Neutrophil Elastase Regulates Emergency Myelopoiesis Preceding Systemic Inflammation in Diet-induced Obesity.

Inflammation plays a significant role in the development of obesity-related complications, but the molecular events that initiate and propagate such inflammation remain unclear. Here, we report that mice fed a high-fat diet (HFD) for as little as 1-3 days show increased differentiation of myeloid progenitors into neutrophils and monocytes but reduced B lymphocyte production in the bone marrow. Levels of neutrophil elastase (NE) and the nuclear factors CCAAT/enhancer-binding protein α (C/EBPα) and growth factor-independent 1 (GFI-1) are elevated in hematopoietic stem and progenitor cells from HFD-fed mice, but mice lacking either NE or C/EBPα are resistant to HFD-induced myelopoiesis. NE deletion increases expression of the inhibitory isoform of p30 C/EBPα, impairs the transcriptional activity of p42 C/EBPα, and reduces expression of the C/EBPα target gene GFI-1 in hematopoietic stem and progenitor cells, suggesting a mechanism by which NE regulates myelopoiesis. Furthermore, NE deletion prevents HFD-induced vascular leakage. Thus, HFD feeding rapidly activates bone marrow myelopoiesis through the NE-dependent C/EBPα-GFI-1 pathway preceding vascular damage and systemic inflammation.

Neutrophil Elastase Increases Vascular Permeability and Leukocyte Transmigration in Cultured Endothelial Cells and Obese Mice.

Neutrophil elastase (NE) plays a pivotal role in inflammation. However, the mechanism underlying NE-mediated inflammation in obesity remains unclear. Here, we report that NE activates protease-activated receptor-2 (PAR2), stimulates actin filament (F-actin) formation, decreases intercellular junction molecule VE-cadherin expression, and increases the permeability of human arterial endothelial cells (hECs). NE also prompts degradation of VE-cadherin and its binding proteins p120- and ß-catenins via MG132-sensitive proteasomes. NE stimulates phosphorylation of myosin light-chain (MLC) and its regulator myosin phosphatase target subunit-1 (MYPT1), a target of Rho kinase (ROCK). Inhibitors of PAR2 and ROCK prohibit NE-induced F-actin formation, MLC phosphorylation, and VE-cadherin reduction in hECs, and impede monocyte transmigration through hEC monolayer pretreated with either neutrophils or NE. Further, administration of an NE inhibitor GW311616A significantly attenuates vascular leakage, leukocyte infiltration, and the expression of proinflammatory cytokines in the white adipose tissue from high-fat diet (HFD)-induced obese mice. Likewise, NE-deficient mice are resistant to HFD-induced vascular leakage in the heart. Together, NE regulates actomyosin cytoskeleton activity and VE-cadherin expression by activating PAR2 signaling in the endothelial cells, leading to increased vascular permeability and leukocyte extravasation. Hence, inhibition of NE is a potential approach to mitigate vascular injury and leukocyte infiltration in obesity-related systemic inflammation.

A novel pleckstrin homology domain-containing protein enhances insulin-stimulated Akt phosphorylation and GLUT4 translocation in adipocytes.

Protein kinase B/Akt protein kinases control an array of diverse functions, including cell growth, survival, proliferation, and metabolism. We report here the identification of pleckstrin homology-like domain family B member 1 (PHLDB1) as an insulin-responsive protein that enhances Akt activation. PHLDB1 contains a pleckstrin homology domain, which we show binds phosphatidylinositol PI(3,4)P(2), PI(3,5)P(2), and PI(3,4,5)P(3), as well as a Forkhead-associated domain and coiled coil regions. PHLDB1 expression is increased during adipocyte differentiation, and it is abundant in many mouse tissues. Both endogenous and HA- or GFP-tagged PHLDB1 displayed a cytoplasmic disposition in unstimulated cultured adipocytes but translocated to the plasma membrane in response to insulin. Depletion of PHLDB1 by siRNA inhibited insulin stimulation of Akt phosphorylation but not tyrosine phosphorylation of IRS-1. RNAi-based silencing of PHLDB1 in cultured adipocytes also attenuated insulin-stimulated deoxyglucose transport and Myc-GLUT4-EGFP translocation to the plasma membrane, whereas knockdown of the PHLDB1 isoform PHLDB2 failed to attenuate insulin-stimulated deoxyglucose transport. Furthermore, adenovirus-mediated expression of PHLDB1 in adipocytes enhanced insulin-stimulated Akt and p70 S6 kinase phosphorylation, as well as GLUT4 translocation. These results indicate that PHLDB1 is a novel modulator of Akt protein kinase activation by insulin.

