2224 Diabetes Volume 65, August 2016 Zhiyong Zhang,1 Louis F. Amorosa,2 Susette M. Coyle,1 Marie A. Macor,1 Morris J. Birnbaum,3 Leonard Y. Lee,1 and Beatrice Haimovich1 Insulin-Dependent Regulation of mTORC2-Akt-FoxO Suppresses TLR4 Signaling in Human Leukocytes: Relevance to Type 2 Diabetes Diabetes 2016;65:2224–2234 | DOI: 10.2337/db16-0027 Leukocyte signaling in patients with systemic insulin re- (1). Modest increases in inflammatory indicators detected in sistance is largely unexplored. We recently discovered the patients have suggested that type 2 diabetes is associated presence of multiple Toll-like receptor 4 (TLR4) signaling with chronic low-grade inflammation (2,3). Human subjects intermediates in leukocytes from patients with type 2 challenged with lipopolysaccharide (LPS), a ligand of Toll-like diabetes or acute insulin resistance associated with cardio- receptor 4 (TLR4) (4), produce acute inflammatory re- pulmonary bypass surgery. We extend this work to show sponses that include insulin resistance (5,6). Mice adminis- that in addition to matrix metalloproteinase 9, hypoxia- tered a low dose of LPS for 1 month develop insulin inducible factor 1a, and cleaved AMPKa, patient leukocytes fi 312 resistance (7). On the other hand, TLR4-de cient mice also express IRS-1 phosphorylated on Ser , Akt phosphor- andmicetransplantedwithbonemarrowcellsfromTLR4- ylated on Thr308, and elevated TLR4 expression. Similar sig- deficient mice were more resistant to high-fat diet–induced naling intermediates were detected in leukocytes and insulin resistance than wild-type mice or mice transplanted neutrophils treated with lipopolysaccharide (LPS), a ligand withbonemarrowcellsfromwild-typemice(8).Theseand of TLR4, in vitro. In contrast, insulin, but not LPS, induced mammalian target of rapamycin complex 2 (mTORC2)– other studies have indicated that immune cells and TLR4 SIGNAL TRANSDUCTION – dependent phosphorylation of Akt on Ser473 and FoxO1/O3a signaling contribute to systemic insulin resistance (8 10). on Thr24/32 in leukocytes and neutrophils. Insulin suppressed We reported that LPS triggers rapid changes in cellu- LPS-induced responses in a dose- and time-dependent lar metabolism in human and mouse leukocytes (11,12) – manner. AS1842856, a FoxO1 inhibitor, also suppressed through a phosphoinositide 3-kinase (PI3K) dependent TLR4 signaling. We propose that insulin is a homeostatic process that involves two metabolic regulators: AMPK regulator of leukocyte responses to LPS/TLR4 and that the and hypoxia-inducible factor 1 (HIF-1) (13). We showed signaling intermediates expressed in leukocytes of patients that LPS increases the expression of matrix metallopro- with type 2 diabetes indicate TLR4 signaling dominance teinase 9 (MMP9) in leukocytes and neutrophils and that and deficient insulin signaling. The data suggest that in- intracellular MMP9 cleaves the catalytic subunit of AMPK sulin suppresses LPS/TLR4 signals in leukocytes through (AMPKa) (13). AMPK inactivation enables dephosphory- the mTORC2-Akt-FoxO signaling axis. Better understand- lation of the regulatory-associated protein of mammalian ing of leukocyte signaling in patients with type 2 diabetes target of rapamycin (mTOR) (Raptor) on Ser792, resulting may shed new light on disease causation and progression. in mTOR complex 1 (mTORC1) activation (14,15). mTORC1 phosphorylates ribosomal S6 kinase (S6K1) on Robust inflammatory responses associated with injury or Thr389 (16,17). In addition, mTORC1 activates the tran- sepsis trigger the onset of insulin resistance in humans scription factor HIF-1, which promotes glycolysis and 1Department of Surgery, Rutgers Robert Wood Johnson Medical School, New This article contains Supplementary Data online at http://diabetes Brunswick, NJ .diabetesjournals.org/lookup/suppl/doi:10.2337/db16-0027/-/DC1. 2 Department of Medicine, Rutgers Robert Wood Johnson Medical School, New M.J.B. is currently affiliated with the Cardiovascular and Metabolic Research Unit, Brunswick, NJ Pfizer, Inc., Cambridge, MA. 3Institute of Diabetes, Obesity, and Metabolism, Perelman School of Medicine, © 2016 by the American Diabetes Association. Readers may use this article as University of Pennsylvania, Philadelphia, PA long as the work is properly cited, the use is educational and not for profit, and Corresponding author: Beatrice Haimovich, [email protected]. the work is not altered. Received 6 January 2016 and accepted 2 May 2016. diabetes.diabetesjournals.org Zhang and Associates 2225 proinflammatory cytokine production in both human and RESEARCH DESIGN AND METHODS mouse leukocytes treated with LPS (18,19). Because nei- Materials a 389 ther HIF-1 nor S6K1 phosphorylated on Thr was de- The following antibodies were used: anti-actin (Sigma- fi tected