IRS1-independent defects define major nodes of insulin resistance

Abstract

Insulin resistance is a common disorder caused by a wide variety of physiological insults, some of which include poor diet, inflammation, anti-inflammatory steroids, hyperinsulinemia, and dyslipidemia. The common link between these diverse insults and insulin resistance is widely considered to involve impaired insulin signaling, particularly at the level of the insulin receptor substrate (IRS). To test this model, we utilized a heterologous system involving the platelet-derived growth factor (PDGF) pathway that recapitulates many aspects of insulin action independently of IRS. We comprehensively analyzed six models of insulin resistance in three experimental systems and consistently observed defects in both insulin and PDGF action despite a range of insult-specific defects within the IRS-Akt nexus. These findings indicate that while insulin resistance is associated with multiple deficiencies, the most deleterious defects and the origin of insulin resistance occur independently of IRS.

Figure 1. Insulin-stimulated GLUT4 translocation is not proportional to Akt phosphorylation

A) 3T3-L1 adipocytes were treated with a dose response of insulin. At each dose, the phosphorylation status of Akt at Ser473, GSK3α/β at Ser9/21, and TBC1D4 at Thr642 were detected by Western blot. B) Insulin dose response for GLUT4 translocation to the plasma membrane in 3T3-L1 adipocytes. C) To directly compare the insulin-stimulated response of each parameter in A) and B), the Western blots and translocation data were quantified and normalized to 0 and 100nM insulin representing minimal and maximal stimulation. An evaluation of submaximal stimulation reveals a distinct non-linearity between Akt phosphorylation and GLUT4 translocation to the plasma membrane. However, phosphorylation of TBC1D4 at submaximal insulin levels is more consistent with GLUT4 translocation than Akt, indicating an increased activity toward this substrate over others like GSK3. Error bars, ± s.e.m. of 3-4 experiments. *P<0.05 relative to Akt phosphorylation at the given dose of insulin, Student's t-test. D) The PI3K inhibitor wortmannin (WT, 100 nM for 10 min prior to insulin) and an Akt inhibitor (at concentrations of 0.1, 1, and 10 μM) inhibit Akt phosphorylation and subsequent phosphorylation of TBC1D4 in 3T3-L1 adipocytes treated with 100 nM insulin for 12 minutes indicating that Akt is the sole insulin-regulated kinase acting on this site. Representative Western blots of 3 separate experiments are shown. E) Insulin (20 minutes) was added to L6 myotubes at the concentrations indicated and the response of Akt phosphorylation and GLUT4 translocation were analyzed as a percent of maximal stimulation. Error bars, ± s.e.m., n= 3. *P<0.05, Student's t-test.

Figure 2. PDGF mimics insulin action while circumventing the IR and IRS

A) The human PDGF Receptor was ectopically over-expressed in mouse 3T3-L1 adipocytes and rat L6 myotubes. Representative Western blots of PDGFR expression are shown. Abbreviations: Pa, preadipocytes; Ad, adipocytes; Mb, myoblasts; and Mt, myotubes. B) PDGFR expressing adipocytes (left column) and myotubes (right column) were serum starved for 90 min prior to acute stimulation with either 100 nM insulin or 20 ng/mL PDGF for 20 min. Representative Western blots of cell and tissue extracts show similar amplification of insulin- and PDGF-stimulated signaling intermediates. C) Insulin- (hashed bars) and PDGF-stimulated (graded bars) GLUT4 translocation to the plasma membrane as a percentage of total HA-GLUT4 in adipocytes (left) and myotubes (right). In both cell types insulin was slightly more potent than PDGF (89±2% and 90±3% of maximal insulin for adipocytes and myotubes respectively). Error bars, ± s.e.m. (n = 5 experiments with >3 measurements each), Student's t-test.

