Leukocyte signaling in patients with systemic insulin resistance is largely unexplored. We recently discovered the presence of multiple Toll-like receptor 4 (TLR4) signaling intermediates in leukocytes from patients with type 2 diabetes or acute insulin resistance associated with cardiopulmonary bypass surgery. We extend this work to show that in addition to matrix metalloproteinase 9, hypoxia-inducible factor 1α, and cleaved AMPKα, patient leukocytes also express IRS-1 phosphorylated on Ser312, Akt phosphorylated on Thr308, and elevated TLR4 expression. Similar signaling intermediates were detected in leukocytes and neutrophils treated with lipopolysaccharide (LPS), a ligand of TLR4, in vitro. In contrast, insulin, but not LPS, induced mammalian target of rapamycin complex 2 (mTORC2)–dependent phosphorylation of Akt on Ser473 and FoxO1/O3a on Thr24/32 in leukocytes and neutrophils. Insulin suppressed LPS-induced responses in a dose- and time-dependent manner. AS1842856, a FoxO1 inhibitor, also suppressed TLR4 signaling. We propose that insulin is a homeostatic regulator of leukocyte responses to LPS/TLR4 and that the signaling intermediates expressed in leukocytes of patients with type 2 diabetes indicate TLR4 signaling dominance and deficient insulin signaling. The data suggest that insulin suppresses LPS/TLR4 signals in leukocytes through the mTORC2-Akt-FoxO signaling axis. Better understanding of leukocyte signaling in patients with type 2 diabetes may shed new light on disease causation and progression.

Robust inflammatory responses associated with injury or sepsis trigger the onset of insulin resistance in humans (1). Modest increases in inflammatory indicators detected in patients have suggested that type 2 diabetes is associated with chronic low-grade inflammation (2,3). Human subjects challenged with lipopolysaccharide (LPS), a ligand of Toll-like receptor 4 (TLR4) (4), produce acute inflammatory responses that include insulin resistance (5,6). Mice administered a low dose of LPS for 1 month develop insulin resistance (7). On the other hand, TLR4-deficient mice and mice transplanted with bone marrow cells from TLR4-deficient mice were more resistant to high-fat diet–induced insulin resistance than wild-type mice or mice transplanted with bone marrow cells from wild-type mice (8). These and other studies have indicated that immune cells and TLR4 signaling contribute to systemic insulin resistance (810).

We reported that LPS triggers rapid changes in cellular metabolism in human and mouse leukocytes (11,12) through a phosphoinositide 3-kinase (PI3K)–dependent process that involves two metabolic regulators: AMPK and hypoxia-inducible factor 1 (HIF-1) (13). We showed that LPS increases the expression of matrix metalloproteinase 9 (MMP9) in leukocytes and neutrophils and that intracellular MMP9 cleaves the catalytic subunit of AMPK (AMPKα) (13). AMPK inactivation enables dephosphorylation of the regulatory-associated protein of mammalian target of rapamycin (mTOR) (Raptor) on Ser792, resulting in mTOR complex 1 (mTORC1) activation (14,15). mTORC1 phosphorylates ribosomal S6 kinase (S6K1) on Thr389 (16,17). In addition, mTORC1 activates the transcription factor HIF-1, which promotes glycolysis and proinflammatory cytokine production in both human and mouse leukocytes treated with LPS (18,19). Because neither HIF-1α nor S6K1 phosphorylated on Thr389 was detected in MMP9-deficient mouse leukocytes treated with LPS, the mechanism by which TLR4 activates mTORC1 might be unique (13).

A second complex involving mTOR, known as mTOR complex 2 (mTORC2), regulates the phosphorylation of Akt on Ser473 (20,21). Activated Akt phosphorylates FoxO1 and FoxO3a, which belong to the forkhead family of transcription factors, on multiple threonine and serine residues (22). Phosphorylated FoxO proteins are excluded from the nucleus and remain inactive (23). FoxO1 regulates leukocyte survival (2426) and TLR4 signaling (27,28). Although activated FoxO1 triggers an increase in TLR4 expression in LPS-treated Raw264.7 cells (28), it also contributes to LPS-dependent TLR4 signaling inactivation in these cells (28). The role of FoxO relative to TLR4 signaling in human leukocytes is currently unknown.

Human leukocytes express an insulin receptor (29). Insulin receptor activation triggers phosphorylation of insulin receptor substrate (IRS) proteins on tyrosine residues. This leads to PI3K and phosphoinositide-dependent kinase 1 activation, the latter of which phosphorylates Akt on Thr308 (30). Insulin induces phosphorylation of Akt on Ser473 through mTORC2, and Akt phosphorylates FoxO (21,31,32). FoxO1 phosphorylation was impaired in macrophages derived from insulin resistant obese mice (33). Despite this general framework, the insulin signaling pathway in leukocytes is undetermined.

We recently showed that leukocytes from a cohort of patients with type 2 diabetes exhibited distinct signaling intermediates (13). From these observations, we hypothesized that characterization of signaling intermediates expressed in patient leukocytes could have utility in determining disease etiology and progression. The data showed that leukocytes from patients with type 2 diabetes exhibit two patterns of signaling intermediates. The presence of MMP9 and HIF-1α as well as cleaved AMPKα define the first pattern (pattern 1), whereas the presence of Akt phosphorylated on Ser473 defines the second (pattern 2). IRS-1 phosphorylated on Ser312 and S6K1 phosphorylated on Thr389 were seen in leukocytes expressing either pattern 1 or pattern 2. The signaling intermediates associated with pattern 1 were reproduced in leukocytes treated with LPS in vitro. Insulin but not LPS induced phosphorylation of Akt on Ser473 and FoxO1/FoxO3a on Thr24/Thr32 in leukocytes and neutrophils and suppressed LPS-induced TLR4 signaling in a dose- and time-dependent manner. The data suggested that TLR4 signaling is linked to pattern 1. We propose that leukocytes with pattern 2 are partially responsive to insulin, which is no longer able to fully suppress the inflammatory signals present in these patients’ leukocytes/neutrophils.

Materials

The following antibodies were used: anti-actin (Sigma-Aldrich); anti-HIF-1α, AMPKα, MMP9, IκB-α, total Akt, and TLR4 (Santa Cruz Biotechnology); and anti-Raptor Ser792, total Raptor, S6K1 Thr389, Akt Thr308, Akt Ser473, IRS-1 Ser312 (Ser307 in mice), IRS-1, and FoxO1/FoxO3a Thr24/Thr32 (Cell Signaling Technology). Source and final concentration of pharmacologic agents were as follows: LPS from Escherichia coli 0111:B4 (Sigma-Aldrich); insulin (Humalog; Eli Lilly), rapamycin (100 nmol/L), Torin (50 nmol/L), and PP242 (100 nmol/L) (Tocris); and AS1842856 (100 nmol/L) (Millipore).

Human Subjects

The Rutgers Health Sciences Institutional Review Board approved the study. Patients with type 2 diabetes and patients without diabetes were recruited from endocrinology clinics at Rutgers Robert Wood Johnson Medical School during a scheduled visit. Written informed consent was obtained from all subjects before inclusion in the study. Twelve patients with type 2 diabetes were recruited, and their demographics are shown in Table 1. Patient clinical records are presented in Supplementary Table 1. Cardiac surgery patients were recruited from Rutgers Robert Wood Johnson University Hospital. Inclusion and exclusion criteria were described previously (13) and are included in the Supplementary Data.

