5.

The death domain of p75^NTR directly associates with Rab5 and Rab31 GTPases. (A) Immunoprecipitation of HA-FLp75^NTR with myc-Rab31 transfected in HEK293T cells. (B) Immunoprecipitation of HA-FLp75^NTR with GFP-Rab5 transfected in HEK293T cells. Lysates were immunoprecipitated with anti-HA antibody, and Western blots were developed with anti-GFP and anti-HA antibodies. (C) Immunoprecipitation of p75^NTR with Rab31 and Rab5 in 3T3L1 adipocytes stimulated with insulin. Lysates were immunoprecipitated with anti-p75^NTR antibody, and Western blots were developed with anti-Rab31 and anti-Rab5 antibodies. (D) Mapping of the p75^NTR sites required for interaction with Rab31. Immunoprecipitation of myc-Rab31 with truncated forms of HA-p75^NTR transfected in HEK293T cells. Lysates were immunoprecipitated with anti-myc antibody, and Western blots were developed with anti-HA and anti-myc antibodies. (E) Mapping of the p75^NTR sites required for interaction with Rab5. Immunoprecipitation of truncated forms of HA-p75^NTR with GFP-Rab5 in HEK293T cells. Lysates were immunoprecipitated with anti-HA antibody, and Western blots were developed with anti-GFP and anti-HA antibodies. (F) Basal and insulin-stimulated glucose uptake of WT MEF-derived adipocytes electroporated with p75FL, p75Δ83, or GFP control expression vectors. After 72 h, cells were serum starved for 3 h and then stimulated with 100 nM insulin (**P < 0.01; ***P < 0.001; NS, not significant). Data are shown as the means ± SEM of two different experiments performed in triplicate. Statistical comparisons between means were made with one-way ANOVA. (G) Peptide array mapping of the p75^NTR ICD sites required for the interaction with Rab5 and Rab31. Schematic diagram of the p75^NTR ICD shows the domain organization. The six helixes within the DD are highlighted in yellow. Peptide array screened with recombinant GST-Rab5 and GST-Rab31 revealed helix 4 of p75^NTR ICD as the strongest region that interacts with Rab5 and Rab31. Peptide location, length, and sequences are shown. Peptide library was also screened with recombinant GST as a control. (H) Schematic representation of the binding of Rab5 family GTPases and RhoGDI to helixes 4 and 5 of the death domain of p75^NTR, respectively. Coimmunoprecipitation of p75ICD and activated GST-Rab5 recombinant proteins. Tat-Pep5 or control peptide (Tat-ctrl) were added as indicated. GST-Rab5 was immunoprecipitated with anti-GST antibody, and Western blots were developed with anti-p75^NTR and anti-GST antibodies. (I) Coimmunoprecipitation of p75ICD with activated GST-Rab5 or GST-RhoGDI recombinant proteins. Pep5 was added as indicated. GST-Rab5 and GST-RhoGDI were immunoprecipitated with anti-GST antibody, and Western blots were developed with anti-p75^NTR and anti-GST antibodies. Western blots were performed in triplicates and in all panels, representative blots are shown.