Akt substrate TBC1D1 regulates GLUT1 expression through the mTOR pathway in 3T3-L1 adipocytes.

Multiple studies have suggested that the protein kinase Akt/PKB (protein kinase B) is required for insulin-stimulated glucose transport in skeletal muscle and adipose cells. In an attempt to understand links between Akt activation and glucose transport regulation, we applied mass spectrometry-based proteomics and bioinformatics approaches to identify potential Akt substrates containing the phospho-Akt substrate motif RXRXXpS/T. The present study describes the identification of the Rab GAP (GTPase-activating protein)-domain containing protein TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1], which is closely related to TBC1D4 [TBC domain family, member 4, also denoted AS160 (Akt substrate of 160 kDa)], as an Akt substrate that is phosphorylated at Thr(590). RNAi (RNA interference)-mediated silencing of TBC1D1 elevated basal deoxyglucose uptake by approx. 61% in 3T3-L1 mouse embryo adipocytes, while the suppression of TBC1D4 and RapGAP220 under the same conditions had little effect on basal and insulin-stimulated deoxyglucose uptake. Silencing of TBC1D1 strongly increased expression of the GLUT1 glucose transporter but not GLUT4 in cultured adipocytes, whereas the decrease in TBC1D4 had no effect. Remarkably, loss of TBC1D1 in 3T3-L1 adipocytes activated the mTOR (mammalian target of rapamycin)-p70 S6 protein kinase pathway, and the increase in GLUT1 expression in the cells treated with TBC1D1 siRNA (small interfering RNA) was blocked by the mTOR inhibitor rapamycin. Furthermore, overexpression of the mutant TBC1D1-T590A, lacking the putative Akt/PKB phosphorylation site, inhibited insulin stimulation of p70 S6 kinase phosphorylation at Thr(389), a phosphorylation induced by mTOR. Taken together, our data suggest that TBC1D1 may be involved in controlling GLUT1 glucose transporter expression through the mTOR-p70 S6 kinase pathway.

Lipidomic analysis reveals significant lipogenesis and accumulation of lipotoxic components in ob/ob mouse organs.

To further understand the role of lipogenesis and lipotoxicity in the development of obesity and diabetes, lipidomes of various organs from ob/ob mice and their wild type controls were analyzed by shotgun lipidomics at 10, 12, and 16 weeks of age. We observed that the amounts of fatty acyl (FA) chains corresponding to those from de novo synthesis (e.g., 16:0, 16:1, and 18:1 FA) were substantially elevated in ob/ob mice, consistent with increased expression of genes and proteins involved in biosynthesis. Polyunsaturated fatty acid species were moderately increased in the examined tissues of ob/ob mice, since they can only be absorbed from diets or elongated from the ingested n-3 or n-6 FA. Different profiles of FA chains between ob/ob mouse liver and skeletal muscle reflect diverging lipogenesis pathways in these organs. Amounts of vaccenic acids (i.e., 18:1(n-7) FA) in 12- and 16-week ob/ob mouse liver were significantly increased compared to their controls, indicating enhanced de novo synthesis of this acid through 16:1(n-7) FA in the liver starting at 12 weeks of age. Coincidentally, synthesis of triacylglycerol from monoacylglycerol in the liver was also increased in ob/ob mice starting at 12 weeks of age, as revealed by simulation of triacylglycerol synthesis. Moreover, levels of lipotoxic lipid classes were significantly higher in ob/ob mice than their age-matched controls, supporting the notion that elevated lipotoxic components are tightly associated with insulin resistance in ob/ob mice. Taken together, the current study revealed that lipogenesis and lipotoxicity in ob/ob mice likely contribute to insulin resistance and provides great insights into the underlying mechanisms of diabetes and obesity.