Table 1

Patient characteristics

Without diabetesWith type 2 diabetes
No. of patients 10 12 
Age (years) 61 ± 4 61 ± 2 
Sex   
 Male 
 Female 
BMI (kg/m234 ± 3 29 ± 1 
Fasting plasma glucose (mg/dL) 101 ± 5 147 ± 14* 
HbA1c   
 % 5.9 ± 0.1 7.7 ± 0.3** 
 mmol/mol 41 60.7 
Cholesterol (mg/dL) 157 ± 14 171 ± 16 
HDL cholesterol (mg/dL) 49 ± 4 50 ± 2 
LDL cholesterol (mg/dL) 89 ± 10 96 ± 14 
Triglycerides (mg/dL) 94 ± 20 127 ± 12 
Alanine aminotransferase (IU/L) 28 ± 9 30 ± 6 
Aspartate aminotransferase (IU/L) 18 ± 2 27 ± 3 
Without diabetesWith type 2 diabetes
No. of patients 10 12 
Age (years) 61 ± 4 61 ± 2 
Sex   
 Male 
 Female 
BMI (kg/m234 ± 3 29 ± 1 
Fasting plasma glucose (mg/dL) 101 ± 5 147 ± 14* 
HbA1c   
 % 5.9 ± 0.1 7.7 ± 0.3** 
 mmol/mol 41 60.7 
Cholesterol (mg/dL) 157 ± 14 171 ± 16 
HDL cholesterol (mg/dL) 49 ± 4 50 ± 2 
LDL cholesterol (mg/dL) 89 ± 10 96 ± 14 
Triglycerides (mg/dL) 94 ± 20 127 ± 12 
Alanine aminotransferase (IU/L) 28 ± 9 30 ± 6 
Aspartate aminotransferase (IU/L) 18 ± 2 27 ± 3 

Data are mean ± SEM unless otherwise indicated.

*P = 0.024.

**P = 0.002.

Leukocytes Isolation

Blood drawn into heparin-containing tubes was separated into aliquots and treated with LPS (10 ng/mL) or insulin (1 unit/mL), unless otherwise indicated. The leukocytes were isolated as previously described (12). Lysates containing equal protein amounts were analyzed by immunoblotting.

Neutrophil and Mononucleated Cell Isolation

Neutrophils and mononucleated cells (which include monocytes and lymphocytes) were first separated by using Ficoll-Hypaque (Sigma-Aldrich). The mononuclear cell fraction was washed twice with PBS and then suspended in DMEM. The neutrophil fraction was separated from the red blood cells by using dextran (molecular weight 500,000) sedimentation. After lysis of residual red blood cells and two washes with PBS, the neutrophils were suspended in DMEM. Isolated neutrophil and mononucleated cell suspensions were treated with LPS and/or insulin and then lysed. Lysates containing equal protein amounts were analyzed by immunoblotting.

Akt Phosphorylation on Thr308 and/or Ser473, IRS-1 Phosphorylation on Ser312, and Increased TLR4 Expression Are Detected in Leukocytes From Patients With Type 2 Diabetes

We previously characterized signaling intermediates found in peripheral blood leukocytes from 12 patients with type 2 diabetes as defined by hemoglobin A1c (HbA1c) ≥6.4% (≥46.4 mmol/mol) and/or concurrent treatment for diabetes (13). The demographics of patients with and without diabetes are reported in Table 1. All 12 samples from patients with type 2 diabetes exhibited S6K1 phosphorylation on Thr389 and dephosphorylated Raptor on Ser792 (12) (Fig. 1). In addition to these markers, patient leukocytes expressed two predominant patterns of signaling intermediates. Pattern 1, seen in leukocytes from 8 of 12 patients, included MMP9, HIF-1α, and cleaved AMPKα (Fig. 1A, lane 1) (13). Leukocytes from 4 of 12 patients exhibited pattern 2. These leukocytes did not express MMP9, HIF-1α, or cleaved AMPKα (Fig. 1A, lane 3) (13). The signaling intermediates associated with pattern 1 were reproduced in leukocytes and neutrophils treated with LPS in vitro to activate TLR4 (13). In the current study, we sought to identify additional leukocyte signaling components that may relate to patient insulin resistance. To this end, we examined whether the expression and/or phosphorylation of three signaling components (Akt, IRS-1, and TLR4) previously linked to insulin resistance (3436) are modified in leukocytes from the cohort of patients described in Table 1 and our previous work (13).

Figure 1

Signaling intermediates expressed in leukocytes from patients with type 2 diabetes. Blood samples were obtained from patients without diabetes (N) (n = 10) and patients with type 2 diabetes (2) (n = 12) seen at Rutgers Robert Wood Johnson Medical School clinics. The researchers were blinded to the clinical characteristics of the study participants (Table 1) while the samples were being analyzed. *, a healthy control sample used multiple times; ▼, a patient without diabetes with an HbA1c of 6% (46.4 mmol/mol) and fasting glucose of 117 mg/dL. A: Signaling intermediates detected in leukocytes from a patient without diabetes (lane 2) and representative patients with type 2 diabetes showing pattern 1 (lane 1) or pattern 2 (lane 3). B: Samples presented in the same order as those in Zhang et al. (13) to enable direct comparison. Leukocytes were isolated, lysed, and analyzed by immunoblotting to determine the expression levels and phosphorylation state of the specified proteins. Immunoblotting for actin was used to confirm equal protein loading. p-S, phosphorylation on serine; p-T, phosphorylation on threonine.

Figure 1

Signaling intermediates expressed in leukocytes from patients with type 2 diabetes. Blood samples were obtained from patients without diabetes (N) (n = 10) and patients with type 2 diabetes (2) (n = 12) seen at Rutgers Robert Wood Johnson Medical School clinics. The researchers were blinded to the clinical characteristics of the study participants (Table 1) while the samples were being analyzed. *, a healthy control sample used multiple times; ▼, a patient without diabetes with an HbA1c of 6% (46.4 mmol/mol) and fasting glucose of 117 mg/dL. A: Signaling intermediates detected in leukocytes from a patient without diabetes (lane 2) and representative patients with type 2 diabetes showing pattern 1 (lane 1) or pattern 2 (lane 3). B: Samples presented in the same order as those in Zhang et al. (13) to enable direct comparison. Leukocytes were isolated, lysed, and analyzed by immunoblotting to determine the expression levels and phosphorylation state of the specified proteins. Immunoblotting for actin was used to confirm equal protein loading. p-S, phosphorylation on serine; p-T, phosphorylation on threonine.

Optimal Akt activation is suggested to require phosphorylation on both Thr308 and Ser473 (37). Leukocytes from 10 of the 12 patients with type 2 diabetes showed Akt phosphorylated on Thr308 (Fig. 1B), as did leukocytes from one patient with prediabetes (subject 16: HbA1c 6.0% [46.4 mmol/mol], fasting glucose 117 mg/dL). However, Akt phosphorylated on Ser473 was only detected in leukocytes showing pattern 2 (Fig. 1B, lanes 4, 7, 17, and 21) (13), suggesting differential regulation of Akt phosphorylation on residues Thr308 and Ser473 in leukocytes of patients with type 2 diabetes.

