A role for protein kinase Bbeta/Akt2 in insulin-stimulated GLUT4 translocation in adipocytes

Abstract

Insulin stimulates glucose uptake into muscle and fat cells by promoting the translocation of glucose transporter 4 (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3K) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt, a downstream target of PI3K in regulation of GLUT4 translocation, has been controversial. Here we report that microinjection of a PKB substrate peptide or an antibody to PKB inhibited insulin-stimulated GLUT4 translocation to the plasma membrane by 66 or 56%, respectively. We further examined the activation of PKB isoforms following treatment of cells with insulin or platelet-derived growth factor (PDGF) and found that PKBbeta is preferentially expressed in both rat and 3T3-L1 adipocytes, whereas PKBalpha expression is down-regulated in 3T3-L1 adipocytes. A switch in growth factor response was also observed when 3T3-L1 fibroblasts were differentiated into adipocytes. While PDGF was more efficacious than insulin in stimulating PKB phosphorylation in fibroblasts, PDGF did not stimulate PKBbeta phosphorylation to any significant extent in adipocytes, as assessed by several methods. Moreover, insulin, but not PDGF, stimulated the translocation of PKBbeta to the plasma membrane and high-density microsome fractions of 3T3-L1 adipocytes. These results support a role for PKBbeta in insulin-stimulated glucose transport in adipocytes.

FIG. 1

Microinjection of PKB substrate peptide or an N-terminal PKB antibody inhibits insulin-stimulated GLUT4 translocation. 3T3-L1 adipocytes on coverslips were preincubated in Krebs-Ringer bicarbonate–HEPES buffer for 45 to 90 min. (A) Cells were then either not microinjected (Nil), microinjected with PKB substrate peptide (KRPRAATF), or microinjected with control peptide (KRPRAAAF) at 5 mg/ml. (B) 3T3-L1 adipocytes were either not injected (Nil) or microinjected with a purified N-terminal PKB antibody (PKB Ab) (N19; Santa Cruz) or purified rabbit IgG fraction (Control Ab) at 0.2 mg/ml. Bathing buffer was changed, and the cells were allowed to recover for 60 min. Cells were then stimulated or not with 100 nM insulin for 20 min prior to assessment of PM GLUT4 levels by the PM lawn assay as described in Materials and Methods. Results are from four or more experiments in which GLUT4 levels in six or more fields were determined for each condition within each experiment. ∗, P < 0.01 compared with insulin stimulation (paired t test).

FIG. 2

Change in growth factor response accompanies differentiation of 3T3-L1 adipocytes. (A) Serum-starved 3T3-L1 fibroblasts or adipocytes were stimulated with 1 μM insulin (I) or 50 ng of PDGF per ml (P) for 15 min or left untreated (B). Cell lysates (30 μg) were analyzed by SDS-PAGE and immunoblotting with phospho-Ser473 (pSer473) or phospho-Thr308 (pThr308) PKB antibodies. (B) HA-PKBα or HA-PKBβ was immunoprecipitated from lysates (100 μg) of 3T3-L1 fibroblasts expressing either HA-PKBα or HA-PKBβ and analyzed by SDS-PAGE. Immunoblotting was performed with the sheep PKBα, the sheep PKBβ, or the rabbit PKBβ antibodies. (C) Cell lysates (30 μg) prepared as described for panel A were analyzed by SDS-PAGE and immunoblotting with the sheep PKBα or the sheep PKBβ antibody. Bands labeled as band 1 exhibited the same electrophoretic mobility.

FIG. 3

PKBβ expression is induced upon adipocyte differentiation. (A) 3T3-L1 cells were harvested on each day of the differentiation procedure and analyzed for the expression of PKBβ (●) or GLUT4 (○) by immunoblotting with the sheep PKBβ antibody or a rabbit anti-GLUT4 antibody (R017), respectively. Immunoreactive signal (obtained as Lumi-Imager units) was adjusted for total protein obtained per sample and then expressed as a percentage of the maximum. Results are representative of two separate experiments. (B) 3T3-L1 fibroblasts were harvested at subconfluence (50% or 90%) or 1 day after reaching confluence (post-conf.). 3T3-L1 adipocytes were harvested after completion of differentiation, at day 8 (adip). Cell lysates (30 μg) were analyzed for the level of PKBβ expression by SDS-PAGE and immunoblotting with the sheep PKBβ antibody.

