Role of TAPP1 and TAPP2 adaptor binding to PtdIns(3,4)P2 in regulating insulin sensitivity defined by knock-in analysis

Insulin sensitivity is critically dependent on the activity of PI3K (phosphoinositide 3-kinase) and generation of the PtdIns(3,4,5)P3 second messenger. PtdIns(3,4,5)P3 can be broken down to PtdIns(3,4)P2 through the action of the SHIPs (Src-homology-2-domain-containing inositol phosphatases). As PtdIns(3,4)P2 levels peak after those of PtdIns(3,4,5)P3, it has been proposed that PtdIns(3,4)P2 controls a negative-feedback loop that down-regulates the insulin and PI3K network. Previously, we identified two related adaptor proteins termed TAPP [tandem PH (pleckstrin homology)-domain-containing protein] 1 and TAPP2 that specifically bind to PtdIns(3,4)P2 through their C-terminal PH domain. To determine whether TAPP1 and TAPP2 play a role in regulating insulin sensitivity, we generated knock-in mice that express normal endogenous levels of mutant TAPP1 and TAPP2 that are incapable of binding PtdIns(3,4)P2. These homozygous TAPP1R211L/R211LTAPP2R218L/R218L double knock-in mice are viable and exhibit significantly enhanced activation of Akt, a key downstream mediator of insulin signalling. Consistent with increased PI3K and Akt activity, the double knock-in mice display enhanced whole body insulin sensitivity and disposal of glucose uptake into muscle tissues. We also generated wild-type and double TAPP1R211L/R211LTAPP2R218L/R218L knock-in embryonic fibroblasts and found that insulin triggered enhanced production of PtdIns(3,4,5)P3 and Akt activity in the double knock-in fibroblasts. These observations provide the first genetic evidence to support the notion that binding of TAPP1 and TAPP2 adap-tors to PtdIns(3,4)P2 function as negative regulators of the insulin and PI3K signalling pathways.

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The dietary flavonoids naringenin and quercetin acutely impair glucose metabolism in rodents possibly via inhibition of hypothalamic insulin signalling

British Journal Of Nutrition ◽

10.1017/s0007114512003005 ◽

2012 ◽

Vol 109 (6) ◽

pp. 1040-1051 ◽

Cited By ~ 15

Author(s):

Christiane E. Koch ◽

Goutham K. Ganjam ◽

Juliane Steger ◽

Karen Legler ◽

Sigrid Stöhr ◽

...

Keyword(s):

Glucose Tolerance ◽

Insulin Signalling ◽

Inhibitory Effect ◽

Whole Body ◽

Downstream Target ◽

Glucose Homoeostasis ◽

Pi3k Signalling ◽

Phosphoinositide 3 Kinase

Secondary metabolites of herbs and spices are widely used as an alternative strategy in the therapy of various diseases. The polyphenols naringenin, quercetin and curcumin have been characterised as anti-diabetic agents. Conversely, in vitro, naringenin and quercetin are described to inhibit phosphoinositide-3-kinase (PI3K), an enzyme that is essential for the neuronal control of whole body glucose homoeostasis. Using both in vitro and in vivo experiments, we tested whether the inhibitory effect on PI3K occurs in neurons and if it might affect whole body glucose homoeostasis. Quercetin was found to inhibit basal and insulin-induced phosphorylation of Akt (Ser473), a downstream target of PI3K, in HT-22 cells, whereas naringenin and curcumin had no effect. In Djungarian hamsters (Phodopus sungorus) naringenin and quercetin (10 mg/kg administered orally) diminished insulin-induced phosphorylation of Akt (Ser473) in the arcuate nucleus, indicating a reduction in hypothalamic PI3K activity. In agreement with this finding, glucose tolerance in naringenin-treated hamsters (oral) and mice (oral and intracerebroventricular) was reduced compared with controls. Dietary quercetin also impaired glucose tolerance, whereas curcumin was ineffective. Circulating levels of insulin and insulin-like growth factor-binding protein were not affected by the polyphenols. Oral quercetin reduced the respiratory quotient, suggesting that glucose utilisation was impaired after treatment. These data demonstrate that low doses of naringenin and quercetin acutely and potently impair glucose homoeostasis. This effect may be mediated by inhibition of hypothalamic PI3K signalling. Whether chronic impairments in glucose homoeostasis occur after long-term application remains to be identified.