Insulin resistance is frequently associated with either a decline in IRS protein expression or increased IRS phosphorylation on serine residues (38,39). Leukocytes from patients with and without diabetes expressed similar IRS-1 protein levels (Fig. 1B). However, phosphorylation of IRS-1 on Ser312 (equivalent to murine IRS-1 Ser307) was detected in all 12 patients with diabetes (Fig. 1B) as well as in the one patient with prediabetes (subject 16).

Skeletal muscle biopsy specimens and monocytes from patients with type 2 diabetes express higher TLR4 protein levels than control subjects (34,35). These findings motivated us to examine the expression of TLR4 in patient leukocytes. Increased TLR4 expression was detected in 10 of the 12 leukocyte samples of patients with type 2 diabetes (Fig. 1B).

Phosphorylation of Akt on Thr308 but Not Ser473, Phosphorylation of IRS-1 on Ser312, and Increased TLR4 Expression Are Detected in Leukocytes Treated With LPS In Vitro

We have previously shown that signaling intermediates expressed in leukocytes from patients with type 2 diabetes were reproduced in leukocytes treated with LPS in vitro (13) (Fig. 2A), implicating TLR4 in the cascade of aberrant signaling events associated with clinical insulin resistance. We therefore sought to determine whether LPS/TLR4 signaling could also account for the phosphorylation of Akt on Thr308 and Ser473, the phosphorylation of IRS-1 on Ser312, and/or the increase in TLR4 expression seen in leukocytes from patients with type 2 diabetes. Akt phosphorylated on Thr308 but not Ser473 was detected within 10 min of LPS stimulation, and the phosphorylation signal remained strong for at least 4 h (Fig. 2B). Though initially weak, phosphorylation of IRS-1 on Ser312 was seen by 30 min (Fig. 2B). LPS also triggered a significant increase in TLR4 expression. These data suggest that the signaling intermediates associated with pattern 1 are governed by a common mechanism linked to TLR4.

Figure 2

LPS- and insulin-induced responses in human leukocytes. A, B, and E: Blood samples were treated with LPS for the indicated time. The sample shown in panel B, lane 8, was treated with insulin (1 unit/mL) for 60 min and served as a positive control. C, D, and F: Blood samples were treated with insulin (1 unit/mL) for the indicated time. The sample shown in panel D, lane 8, was treated with LPS (10 ng/mL) for 90 min and served as a positive control. G: Leukocytes from patients with type 2 diabetes exhibiting either pattern 1 or pattern 2 were analyzed for Akt phosphorylation on Ser473 and FoxO1/FoxO3a phosphorylation on Thr24/Thr32. The patient IDs correspond to those presented in Fig. 1B. H: Blood samples were obtained from a CPB patient on the morning of the surgery, in the recovery room, and on days 1–4 postsurgery. Similar data were obtained for two other patients. D, day; P, presurgery; p-S, phosphorylation on serine; p-T, phosphorylation on threonine; R, recovery room.

Figure 2

LPS- and insulin-induced responses in human leukocytes. A, B, and E: Blood samples were treated with LPS for the indicated time. The sample shown in panel B, lane 8, was treated with insulin (1 unit/mL) for 60 min and served as a positive control. C, D, and F: Blood samples were treated with insulin (1 unit/mL) for the indicated time. The sample shown in panel D, lane 8, was treated with LPS (10 ng/mL) for 90 min and served as a positive control. G: Leukocytes from patients with type 2 diabetes exhibiting either pattern 1 or pattern 2 were analyzed for Akt phosphorylation on Ser473 and FoxO1/FoxO3a phosphorylation on Thr24/Thr32. The patient IDs correspond to those presented in Fig. 1B. H: Blood samples were obtained from a CPB patient on the morning of the surgery, in the recovery room, and on days 1–4 postsurgery. Similar data were obtained for two other patients. D, day; P, presurgery; p-S, phosphorylation on serine; p-T, phosphorylation on threonine; R, recovery room.

Insulin Suppresses LPS-Induced TLR4 Signaling in Leukocytes

Having observed Akt phosphorylation on Thr308 but not Ser473 in our LPS model, we sought to identify clinical factors determining the presence of Ser473 phosphorylation in leukocytes. Examination of patient medical records revealed that the four patients with type 2 diabetes expressing Akt phosphorylated on Ser473 were treated with insulin (Supplementary Table 1). Because insulin triggers phosphorylation of Akt on Ser473 and Thr308 and S6K1 on Thr389 in various model cell systems (36), we examined whether insulin can induce similar responses in leukocytes. Leukocytes treated with insulin in vitro exhibited a transient increase in S6K1 phosphorylation on Thr389 that peaked by 60 min (Fig. 2C). Insulin also triggered transient increases in Akt phosphorylation on Thr308 and Ser473 (Fig. 2D). Akt remained phosphorylated on Ser473 for at least 120 min, whereas the phosphorylation on Thr308 decreased below the detection level after 90 min.

In general, Akt phosphorylated on Ser473 phosphorylates FoxO proteins, which are transcription factors. Phosphorylated FoxO proteins are excluded from the nucleus and therefore inactive. Human neutrophils express FoxO1, FoxO3a, and FoxO4 (24). The presence of Akt phosphorylated on Ser473 in leukocytes treated with insulin suggested that insulin might also induce phosphorylation of FoxO proteins. By using an antibody that reacts with both FoxO1 and FoxO3a when phosphorylated on Thr24 and Thr32, respectively, we determined that insulin induces FoxO1/FoxO3a phosphorylation in leukocytes (Fig. 2F). LPS failed to induce a similar response (Fig. 2E)

This finding raised the question about whether FoxO1 and FoxO3a are phosphorylated in the leukocytes of patients with type 2 diabetes. To this end, we reanalyzed 8 of the 12 samples shown in Fig. 1B. The patient ID numbers shown in Fig. 2G correspond to those in Fig. 1B. FoxO1 and FoxO3a were not phosphorylated in leukocytes with pattern 1. Two of the four samples with pattern 2 showed no FoxO1/FoxO3a phosphorylation (subjects 7 and 4). The other two exhibited weak phosphorylation (subjects 17 and 21). Of note, although subject samples 7 and 4 exhibited elevated TLR4 expression, samples 17 and 21 did not. Collectively, these data indicate that the phosphorylation of FoxO1/FoxO3a is impaired in leukocytes from patients with type 2 diabetes.