Membrane Trafficking Protein CDP138 Regulates Fat Browning and Insulin Sensitivity through Controlling Catecholamine Release.

CDP138 is a calcium- and lipid-binding protein that is involved in membrane trafficking. Here, we report that mice without CDP138 develop obesity under normal chow diet (NCD) or high-fat diet (HFD) conditions. CDP138-/- mice have lower energy expenditure, oxygen consumption, and body temperature than wild-type (WT) mice. CDP138 is exclusively expressed in adrenal medulla and is colocalized with tyrosine hydroxylase (TH), a marker of sympathetic nervous terminals, in the inguinal fat. Compared with WT controls, CDP138-/- mice had altered catecholamine levels in circulation, adrenal gland, and inguinal fat. Adrenergic signaling on cyclic AMP (cAMP) formation and hormone-sensitive lipase (HSL) phosphorylation induced by cold challenge but not by an exogenous ß3 adrenoceptor against CL316243 were decreased in adipose tissues of CDP138-/- mice. Cold-induced beige fat browning, fatty acid oxidation, thermogenesis, and related gene expression were reduced in CDP138-/- mice. CDP138-/- mice are also prone to HFD-induced insulin resistance, as assessed by Akt phosphorylation and glucose transport in skeletal muscles. Our data indicate that CDP138 is a regulator of stress response and plays a significant role in adipose tissue browning, energy balance, and insulin sensitivity through regulating catecholamine secretion from the sympathetic nervous terminals and adrenal gland.

Imbalance between neutrophil elastase and its inhibitor α1-antitrypsin in obesity alters insulin sensitivity, inflammation, and energy expenditure.

The molecular mechanisms involved in the development of obesity and related complications remain unclear. Here, we report that obese mice and human subjects have increased activity of neutrophil elastase (NE) and decreased serum levels of the NE inhibitor α1-antitrypsin (A1AT, SerpinA1). NE null (Ela2(-/-)) mice and A1AT transgenic mice were resistant to high-fat diet (HFD)-induced body weight gain, insulin resistance, inflammation, and fatty liver. NE inhibitor GW311616A reversed insulin resistance and body weight gain in HFD-fed mice. Ela2(-/-) mice also augmented circulating high molecular weight (HMW) adiponectin levels, phosphorylation of AMP-activated protein kinase (AMPK), and fatty acid oxidation (FAO) in the liver and brown adipose tissue (BAT) and uncoupling protein (UCP1) levels in the BAT. These data suggest that the A1AT-NE system regulates AMPK signaling, FAO, and energy expenditure. The imbalance between A1AT and NE contributes to the development of obesity and related inflammation, insulin resistance, and liver steatosis.

C2 domain-containing phosphoprotein CDP138 regulates GLUT4 insertion into the plasma membrane.

The protein kinase B(ß) (Akt2) pathway is known to mediate insulin-stimulated glucose transport through increasing glucose transporter GLUT4 translocation from intracellular stores to the plasma membrane (PM). Combining quantitative phosphoproteomics with RNAi-based functional analyses, we show that a previously uncharacterized 138 kDa C2 domain-containing phosphoprotein (CDP138) is a substrate for Akt2, and is required for optimal insulin-stimulated glucose transport, GLUT4 translocation, and fusion of GLUT4 vesicles with the PM in live adipocytes. The purified C2 domain is capable of binding Ca(2+) and lipid membranes. CDP138 mutants lacking the Ca(2+)-binding sites in the C2 domain or Akt2 phosphorylation site S197 inhibit insulin-stimulated GLUT4 insertion into the PM, a rate-limiting step of GLUT4 translocation. Interestingly, CDP138 is dynamically associated with the PM and GLUT4-containing vesicles in response to insulin stimulation. Together, these results suggest that CDP138 is a key molecule linking the Akt2 pathway to the regulation of GLUT4 vesicle-PM fusion.

