Insulin-Dependent Regulation of mTORC2-Akt-FoxO Suppresses TLR4 Signaling in Human Leukocytes: Relevance to Type 2 Diabetes

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 (8–10).

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 (24–26) 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.

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).

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.

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.

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.

We previously characterized signaling intermediates found in peripheral blood leukocytes from 12 patients with type 2 diabetes as defined by hemoglobin A_1c (HbA_1c) ≥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 (34–36) are modified in leukocytes from the cohort of patients described in Table 1 and our previous work (13).

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: HbA_1c 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).

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.

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.

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.

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).

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.

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.

TLR4 expression was markedly increased in the majority of the type 2 diabetes samples studied and in leukocytes treated with LPS in vitro (Figs. 1–3). 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.

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.

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.