Figure 3. Multiple insults antagonize insulin and PDGF action

A) GLUT4 translocation to the plasma membrane was measured after glucose oxidase (GO, 50 mU/mL for 2 h), chronic insulin (CI, 10 nM for 24 h), TNFα (TNF, 2 ng/mL for 4 d), or dexamethasone (DEX, 20 nM for 8 d) treatment in 3T3-L1 adipocytes stimulated with insulin and PDGF. B) GLUT4 translocation to the plasma membrane was analyzed in L6 myotubes treated with the above conditions or palmitate (PALM, 150 μM for 18 h). All data were normalized to maximum stimulation with 100nM insulin for each cell type. Results are displayed as means ± s.e.m., *P<0.05 vs. respective insulin- or PDGF-stimulated control (Ø) (n=3-5 experiments for each data set), Student's t-test.

Figure 4. Signal transduction in 3T3-L1 adipocytes and L6 myotubes treated with multiple models of insulin resistance. Cells were left in unstimulated basal conditions (open bars) or stimulated with Insulin (hashed bars) or PDGF (graded bars)

A) Representative Western blots and quantitative bar graphs are shown for the detection of PDGFR and IRS-1 tyrosine phosphorylation and total IRS-1 protein levels. The pY612-IRS-1 antibody detects phosphorylation of the PI3 kinase binding site in both molecules. B) Representative Western blots and quantification of Akt Ser473 and Thr308 phosphorylation and total Akt levels are shown in this section. C) Cell lysates were also probed for TBC1D4 phosphorylation at Thr642 and total TBC1D4 expression. The lower panel represents a quantification of phosphorylated to total (p/t)-TBC1D4. Western blots are representative and statistical values are displayed as means ± s.e.m., *P<0.05 vs. respective control (Ø), Student's t-test, n=3-5 experiments.

Figure 5. Palmitate antagonizes insulin action at GLUT4 without disrupting signal transduction through TBC1D4 in L6 myotubes

A) Basal (white bars) and Insulin-stimulated (black bars) GLUT4 translocation was measured in L6 myotubes incubated with a dose curve of palmitate for 18 h. Error bars, ± s.e.m., (n = 3 independent experiments of >3 measurements each) *P<0.05 between palmitate and carrier control, Student's t-test. B) Representative Western blots of phosphorylated and total IRS-1, Akt, and TBC1D4 in palmitate-treated myotubes. C) No significant difference was observed in the phosphorylation of IRS-1, Akt or TBC1D4 between control myotubes and those treated with up to 300 μM palmitate. However, higher concentrations severely antagonized insulin action at these molecules as shown.

Figure 6. Modulation of Insulin and PDGF action in high fat fed PDGFR transgenic mice

A) Intact soleus muscles from transgenic mice were blotted for expression of the PDGFR. B) Isolated solei were incubated in vitro and stimulated with 2 mU/mL insulin or 500 ng/mL PDGF for 30 min and Western blots were performed on whole cell lysates. Representative blots are shown. Phosphorylation of T308-Akt was undetectable. C) PDGF (shaded bars) had a more potent effect on ³H-2-deoxyglucose uptake into soleus muscle than insulin (hashed bars) in transgenic mice (3.59 ± 0.09 vs. 2.78 ± 0.12 μMol ³H-2-deoxy-D-glucose/g/hr) consistent with previous studies (Yuasa et al., 2004). D) Glucose tolerance tests were performed on mice in the 4h fasted state to predict insulin resistance. Both diet durations of 17 and 42 days produced significant reductions in glucose tolerance indicated by differences blood glucose levels of high fat fed mice versus the low fat diet group, n=7-9 mice per curve, individual points are displayed as means ± s.e.m., *P<0.05 between lean diet and test diet groups by students t-test. E) Reduction in insulin- (hashed bars) and PDGF-stimulated (shaded bars) 2DOG uptake in isolated soleus muscles from mice fed a high fat diet for 17 days (-17±6% and -16±5% of control insulin- and PDGF-stimulation, respectively) or 42 days (-29±5% and -15±2% of control insulin- and PDGF-stimulation, respectively). n= 3-7 solei per data set. F) Representative Western blots and bar graphs are shown for soleus muscles stimulated with insulin (hashed bars) or PDGF (shaded bars) for 30 min. Total protein levels are shown in full bars. n = 3-5 solei per experiment. G) Representative Western blots and quantification of GLUT4 expression in mice fed a LFD and HFD for 17 and 42 days. n=4 per group. E-G) Results are displayed as means ± s.e.m., *P<0.05 between lean diet and test diet groups, Student's t-test.