FIG. 4

PKBβ is the predominant isoform in adipocytes. (A) Cell lysates (100 μg) from serum-starved 3T3-L1 fibroblasts (Fib) or 3T3-L1 adipocytes (Ad) were depleted of PKBβ by two consecutive rounds of immunoprecipitation with the sheep PKBβ antibody. Twenty micrograms of lysate (Lysate) and one-fifth of the immunoprecipitation supernatant (After PKBβ-IP) were analyzed by SDS-PAGE and immunoblotting with the sheep PKBα or the sheep PKBβ antibodies. (B) Cell lysates (100 μg) from isolated rat adipocytes treated with 1 μM insulin (I) for 15 min or left basal (B) were depleted of PKBβ and analyzed as described for panel A. (C) Immunoprecipitation with the sheep PKBβ antibody was performed on 100 μg of cell lysates prepared from 3T3-L1 adipocytes overexpressing HA-PKBα. The immunoprecipitate (PKBβ-IP) and 10 μg of lysate (Lysate) were analyzed by SDS-PAGE and immunoblotting for the presence of HA-PKBα, with a monoclonal HA antibody.

FIG. 5

Insulin but not PDGF stimulates phosphorylation of PKBβ in 3T3-L1 adipocytes. (A) PKB isoforms were immunoprecipitated from 100 μg of 3T3-L1 lysates prepared from unstimulated cells (B) or cells treated with 1 μM insulin (I) or 50 ng of PDGF per ml (P) for 5 or 15 min. In the case of the PKBα immunoprecipitation, PKBβ was first depleted from the cell lysate by two consecutive rounds of immunoprecipitation. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with phospho-Ser473 (pSer473) or phospho-Thr308 (pThr308) PKB antibodies or the sheep PKBβ antibody (PKBβ). (B) PKBβ was immunoprecipitated from ³²P-labeled 3T3-L1 adipocytes treated with 1 μM insulin (I) or 50 ng of PDGF per ml (P) for 15 or left basal (B), by using the sheep PKBβ antibody, and analyzed by SDS-PAGE. The polyvinylidene difluoride membrane was subjected to autoradiography (autorad.) and then immunoblotted with the phospho-Ser473 antibody (pSer473).

FIG. 6

Analysis of insulin-stimulated PKBβ phosphorylation in 3T3-L1 adipocytes by 2-DE. 3T3-L1 adipocytes were ³²P labeled and then stimulated without (A) or with (B) 1 μM insulin for 15 min. PKBβ was immunoprecipitated from cell lysates by using the rabbit anti-PKBβ antibody and analyzed by 2-DE and autoradiography. Lysates (150 μg) prepared from untreated 3T3-L1 adipocytes (C) or adipocytes treated with 1 μM insulin for 15 min (D), 50 ng of PDGF per ml for 15 min (E), or 100 nM wortmannin for 40 min with 1 μM insulin added for the last 15 min (F) were analyzed by 2-DE and immunoblotting with the rabbit anti-PKBβ antibody. Immunoblots of the polyvinylidene difluoride membranes from panels A and B yielded results similar to those for panels C and D.

FIG. 7

Insulin, but not PDGF, stimulates the translocation of PKBβ to membrane fractions in 3T3-L1 adipocytes. 3T3-L1 adipocytes were stimulated with insulin (I) or PDGF (P) for 5 or 15 min or left basal (B). Cells were homogenized and subfractionated by differential centrifugation as described in Materials and Methods, to generate the PM, HDM, HSP (also termed LDM), cytosol (CYT), and the mitochondrial-nuclear (M/N) fractions. Twenty micrograms of each fraction was analyzed by SDS-PAGE and immunoblotting with the sheep PKBβ (PKBβ) or the phospho-Ser473 PKB (pSer473) antibody.