Identification of WNK1 as a substrate of Akt/protein kinase B and a negative regulator of insulin-stimulated mitogenesis in 3T3-L1 cells.

Insulin signaling through protein kinase Akt/protein kinase B (PKB), a downstream element of the phosphatidylinositol 3-kinase (PI3K) pathway, regulates diverse cellular functions including metabolic pathways, apoptosis, mitogenesis, and membrane trafficking. To identify Akt/PKB substrates that mediate these effects, we used antibodies that recognize phosphopeptide sites containing the Akt/PKB substrate motif (RXRXX(p)S/T) to immunoprecipitate proteins from insulin-stimulated adipocytes. Tryptic peptides from a 250-kDa immunoprecipitated protein were identified as the protein kinase WNK1 (with no lysine) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, consistent with a recent report that WNK1 is phosphorylated on Thr60 in response to insulin-like growth factor I. Insulin treatment of 3T3-L1 adipocytes stimulated WNK1 phosphorylation, as detected by immunoprecipitation with antibody against WNK1 followed by immunoblotting with the anti-phosphoAkt substrate antibody. WNK1 phosphorylation induced by insulin was unaffected by rapamycin, an inhibitor of p70 S6 kinase pathway but abolished by the PI3K inhibitor wortmannin. RNA interference-directed depletion of Akt1/PKB alpha and Akt2/PKB beta attenuated insulin-stimulated WNK1 phosphorylation, but depletion of protein kinase C lambda did not. Whereas small interfering RNA-induced loss of WNK1 protein did not significantly affect insulin-stimulated glucose transport in 3T3-L1 adipocytes, it significantly enhanced insulin-stimulated thymidine incorporation by about 2-fold. Furthermore, depletion of WNK1 promoted serum-stimulated cell proliferation of 3T3-L1 preadipocytes, as evidenced by a 36% increase in cell number after 48 h in culture. These data suggest that WNK1 is a physiologically relevant target of insulin signaling through PI3K and Akt/PKB and functions as a negative regulator of insulin-stimulated mitogenesis.

A phosphatidylinositol 3-kinase-independent insulin signaling pathway to N-WASP/Arp2/3/F-actin required for GLUT4 glucose transporter recycling.

Recruitment of intracellular glucose transporter 4 (GLUT4) to the plasma membrane of fat and muscle cells in response to insulin requires phosphatidylinositol (PI) 3-kinase as well as a proposed PI 3-kinase-independent pathway leading to activation of the small GTPase TC10. Here we show that in cultured adipocytes insulin causes acute cortical localization of the actin-regulatory neural Wiskott-Aldrich syndrome protein (N-WASP) and actin-related protein-3 (Arp3) as well as cortical F-actin polymerization by a mechanism that is insensitive to the PI 3-kinase inhibitor wortmannin. Expression of the dominant inhibitory N-WASP-DeltaWA protein lacking the Arp and actin binding regions attenuates the cortical F-actin rearrangements by insulin in these cells. Remarkably, the N-WASP-DeltaWA protein also inhibits insulin action on GLUT4 translocation, indicating dependence of GLUT4 recycling on N-WASP-directed cortical F-actin assembly. TC10 exhibits sequence similarity to Cdc42 and has been reported to bind N-WASP. We show the inhibitory TC10 (T31N) mutant, which abrogates insulin-stimulated GLUT4 translocation and glucose transport, also inhibits both cortical localization of N-WASP and F-actin formation in response to insulin. These findings reveal that N-WASP likely functions downstream of TC10 in a PI 3-kinase-independent insulin signaling pathway to mobilize cortical F-actin, which in turn promotes GLUT4 responsiveness to insulin.

Insulin signaling through Akt/protein kinase B analyzed by small interfering RNA-mediated gene silencing.