in MMP9-de cient mouse leukocytes treated with Aldrich); anti-HIF-1a, AMPKa, MMP9, IkB-a, total Akt, LPS, the mechanism by which TLR4 activates mTORC1 and TLR4 (Santa Cruz Biotechnology); and anti-Raptor might be unique (13). Ser792, total Raptor, S6K1 Thr389,AktThr308, Akt Ser473, A second complex involving mTOR, known as mTOR IRS-1 Ser312 (Ser307 in mice), IRS-1, and FoxO1/FoxO3a complex 2 (mTORC2), regulates the phosphorylation of Thr24/Thr32 (Cell Signaling Technology). Source and final 473 Akt on Ser (20,21). Activated Akt phosphorylates concentration of pharmacologic agents were as follows: FoxO1 and FoxO3a, which belong to the forkhead family LPS from Escherichia coli 0111:B4 (Sigma-Aldrich); insulin of transcription factors, on multiple threonine and ser- (Humalog; Eli Lilly), rapamycin (100 nmol/L), Torin ine residues (22). Phosphorylated FoxO proteins are ex- (50 nmol/L), and PP242 (100 nmol/L) (Tocris); and AS1842856 cluded from the nucleus and remain inactive (23). FoxO1 (100 nmol/L) (Millipore). regulates leukocyte survival (24–26) and TLR4 signaling (27,28). Although activated FoxO1 triggers an increase Human Subjects in TLR4 expression in LPS-treated Raw264.7 cells (28), The Rutgers Health Sciences Institutional Review Board it also contributes to LPS-dependent TLR4 signaling approved the study. Patients with type 2 diabetes and inactivation in these cells (28). The role of FoxO rela- patients without diabetes were recruited from endocri- tive to TLR4 signaling in human leukocytes is currently nology clinics at Rutgers Robert Wood Johnson Medical unknown. School during a scheduled visit. Written informed consent Human leukocytes express an insulin receptor (29). was obtained from all subjects before inclusion in the Insulin receptor activation triggers phosphorylation of study. Twelve patients with type 2 diabetes were recruited, insulin receptor substrate (IRS) proteins on tyrosine and their demographics are shown in Table 1. Patient clinical residues. This leads to PI3K and phosphoinositide- records are presented in Supplementary Table 1. Cardiac dependent kinase 1 activation, the latter of which phosphor- surgery patients were recruited from Rutgers Robert Wood ylates Akt on Thr308 (30). Insulin induces phosphorylation Johnson University Hospital. Inclusion and exclusion crite- of Akt on Ser473 through mTORC2, and Akt phosphor- ria were described previously (13) and are included in the ylates FoxO (21,31,32). FoxO1 phosphorylation was Supplementary Data. impaired in macrophages derived from insulin resis- Leukocytes Isolation tant obese mice (33). Despite this general frame- Blood drawn into heparin-containing tubes was sepa- work,theinsulinsignalingpathwayinleukocytesis rated into aliquots and treated with LPS (10 ng/mL) undetermined. or insulin (1 unit/mL), unless otherwise indicated. The We recently showed that leukocytes from a cohort of patients with type 2 diabetes exhibited distinct signaling intermediates (13). From these observations, we hypoth- esized that characterization of signaling intermediates Table 1—Patient characteristics expressed in patient leukocytes could have utility in deter- Without With type 2 diabetes diabetes mining disease etiology and progression. The data showed that leukocytes from patients with type 2 diabetes exhibit No. of patients 10 12 two patterns of signaling intermediates. The presence Age (years) 61 6 4616 2 of MMP9 and HIF-1a as well as cleaved AMPKa define Sex the first pattern (pattern 1), whereas the presence of Male 5 9 Akt phosphorylated on Ser473 defines the second (pattern 2). Female 5 3 2 IRS-1 phosphorylated on Ser312 and S6K1 phosphorylated BMI (kg/m )346 3296 1 on Thr389 were seen in leukocytes expressing either pat- Fasting plasma glucose (mg/dL) 101 6 5 147 6 14* tern 1 or pattern 2. The signaling intermediates asso- HbA1c ciated with pattern 1 were reproduced in leukocytes % 5.9 6 0.1 7.7 6 0.3** treated with LPS in vitro. Insulin but not LPS induced mmol/mol 41 60.7 phosphorylation of Akt on Ser473 and FoxO1/FoxO3a Cholesterol (mg/dL) 157 6 14 171 6 16 on Thr24/Thr32 in leukocytes and neutrophils and HDL cholesterol (mg/dL) 49 6 4506 2 suppressed LPS-induced TLR4 signaling in a dose- and LDL cholesterol (mg/dL) 89 6 10 96 6 14 time-dependent manner. The data suggested that TLR4 Triglycerides (mg/dL) 94 6 20 127 6 12 signaling is linked to pattern 1. We propose that leuko- Alanine aminotransferase (IU/L) 28 6 9306 6 cytes with pattern 2 are partially responsive to insulin, Aspartate aminotransferase (IU/L) 18 6 2276 3 which is no longer able to fully suppress the inflam- 6 P matory signals present in these patients’ leukocytes/ Data are mean SEM unless otherwise indicated. * = 0.024. **P = 0.002. neutrophils. 2226 Insulin and TLR4 Signaling