Regardless of diabetes history, the majority of patients who undergo cardiopulmonary bypass (CPB) surgery experience an acute phase of insulin resistance immediately after surgery and require insulin infusion to maintain glucose within a normal range (40). We reported that leukocytes from patients who underwent CPB surgery express signaling intermediates postsurgery similar to those seen in leukocytes from patients with type 2 diabetes (13). Here, we studied temporal changes in Akt, IRS-1, and FoxO1/FoxO3a phosphorylation in leukocytes from patients who underwent CPB. Signaling intermediates seen in leukocytes from one of three patients studied are presented in Fig. 2H. Robust Akt phosphorylation on Thr308 and IRS-1 phosphorylation on Ser312 were seen between days 1 and 3 postsurgery (Fig. 2H), whereas total Akt and IRS-1 expression remained constant throughout. S6K1 phosphorylation on Thr389 was detected on days 1–3 postsurgery, and this coincided with the presence of cleaved AMPKα, Raptor dephosphorylated on Ser792, and MMP9 expression (13). Of note, as glucose homeostasis improved by days 3 and 4, the expression of the signaling intermediates associated with pattern 1 returned to baseline, whereas Akt phosphorylated on Ser473 and FoxO1/FoxO3a on Thr24/Thr32 appeared. Thus, the leukocytes exhibited pattern 1 while the patient was insulin resistant. The signaling mediators associated with patterns 1 and 2 detected in patients with type 2 diabetes (Figs. 1B and 2G) and in patients after CPB surgery (Fig. 2H) are presented in Table 2.

Table 2

Summary of signaling intermediates detected in leukocytes from patients without and with type 2 diabetes with pattern 1 or pattern 2

With type 2 diabetes
Without diabetesPattern 1 (8 of 12 patients)Pattern 2 (4 of 12 patients)
MMP9 expressed 
AMPKα cleaved 
p-Raptor Ser792 
HIF-1α expressed 
p-S6K1 Thr389 
p-Akt Thr308 Y or N 
p-Akt Ser473 
p-FoxO1/FoxO3a Thr24/Thr32 Y or N 
p-IRS-1 Ser312 
Increased TLR4 Y or N 
With type 2 diabetes
Without diabetesPattern 1 (8 of 12 patients)Pattern 2 (4 of 12 patients)
MMP9 expressed 
AMPKα cleaved 
p-Raptor Ser792 
HIF-1α expressed 
p-S6K1 Thr389 
p-Akt Thr308 Y or N 
p-Akt Ser473 
p-FoxO1/FoxO3a Thr24/Thr32 Y or N 
p-IRS-1 Ser312 
Increased TLR4 Y or N 

N, no; p, phosphorylated; Y, yes.

Insulin Suppresses LPS-Induced Responses in a Dose- and Time-Dependent Manner

Studies have suggested that even a single meal high in fat can trigger release of LPS from the gut into the circulation (41). Because insulin levels increase after food intake, it is possible that human leukocytes are exposed periodically and transiently to LPS plus insulin. For this reason, we next examined the combined effects of LPS and insulin on leukocyte signaling. Although LPS-induced signals were detected within minutes (Fig. 2A and B), combined treatment with LPS and insulin delayed the appearance of TLR4 signaling mediators until at least 90 min (Fig. 3A and B). Furthermore, the appearance of TLR4 signaling mediators after ∼90 min coincided with a decline in Akt phosphorylation on Ser473 and FoxO1/FoxO3a on Thr24/Thr32. In complementary dose-dependent studies, leukocytes were treated for 1 h with decreasing insulin concentrations ranging from 1 to 0.05 units/mL (Fig. 3C and D) in combination with LPS (10 ng/mL). Control samples were treated with insulin alone for 1 or 2 h. Akt phosphorylation on Ser473, but not on Thr308, was detected in leukocytes treated for 2 h with insulin alone (1 unit/mL) (Fig. 3D, lane 4). As the phosphorylation of Akt on Ser473 declined in leukocytes treated with insulin at a concentration <0.25 units/mL, TLR4 signaling intermediates became visible (Fig. 3C and D, lanes 8 and 9). These studies establish that insulin can suppress leukocyte responses to LPS in a time- and dose-dependent manner.

Figure 3

Insulin suppresses LPS-induced signaling in leukocytes in a time- and dose-dependent manner. A and B: Blood samples were treated with LPS (10 ng/mL) plus insulin (1 unit/mL) for the indicated time. The sample in panel B, lane 8, was treated with LPS alone (10 ng/mL) for 90 min and served as a positive control. C and D: Blood samples were untreated (lane 1), treated with LPS for 2 h (lane 2), or treated with insulin (1 unit/mL) for 1 h (lane 3) or 2 h (lane 4). The samples shown in lanes 5–9 were treated for 1 h with LPS (10 ng/mL) in combination with insulin at the indicated concentration. p-S, phosphorylation on serine; p-T, phosphorylation on threonine; U, unit; UN, untreated.

Figure 3

Insulin suppresses LPS-induced signaling in leukocytes in a time- and dose-dependent manner. A and B: Blood samples were treated with LPS (10 ng/mL) plus insulin (1 unit/mL) for the indicated time. The sample in panel B, lane 8, was treated with LPS alone (10 ng/mL) for 90 min and served as a positive control. C and D: Blood samples were untreated (lane 1), treated with LPS for 2 h (lane 2), or treated with insulin (1 unit/mL) for 1 h (lane 3) or 2 h (lane 4). The samples shown in lanes 5–9 were treated for 1 h with LPS (10 ng/mL) in combination with insulin at the indicated concentration. p-S, phosphorylation on serine; p-T, phosphorylation on threonine; U, unit; UN, untreated.

LPS and Insulin-Induced Responses Seen in Leukocytes Are Reproduced in Neutrophils

Neutrophils, lymphocytes, and monocytes constitute, respectively, ∼50–65%, ∼30%, and ∼5% of all leukocytes in human blood. We showed that the temporal changes in MMP9, HIF-1α, and AMPKα expression seen in leukocytes treated with LPS are also seen in neutrophils (13). In addition, neutrophils and leukocytes isolated from patients after CPB surgery displayed similar signaling intermediates (13). In the current study, we first sought to determine whether LPS might also induce, as seen in leukocytes (Fig. 2A), Akt phosphorylation on Thr308 and IRS-1 phosphorylation on Ser312 in isolated neutrophils (Fig. 4A). We then asked whether neutrophils respond to insulin. As shown in Fig. 4A, insulin triggered Akt phosphorylation on Ser473 and Thr308 and S6K1 phosphorylation on Thr389 in neutrophils and suppressed LPS-induced signaling in a dose-dependent manner. Both LPS and insulin failed to trigger similar responses in mononucleated blood cells (Fig. 4B).

Figure 4

Insulin suppresses LPS-induced signaling in neutrophils but not mononucleated cells in a dose-dependent manner. After isolation, neutrophils (A) and mononucleated cells (B) were treated with LPS, insulin, or a combination of the two as described in Fig. 2C and D. All the samples shown had equal protein amounts. In panel B, lane 10 was loaded with lysates of leukocytes treated with LPS or insulin (for the blots probed for p-S473 Akt and p-T24/32 FoxO1/FoxO3a) and served as a positive control. p-S, phosphorylation on serine; p-T, phosphorylation on threonine; U, unit; UN, untreated.

Figure 4

Insulin suppresses LPS-induced signaling in neutrophils but not mononucleated cells in a dose-dependent manner. After isolation, neutrophils (A) and mononucleated cells (B) were treated with LPS, insulin, or a combination of the two as described in Fig. 2C and D. All the samples shown had equal protein amounts. In panel B, lane 10 was loaded with lysates of leukocytes treated with LPS or insulin (for the blots probed for p-S473 Akt and p-T24/32 FoxO1/FoxO3a) and served as a positive control. p-S, phosphorylation on serine; p-T, phosphorylation on threonine; U, unit; UN, untreated.