Glucose homeostasis is controlled by insulin in part through the translocation of intracellular glucose transporter 4 to the plasma membrane in muscle and fat cells. Akt/protein kinase B downstream of phosphatidylinositol 3-kinase has been implicated in this insulin-signaling pathway, but results with a variety of reagents including Akt1-/- and Akt2-/- mice have been equivocal. Here we report the application of small interfering RNA-directed gene silencing to deplete both Akt1 and Akt2 in cultured 3T3-L1 adipocytes. Loss of Akt1 alone slightly impaired insulin-mediated hexose transport activity but had no detectable effect on glycogen synthase kinase (GSK)-3 phosphorylation. In contrast, depletion of Akt2 alone by 70% inhibited approximately half of the insulin responsiveness. Combined depletions of Akt1 plus Akt2 in these cells even more markedly attenuated insulin action on glucose transporter 4 movements, hexose transport activity, and GSK-3 phosphorylation. These data demonstrate a primary role of Akt2 in insulin signaling, significant functional redundancy of Akt1 and Akt2 isoforms in this pathway, and an absolute requirement of Akt protein kinases for regulation of glucose transport and GSK-3 in cultured adipocytes.

Glucose transporter recycling in response to insulin is facilitated by myosin Myo1c.

Insulin stimulates glucose uptake in muscle and adipocytes by signalling the translocation of GLUT4 glucose transporters from intracellular membranes to the cell surface. The translocation of GLUT4 may involve signalling pathways that are both independent of and dependent on phosphatidylinositol-3-OH kinase (PI(3)K). This translocation also requires the actin cytoskeleton, and the rapid movement of GLUT4 along linear tracks may be mediated by molecular motors. Here we report that the unconventional myosin Myo1c is present in GLUT4-containing vesicles purified from 3T3-L1 adipocytes. Myo1c, which contains a motor domain, three IQ motifs and a carboxy-terminal cargo domain, is highly expressed in primary and cultured adipocytes. Insulin enhances the localization of Myo1c with GLUT4 in cortical tubulovesicular structures associated with actin filaments, and this colocalization is insensitive to wortmannin. Insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane is augmented by the expression of wild-type Myo1c and inhibited by a dominant-negative cargo domain of Myo1c. A decrease in the expression of endogenous Myo1c mediated by small interfering RNAs inhibits insulin-stimulated uptake of 2-deoxyglucose. Thus, myosin Myo1c functions in a PI(3)K-independent insulin signalling pathway that controls the movement of intracellular GLUT4-containing vesicles to the plasma membrane.

Characterization of multiple signaling pathways of insulin in the regulation of vascular endothelial growth factor expression in vascular cells and angiogenesis.

The effects of insulin on vascular endothelial growth factor (VEGF) expression in cultured vascular cells and in angiogenesis were characterized. Insulin increased VEGF mRNA levels in mouse aortic smooth muscle cells from 10(-9) to 10(-7) m with an initial peak of 3.7-fold increases at 1 h and a second peak of 2.8-fold after 12 h. The first peak of VEGF expression was inhibited by LY294002, an inhibitor of phosphatidylinositol (PI) 3-kinase, and by the overexpression of dominant negative forms of p85 subunit of PI 3-kinase or Akt. Inhibitors of MEK kinase, PD98059, or overexpression of dominant negative forms of Ras was ineffective. In contrast, the chronic effect of insulin on VEGF expression was partially inhibited by both LY294002 or PD98059 as well as by the overexpression of dominant negatives of PI 3-kinase or Ras. The importance of PI 3-kinase-Akt pathway on VEGF expression was confirmed in mouse aortic smooth muscle cells isolated from insulin receptor substrate -1 knockout (IRS-1-/-) mice that showed parallel reductions of 46-49% in insulin-stimulated VEGF expression and PI 3-kinase-Akt activation. Insulin-induced activation of PI 3-kinase-Akt on hypoxia-induced VEGF expression and neovascularization was reduced by 40% in the retina of neonatal hypoxia model using IRS-1-/- mice. Thus, unlike other cells, insulin can regulate VEGF expression by both IRS-1/PI 3-kinase-Akt cascade and Ras-MAPK pathways in aortic smooth muscle cells. The in vivo results provide direct evidence that insulin can modulate hypoxia-induced angiogenesis via reduction in VEGF expression in vivo.