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Small intestinal metabolism is central to whole-body insulin resistance

Gut ◽

10.1136/gutjnl-2020-322073 ◽

2020 ◽

pp. gutjnl-2020-322073

Author(s):

Giulia Angelini ◽

Serenella Salinari ◽

Lidia Castagneto-Gissey ◽

Alessandro Bertuzzi ◽

James Casella-Mariolo ◽

...

Keyword(s):

Insulin Resistance ◽

Insulin Sensitivity ◽

Glucose Uptake ◽

Insulin Signalling ◽

Whole Body ◽

Glucose Disposal ◽

Jejunal Loop ◽

Oral Glucose ◽

Metabolic Signature ◽

Jejunal Bypass

ObjectiveTo assess the role of jejunum in insulin resistance in humans and in experimental animals.DesignTwenty-four subjects undergoing biliopancreatic diversion (BPD) or Roux-en-Y gastric bypass (RYGB) were enrolled. Insulin sensitivity was measured at baseline and at 1 week after surgery using oral glucose minimal model.We excluded the jejunum from intestinal continuity in pigs and created a jejunal loop with its vascular and nerve supply intact accessible from two cutaneous stomas, and reconnected the bowel with an end-to-end anastomosis. Glucose stable isotopes were given in the stomach or in the jejunal loop.In vitro studies using primary porcine and human hepatocytes or myoblasts tested the effects of plasma on gluconeogenesis or glucose uptake and insulin signalling.ResultsWhole-body insulin sensitivity (SI∙104: 0.54±0.12 before vs 0.82±0.11 after BPD, p=0.024 and 0.41±0.09 before vs 0.65±0.09/pM/min after RYGB, p=not significant) and Glucose Disposition Index increased only after BPD. In pigs, insulin sensitivity was significantly lower when glucose was administered in the jejunal loop than in the stomach (glucose rate of disappearance (Rd) area under the curve (AUC)/insulin AUC∙10: 1.82±0.31 vs 2.96±0.33 mmol/pM/min, p=0.0017).Metabolomics showed a similar pattern before surgery and during jejunal-loop stimulation, pointing to a higher expression of gluconeogenetic substrates, a metabolic signature of impaired insulin sensitivity.A greater hepatocyte phosphoenolpyruvate-carboxykinase and glucose-6-phosphatase gene expression was elicited with plasma from porcine jejunal loop or before surgery compared with plasma from jejunectomy in pigs or jejunal bypass in humans.Stimulation of myoblasts with plasma from porcine jejunal loop or before surgery reduced glucose uptake, Ser473-Akt phosphorylation and GLUT4 expression compared with plasma obtained during gastric glucose administration after jejunectomy in pigs or after jejunal bypass in humans.ConclusionProximal gut plays a crucial role in controlling insulin sensitivity through a distinctive metabolic signature involving hepatic gluconeogenesis and muscle insulin resistance. Bypassing the jejunum is beneficial in terms of insulin-mediated glucose disposal in obesity.Trial registration numberNCT03111953.