LPS and Insulin Signaling to mTORC1 and mTORC2 in Leukocytes

We further explored the LPS and insulin signaling pathways in leukocytes. mTORC1 phosphorylates ribosomal S6K1 on Thr389 (16,17). The detection of S6K1 phosphorylated on Thr389 in leukocytes treated with either LPS or insulin (Fig. 2) suggested that both pathways activate mTORC1. Treatment of leukocytes with the mTORC1 inhibitor rapamycin before treatment with LPS blocked the phosphorylation of S6K on Thr389 and the phosphorylation of Akt on Thr308 (Fig. 5A, lane 3). Torin and PP242, which inhibit both mTORC1 and mTORC2, reproduced the effect of rapamycin (42). The phosphorylation of IRS-1 on Ser312 was partially reduced, but TLR4 expression was not affected by rapamycin, Torin, or PP242. We then examined the effect of these inhibitors on leukocyte responses to insulin. Rapamycin, Torin, and PP242 suppressed the phosphorylation of Akt on Thr308 and the phosphorylation of S6K on Thr389. Akt phosphorylation on Ser473, induced by insulin, was inhibited by both Torin and PP242, whereas rapamycin had no effect. These studies show that the increases in TLR4 expression and IRS-1 phosphorylation on Ser312 induced by LPS are partially mTORC1/mTORC2 independent.

Figure 5

LPS and insulin signaling pathways in leukocytes. A: Blood samples were not treated (−) or pretreated for 1 h (+) with rapamycin, Torin, or PP242. The samples were next treated for 90 min with LPS (lanes 2–5) or for 60 min with insulin (lanes 6–9). B: Blood samples were not treated (−) or treated for 1 h (+) with AS1842856 (AS), a FoxO1 inhibitor. The samples were then untreated or treated with LPS for the indicated time. Leukocytes were isolated, lysed, and analyzed by immunoblotting. C: The working models for insulin and TLR4 signaling in leukocytes. Insulin activates mTORC1, triggering phosphorylation of S6K1 on Thr389 and Akt on Thr308. Insulin also activates mTORC2. This leads to phosphorylation of Akt on Ser473 and FoxO1/FoxO3a on Thr24/Thr32. TLR4 activation triggers an increase in MMP9 expression through FoxO proteins (51). MMP9 cleaves AMPKα. AMPK inactivation enables Raptor dephosphorylation on Ser792 and mTORC1 activation. mTORC1 regulates the phosphorylation of S6K1 on Thr389 and Akt on Thr308. FoxO1/FoxO3a also upregulates the expression of TLR4. TLR4 triggers IRS-1 phosphorylation on Ser312 by a mechanism that is only partially regulated by FoxO1/FoxO3a. When insulin and LPS are combined, insulin-dependent activation of mTORC2, Akt phosphorylation on Ser473, and FoxO1/FoxO3a phosphorylation on Thr24/Thr32 suppresses TLR4 signaling in a dose- and time-dependent manner. P, phosphorylation; p-S, phosphorylation on serine; p-T, phosphorylation on threonine; Rapa, rapamycin.

Figure 5

LPS and insulin signaling pathways in leukocytes. A: Blood samples were not treated (−) or pretreated for 1 h (+) with rapamycin, Torin, or PP242. The samples were next treated for 90 min with LPS (lanes 2–5) or for 60 min with insulin (lanes 6–9). B: Blood samples were not treated (−) or treated for 1 h (+) with AS1842856 (AS), a FoxO1 inhibitor. The samples were then untreated or treated with LPS for the indicated time. Leukocytes were isolated, lysed, and analyzed by immunoblotting. C: The working models for insulin and TLR4 signaling in leukocytes. Insulin activates mTORC1, triggering phosphorylation of S6K1 on Thr389 and Akt on Thr308. Insulin also activates mTORC2. This leads to phosphorylation of Akt on Ser473 and FoxO1/FoxO3a on Thr24/Thr32. TLR4 activation triggers an increase in MMP9 expression through FoxO proteins (51). MMP9 cleaves AMPKα. AMPK inactivation enables Raptor dephosphorylation on Ser792 and mTORC1 activation. mTORC1 regulates the phosphorylation of S6K1 on Thr389 and Akt on Thr308. FoxO1/FoxO3a also upregulates the expression of TLR4. TLR4 triggers IRS-1 phosphorylation on Ser312 by a mechanism that is only partially regulated by FoxO1/FoxO3a. When insulin and LPS are combined, insulin-dependent activation of mTORC2, Akt phosphorylation on Ser473, and FoxO1/FoxO3a phosphorylation on Thr24/Thr32 suppresses TLR4 signaling in a dose- and time-dependent manner. P, phosphorylation; p-S, phosphorylation on serine; p-T, phosphorylation on threonine; Rapa, rapamycin.

To assess the role of FoxO1 relative to leukocyte TLR4 signaling, we next used a FoxO1 inhibitor, AS1842856 (43). LPS failed to induce MMP9, AMPKα cleavage, or the increase in TLR4 expression in leukocytes pretreated with AS1842856 (Fig. 5B). These data identify FoxO1 as a critical upstream regulator of TLR4 signaling in human leukocytes. The residual phosphorylation of IRS-1 on Ser312 detected in leukocytes treated with AS1842856, Torin, or PP242 (Fig. 4) suggests that an additional FoxO1/FoxO3a-independent regulatory mechanism contributes to this phosphorylation event. The working models are presented in Fig. 5C.

Insulin Regulates Leukocyte TLR4 Expression and Sensitivity to LPS

TLR4 expression was markedly increased in the majority of the type 2 diabetes samples studied and in leukocytes treated with LPS in vitro (Figs. 13). Leukocytes from patients after CPB also exhibited a significant increase in TLR4 expression postsurgery (Fig. 6A). We next asked whether the increase in TLR4 expression in leukocytes alters their sensitivity to LPS by using patient leukocytes before and after CPB surgery. Leukocyte activation triggers IκBα degradation, enabling translocation of nuclear factor-κB to the nucleus (9). We used IκBα degradation as a proxy for assessing TLR4 sensitivity. Leukocytes obtained either before or after CPB surgery showed IκBα degradation within 5 min of LPS treatment in vitro (Fig. 6B). However, although IκBα degradation in leukocytes from three patients required LPS at 10 ng/mL presurgery, a concentration of 2–5 ng/mL was sufficient to trigger IκBα degradation in leukocytes from the same patients postsurgery (Fig. 6C). These data indicate that the increase in TLR4 expression after CPB surgery correlates with a two- to fivefold increase in leukocyte sensitivity to LPS. Consistent with the observation that FoxO1 regulates the expression of TRL4 in Raw264.7 cells (28), the FoxO1 inhibitor AS1842856 suppressed TLR4 expression in leukocytes treated with LPS (Fig. 5B). Furthermore, insulin suppressed the expression of TLR4 in a time- and dose-dependent fashion (Fig. 6D and E). Together, these data demonstrate that insulin not only suppresses TLR4 signaling but also diminishes leukocyte responsiveness to LPS.

Figure 6

An increase in leukocyte TLR4 expression enhances leukocyte responsiveness to LPS. A: Blood samples were obtained from three CPB patients presurgery, in the recovery room, and on day 1 postsurgery. B: Blood samples obtained from a CPB patient presurgery and on day 1 postsurgery were treated with LPS (10 ng/mL) for the indicated time. C: Blood samples obtained from three CPB patients presurgery and on day 1 postsurgery were treated for 5 min with LPS at the indicated concentration. D: Blood samples were treated with LPS plus insulin for the indicated time. E: Blood samples were untreated (lane 1), treated with LPS for 2 h (lane 2), or treated with insulin (1 unit/mL) for 1 h (lane 3) or 2 h (lane 4). The samples shown in lanes 5–9 were first treated for 1 h with insulin at the indicated concentration and then for 1 h with LPS (10 ng/mL). Leukocytes were isolated, lysed, and analyzed by immunoblotting. D1, day 1; P, presurgery; R, recovery room; U, unit; UN, untreated.

Figure 6

An increase in leukocyte TLR4 expression enhances leukocyte responsiveness to LPS. A: Blood samples were obtained from three CPB patients presurgery, in the recovery room, and on day 1 postsurgery. B: Blood samples obtained from a CPB patient presurgery and on day 1 postsurgery were treated with LPS (10 ng/mL) for the indicated time. C: Blood samples obtained from three CPB patients presurgery and on day 1 postsurgery were treated for 5 min with LPS at the indicated concentration. D: Blood samples were treated with LPS plus insulin for the indicated time. E: Blood samples were untreated (lane 1), treated with LPS for 2 h (lane 2), or treated with insulin (1 unit/mL) for 1 h (lane 3) or 2 h (lane 4). The samples shown in lanes 5–9 were first treated for 1 h with insulin at the indicated concentration and then for 1 h with LPS (10 ng/mL). Leukocytes were isolated, lysed, and analyzed by immunoblotting. D1, day 1; P, presurgery; R, recovery room; U, unit; UN, untreated.

To gain a better understanding of leukocyte signaling in the context of insulin resistance, we studied leukocytes from two patient cohorts: patients with type 2 diabetes (chronic insulin resistance) and patients undergoing CPB surgery (acute insulin resistance). In parallel studies, we analyzed responses of leukocytes and neutrophils treated in vitro with LPS and/or insulin, in combination with well-characterized pharmacologic response modulators. The leukocytes from the cohort of 12 patients with type 2 diabetes exhibited two patterns of signaling intermediates. Pattern 1 included MMP9, cleaved AMPKα, HIF-1α, and enhanced TLR4 expression. Leukocytes with pattern 1 expressed Akt phosphorylated on Thr308 but not Ser473. Three additional signaling intermediates, Raptor dephosphorylated on Ser792, S6K1 phosphorylated on Thr389, and IRS-1 phosphorylated on Ser312, were detected in leukocytes with pattern 1 and pattern 2. Patient leukocytes obtained on days 1–3 after CPB surgery also exhibited pattern 1. We showed that plasma post-CPB surgery contained a factor that induced TLR4 activation in healthy donor leukocytes (13). Furthermore, the entire repertoire of signaling mediators associated with pattern 1 was reproduced in leukocytes treated with LPS in vitro. The FoxO1 inhibitor AS1842856 suppressed MMP9 expression, an increase in TLR4, and AMPKα cleavage, all of which are induced by LPS, indicating that FoxO1 is an essential mediator of leukocyte TLR4 signaling. In line with this finding, others have shown that FoxO1 bound multiple enhancer-like elements in the TLR4 gene and increased the expression of TLR4 in LPS-treated Raw264.7 cells (28). When taken as a whole, the current data provide compelling evidence that pattern 1 reflects TLR4 signaling. Others reported that the concentration of TLR4 ligands is higher in the blood of patients with type 2 diabetes than in patients without diabetes or healthy control subjects (34,44). Persistent presence of TLR4 ligands could sustain chronic TLR4 signaling activation in leukocytes from patients with type 2 diabetes.

To our knowledge, the current data are the first to demonstrate that insulin activates a canonical signaling pathway that includes mTORC2, Akt phosphorylated on Thr308 and Ser473, S6K1 phosphorylated on Thr389, and FoxO1/FoxO3a phosphorylated on Thr24/Thr32 in human leukocytes and neutrophils. The detection of Akt phosphorylated on Ser473 in leukocytes from patients with type 2 diabetes receiving insulin suggests that leukocytes respond to insulin in vivo. Analyses of CPB patient leukocytes led to a similar conclusion. Acute insulin resistance develops in patients after CPB, requiring insulin infusion for several days postsurgery. While being treated with insulin on days 1–3 post-CPB surgery, patient leukocytes exhibited the entire repertoire of signaling mediators associated with pattern 1. By days 3 and 4, as patients regained insulin sensitivity, pattern 1 was replaced by Akt phosphorylated on Ser473 and FoxO1/FoxO3a phosphorylated on Thr24/Thr32. Thus, both the in vitro and the in vivo data suggest that insulin triggers Akt phosphorylation on Ser473 and FoxO1/FoxO3a phosphorylation on Thr24/Thr32 in leukocytes and neutrophils and that in the presence of these signaling mediators, TLR4 signals are suppressed. The data also suggest that insulin fails to suppress TLR4 signals in patients with insulin resistance. A number of studies have suggested that insulin has anti-inflammatory effects, which is consistent with the current findings. Of note, the administration of insulin to subjects challenged with LPS suppressed inflammatory responses in vivo (45). Prior studies that used mouse embryonic fibroblasts and bone marrow–derived dendritic cells showed that TLR4 not only activates FoxO1 but also triggers FoxO1 inactivation through mTORC2 and Akt phosphorylation on Ser473 (46). In marked contrast, TLR4 signaling in leukocytes is limited to FoxO1 activation, indicating that FoxO1 inactivation in the context of TLR4 signaling requires input from another signaling mediator, such as insulin.

The current study also established that TLR4 signaling induces specific phosphorylation of Akt on Thr308. Although phosphorylation of Akt on residue Thr308 or Ser473 enhances the catalytic activity of Akt by ≥10-fold, it is generally believed that the catalytic activity of Akt is regulated synergistically and requires both Thr308 and Ser473 phosphorylation for optimal Akt activation (30,37). Akt phosphorylation on Ser473 is assumed to represent Akt activation. However, in non–small cell lung cancer cells, the phosphorylation of Akt at Thr308 rather than at Ser473 showed a better correlation with Akt activation (47). Studies in human platelets have similarly demonstrated that the phosphorylation of Akt on Ser473 was dispensable for Akt phosphorylation on Thr308 and Akt1 activity (48). Although in many instances PI3K activates Akt, which then activates mTORC1, the current data suggest that mTORC1 regulates the phosphorylation of Akt on Thr308 in leukocytes treated with LPS or insulin. The role of Akt phosphorylated on Thr308 in leukocytes is unclear.

Leukocytes from 4 of 12 patients with type 2 diabetes exhibited pattern 2, which included Akt phosphorylated on Ser473 but not MMP9, HIF-1α, or cleaved AMPKα. The phosphorylation of FoxO1/FoxO3a on Thr24/Thr32 in these samples was either trace or absent. Given that FoxO1 phosphorylation/inactivation was impaired in liver, adipose, and muscle from insulin resistant mice (49), the current data suggest that this might also apply to the leukocytes of patients with type 2 diabetes. Because the four patients were treated with insulin, it is plausible that insulin was able to suppress a subset but not the entire panel of inflammatory signaling mediators in these patients’ leukocytes.

In summary, the current data provide novel insight into chronic human leukocyte inflammatory responses and compelling evidence that TLR4 signaling is activated in leukocytes from patients with type 2 diabetes. Studies have suggested that food intake, particularly when rich in fat, might trigger an increase in TLR4 ligand abundance in blood (50). If such physiologic responses exist, leukocytes might be exposed frequently and simultaneously to insulin and TLR4 ligands. The dynamic and highly competitive interaction between insulin and TLR4 signaling pathways in leukocytes could serve an important homeostatic function, namely, to prevent unwanted leukocyte activation. Chronic exposure to low-dose TLR4 ligands could contribute to the derailment of such physiologic responses, ultimately leading to insulin resistance and type 2 diabetes because leukocyte activation increases reliance on systemic glucose availability. Under conditions of chronic low-grade inflammation, leukocytes most likely contribute to systemic insulin resistance to ensure the glucose availability essential for their survival.

M.J.B. is currently affiliated with the Cardiovascular and Metabolic Research Unit, Pfizer, Inc., Cambridge, MA.

Funding. The study was supported in part by funds from the New Jersey Health Foundation (to L.Y.L. and B.H.).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. Z.Z. contributed to the study design, performance of experiments, and preparation of the manuscript. L.F.A. and L.Y.L. contributed to the patient recruitment and data analysis and interpretation. S.M.C. and M.A.M. contributed to the patient recruitment and data analysis. M.J.B. contributed to the data interpretation. B.H. contributed to the study design, data analysis and interpretation, and preparation of the manuscript. B.H. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and accuracy of the data analyses.

1.
Black
PR
,
Brooks
DC
,
Bessey
PQ
,
Wolfe
RR
,
Wilmore
DW
.
Mechanisms of insulin resistance following injury
.
Ann Surg
1982
;
196
:
420
435
[PubMed]
2.
Duncan
BB
,
Schmidt
MI
,
Pankow
JS
, et al.;
Atherosclerosis Risk in Communities Study
.
Low-grade systemic inflammation and the development of type 2 diabetes: the atherosclerosis risk in communities study
.
Diabetes
2003
;
52
:
1799
1805
[PubMed]
3.
Gregor
MF
,
Hotamisligil
GS
.
Inflammatory mechanisms in obesity
.
Annu Rev Immunol
2011
;
29
:
415
445
[PubMed]
4.
Hoshino
K
,
Takeuchi
O
,
Kawai
T
, et al
.
Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product
.
J Immunol
1999
;
162
:
3749
3752
[PubMed]
5.
Agwunobi
AO
,
Reid
C
,
Maycock
P
,
Little
RA
,
Carlson
GL
.
Insulin resistance and substrate utilization in human endotoxemia
.
J Clin Endocrinol Metab
2000
;
85
:
3770
3778
[PubMed]
6.
Lowry
SF
.
Human endotoxemia: a model for mechanistic insight and therapeutic targeting
.
Shock
2005
;
24
(
Suppl. 1
):
94
100
[PubMed]
7.
Cani
PD
,
Amar
J
,
Iglesias
MA
, et al
.
Metabolic endotoxemia initiates obesity and insulin resistance
.
Diabetes
2007
;
56
:
1761
1772
[PubMed]
8.
Saberi
M
,
Woods
NB
,
de Luca
C
, et al
.
Hematopoietic cell-specific deletion of toll-like receptor 4 ameliorates hepatic and adipose tissue insulin resistance in high-fat-fed mice
.
Cell Metab
2009
;
10
:
419
429
[PubMed]
9.
Yamamoto
Y
,
Gaynor
RB
.
IkappaB kinases: key regulators of the NF-kappaB pathway
.
Trends Biochem Sci
2004
;
29
:
72
79
[PubMed]
10.
Arkan
MC
,
Hevener
AL
,
Greten
FR
, et al
.
IKK-beta links inflammation to obesity-induced insulin resistance
.
Nat Med
2005
;
11
:
191
198
[PubMed]
11.
Zhang
Z
,
Lowry
SF
,
Guarente
L
,
Haimovich
B
.
Roles of SIRT1 in the acute and restorative phases following induction of inflammation
.
J Biol Chem
2010
;
285
:
41391
41401
[PubMed]
12.
Haimovich
B
,
Zhang
Z
,
Calvano
JE
, et al
.
Cellular metabolic regulators: novel indicators of low-grade inflammation in humans
.
Ann Surg
2014
;259:999–1006
[PubMed]
13.
Zhang
Z
,
Amorosa
LF
,
Coyle
SM
, et al
.
Proteolytic cleavage of AMPKα and intracellular MMP9 expression are both required for TLR4-mediated mTORC1 activation and HIF-1α expression in leukocytes
.
J Immunol
2015
;
195
:
2452
2460
[PubMed]
14.
Towler
MC
,
Hardie
DG
.
AMP-activated protein kinase in metabolic control and insulin signaling
.
Circ Res
2007
;
100
:
328
341
[PubMed]
15.
Laplante
M
,
Sabatini
DM
.
mTOR signaling in growth control and disease
.
Cell
2012
;
149
:
274
293
[PubMed]
16.
Pullen
N
,
Thomas
G
.
The modular phosphorylation and activation of p70s6k
.
FEBS Lett
1997
;
410
:
78
82
[PubMed]
17.
Pearson
RB
,
Dennis
PB
,
Han
JW
, et al
.
The principal target of rapamycin-induced p70s6k inactivation is a novel phosphorylation site within a conserved hydrophobic domain
.
EMBO J
1995
;
14
:
5279
5287
[PubMed]
18.
Cramer
T
,
Yamanishi
Y
,
Clausen
BE
, et al
.
HIF-1alpha is essential for myeloid cell-mediated inflammation
.
Cell
2003
;
112
:
645
657
[PubMed]
19.
Rius
J
,
Guma
M
,
Schachtrup
C
, et al
.
NF-kappaB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1alpha
.
Nature
2008
;
453
:
807
811
[PubMed]
20.
Jacinto
E
,
Loewith
R
,
Schmidt
A
, et al
.
Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive
.
Nat Cell Biol
2004
;
6
:
1122
1128
[PubMed]
21.
Sarbassov
DD
,
Guertin
DA
,
Ali
SM
,
Sabatini
DM
.
Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex
.
Science
2005
;
307
:
1098
1101
[PubMed]
22.
Tzivion G, Dobson M, Ramakrishnan G. FoxO transcription factors; regulation by AKT and 14-3-3 proteins. Biochim Biophys Acta 2011;1813:1938–1945
23.
Biggs
WH
 3rd
,
Meisenhelder
J
,
Hunter
T
,
Cavenee
WK
,
Arden
KC
.
Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1
.
Proc Natl Acad Sci U S A
1999
;
96
:
7421
7426
[PubMed]
24.
Crossley
LJ
.
Neutrophil activation by fMLP regulates FOXO (forkhead) transcription factors by multiple pathways, one of which includes the binding of FOXO to the survival factor Mcl-1
.
J Leukoc Biol
2003
;
74
:
583
592
[PubMed]
25.
Jonsson
H
,
Allen
P
,
Peng
SL
.
Inflammatory arthritis requires Foxo3a to prevent Fas ligand-induced neutrophil apoptosis
.
Nat Med
2005
;
11
:
666
671
[PubMed]
26.
Tothova
Z
,
Kollipara
R
,
Huntly
BJ
, et al
.
FoxOs are critical mediators of hematopoietic stem cell resistance to physiologic oxidative stress
.
Cell
2007
;
128
:
325
339
[PubMed]
27.
Peng
SL
.
Foxo in the immune system
.
Oncogene
2008
;
27
:
2337
2344
[PubMed]
28.
Fan
W
,
Morinaga
H
,
Kim
JJ
, et al
.
FoxO1 regulates Tlr4 inflammatory pathway signalling in macrophages
.
EMBO J
2010
;
29
:
4223
4236
[PubMed]
29.
Walrand
S
,
Guillet
C
,
Boirie
Y
,
Vasson
MP
.
Insulin differentially regulates monocyte and polymorphonuclear neutrophil functions in healthy young and elderly humans
.
J Clin Endocrinol Metab
2006
;
91
:
2738
2748
[PubMed]
30.
Alessi
DR
,
James
SR
,
Downes
CP
, et al
.
Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha
.
Curr Biol
1997
;
7
:
261
269
[PubMed]
31.
Guertin
DA
,
Stevens
DM
,
Thoreen
CC
, et al
.
Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1
.
Dev Cell
2006
;
11
:
859
871
[PubMed]
32.
Brunet
A
,
Bonni
A
,
Zigmond
MJ
, et al
.
Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor
.
Cell
1999
;
96
:
857
868
[PubMed]
33.
Su
D
,
Coudriet
GM
,
Hyun Kim
D
, et al
.
FoxO1 links insulin resistance to proinflammatory cytokine IL-1beta production in macrophages
.
Diabetes
2009
;
58
:
2624
2633
[PubMed]
34.
Dasu
MR
,
Devaraj
S
,
Park
S
,
Jialal
I
.
Increased toll-like receptor (TLR) activation and TLR ligands in recently diagnosed type 2 diabetic subjects
.
Diabetes Care
2010
;
33
:
861
868
[PubMed]
35.
Reyna
SM
,
Ghosh
S
,
Tantiwong
P
, et al
.
Elevated toll-like receptor 4 expression and signaling in muscle from insulin-resistant subjects
.
Diabetes
2008
;
57
:
2595
2602
[PubMed]
36.
Boucher
J
,
Kleinridders
A
,
Kahn
CR
.
Insulin receptor signaling in normal and insulin-resistant states
.
Cold Spring Harb Perspect Biol
2014
;
6
:
6
[PubMed]
37.
Alessi
DR
,
Andjelkovic
M
,
Caudwell
B
, et al
.
Mechanism of activation of protein kinase B by insulin and IGF-1
.
EMBO J
1996
;
15
:
6541
6551
[PubMed]
38.
Friedman
JE
,
Ishizuka
T
,
Liu
S
, et al
.
Reduced insulin receptor signaling in the obese spontaneously hypertensive Koletsky rat
.
Am J Physiol
1997
;
273
:
E1014
E1023
[PubMed]
39.
Hirosumi
J
,
Tuncman
G
,
Chang
L
, et al
.
A central role for JNK in obesity and insulin resistance
.
Nature
2002
;
420
:
333
336
[PubMed]
40.
Doenst
T
,
Wijeysundera
D
,
Karkouti
K
,
Zechner
C
,
Maganti
M
,
Rao
V
,
Borger
MA
:
Hyperglycemia during cardiopulmonary bypass is an independent risk factor for mortality in patients undergoing cardiac surgery
.
J Thorac Cardiovasc Surg
2005
;
130
:
1144
41.
Harte
AL
,
Varma
MC
,
Tripathi
G
, et al
.
High fat intake leads to acute postprandial exposure to circulating endotoxin in type 2 diabetic subjects
.
Diabetes Care
2012
;
35
:
375
382
[PubMed]
42.
Thoreen
CC
,
Kang
SA
,
Chang
JW
, et al
.
An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1
.
J Biol Chem
2009
;
284
:
8023
8032
[PubMed]
43.
Nagashima
T
,
Shigematsu
N
,
Maruki
R
, et al
.
Discovery of novel forkhead box O1 inhibitors for treating type 2 diabetes: improvement of fasting glycemia in diabetic db/db mice
.
Mol Pharmacol
2010
;
78
:
961
970
[PubMed]
44.
Pussinen
PJ
,
Havulinna
AS
,
Lehto
M
,
Sundvall
J
,
Salomaa
V
.
Endotoxemia is associated with an increased risk of incident diabetes
.
Diabetes Care
2011
;
34
:
392
397
[PubMed]
45.
Dandona
P
,
Ghanim
H
,
Bandyopadhyay
A
, et al
.
Insulin suppresses endotoxin-induced oxidative, nitrosative, and inflammatory stress in humans
.
Diabetes Care
2010
;
33
:
2416
2423
[PubMed]
46.
Brown
J
,
Wang
H
,
Suttles
J
,
Graves
DT
,
Martin
M
.
Mammalian target of rapamycin complex 2 (mTORC2) negatively regulates Toll-like receptor 4-mediated inflammatory response via FoxO1
.
J Biol Chem
2011
;
286
:
44295
44305
[PubMed]
47.
Vincent
EE
,
Elder
DJ
,
Thomas
EC
, et al
.
Akt phosphorylation on Thr308 but not on Ser473 correlates with Akt protein kinase activity in human non-small cell lung cancer
.
Br J Cancer
2011
;
104
:
1755
1761
[PubMed]
48.
Moore
SF
,
Hunter
RW
,
Hers
I
.
mTORC2 protein complex-mediated Akt (protein kinase B) serine 473 phosphorylation is not required for Akt1 activity in human platelets [published correction appears in J Biol Chem 2011;286:31062]
.
J Biol Chem
2011
;
286
:
24553
24560
[PubMed]
49.
Guo
S
.
Insulin signaling, resistance, and the metabolic syndrome: insights from mouse models into disease mechanisms
.
J Endocrinol
2014
;
220
:
T1
T23
[PubMed]
50.
Erridge
C
,
Attina
T
,
Spickett
CM
,
Webb
DJ
.
A high-fat meal induces low-grade endotoxemia: evidence of a novel mechanism of postprandial inflammation
.
Am J Clin Nutr
2007
;
86
:
1286
1292
[PubMed]
51.
Li
H
,
Liang
J
,
Castrillon
DH
,
DePinho
RA
,
Olson
EN
,
Liu
ZP
.
FoxO4 regulates tumor necrosis factor alpha-directed smooth muscle cell migration by activating matrix metalloproteinase 9 gene transcription
.
Mol Cell Biol
2007
;
27
:
2676
2686
[PubMed]

Supplementary data