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Faculty Opinions recommendation of Long-chain omega-3 fatty acids regulate bovine whole-body protein metabolism by promoting muscle insulin signalling to the Akt-mTOR-S6K1 pathway and insulin sensitivity.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1071837.524763 ◽

2007 ◽

Author(s):

Geraldine Clough

Keyword(s):

Fatty Acids ◽

Insulin Sensitivity ◽

Protein Metabolism ◽

Insulin Signalling ◽

Whole Body ◽

Omega 3 Fatty Acids ◽

Omega 3 ◽

Body Protein ◽

Long Chain

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BCR activation of PI3K is Vav-independent in murine B cells

Biochemical Society Transactions ◽

10.1042/bst0320781 ◽

2004 ◽

Vol 32 (5) ◽

pp. 781-784 ◽

Cited By ~ 2

Author(s):

E. Vigorito ◽

E. Clayton ◽

M. Turner

Keyword(s):

B Cells ◽

Tyrosine Kinases ◽

Adaptor Proteins ◽

Pleckstrin Homology Domain ◽

Pi3k Activation ◽

Lipid Product ◽

Lipid Kinases ◽

Phosphoinositide 3 Kinase

BCR (B-cell antigen receptor)-induced Ca2+ signalling is initiated by activation of tyrosine kinases, which in concert with adaptor proteins and lipid kinases regulate PLC (phospholipase C) γ2 activation. Vav and PI3K (phosphoinositide 3-kinase) are required for optimal Ca2+ responses, although it has not been established, in primary B-cells, if both proteins are components of the same pathway. In vitro evidence suggests that binding of the PI3K lipid product PIP3 to Vav pleckstrin homology domain contributes to Vav activation. However, pharmacological inhibition of PI3K by wortmannin or deletion of the p110δ catalytic subunit has no effect on Vav activation in response to BCR engagement, suggesting that this mechanism does not operate in vivo. We also show that PI3K recruitment to phosphorylated-tyrosine-containing complexes is Vav-independent. Taken together with our previous observation that protein kinase B phosphorylation is normal in Vav-deficient B-cells, we suggest that PI3K activation is Vav-independent in response to strong signals delivered by multivalent cross-linking.

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Phosphoinositide 3-kinase-dependent regulation of phospholipase Cγ

Biochemical Society Transactions ◽

10.1042/bst0350229 ◽

2007 ◽

Vol 35 (2) ◽

pp. 229-230 ◽

Cited By ~ 22

Author(s):

T. Maffucci ◽

M. Falasca

Keyword(s):

Phospholipase C ◽

Tyrosine Kinases ◽

Receptor Tyrosine Kinases ◽

Second Messengers ◽

Pleckstrin Homology Domain ◽

Ph Domain ◽

Pleckstrin Homology ◽

Functional Roles ◽

Phosphoinositide 3 Kinase

Activation of the enzyme PLC (phospholipase C) leads to the formation of second messengers Ins(1,4,5)P3 and diacylglycerol. RTKs (receptor tyrosine kinases) activate this reaction through PLCγ isoenzymes. It has been shown that PI3K (phosphoinositide 3-kinase) may regulate PLCγ activity through the interaction of PI3K product PtdIns(3,4,5)P3 and the PLCγ PH domain (pleckstrin homology domain). Here, we analyse the potential functional roles of the PI3K/PLC pathway.

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When worlds collide: inositol pyrophosphates and phosphoinositides intersect at the plasma membrane

Biochemical Journal ◽

10.1042/bj20130785 ◽

2013 ◽

Vol 453 (3) ◽

pp. e3-e4 ◽

Cited By ~ 1

Author(s):

Christophe Erneux ◽

William's Elong Edimo

Keyword(s):

Plasma Membrane ◽

Growth Factor ◽

Cultured Cells ◽

Insulin Signalling ◽

Growth Factor Receptor ◽

Receptor Activation ◽

Signal Molecules ◽

Pleckstrin Homology Domain ◽

Ph Domain ◽

Inositol Pyrophosphates

Highly phosphorylated inositol pyrophosphates are present in the cells of many organisms such as yeast, Dictyostelium and mammals. They can act as signal molecules in growth factor and insulin signalling both in cultured cells and in intact mice. Their action involves protein pyrophosphorylation or binding to multiple protein interactors such as PH (pleckstrin homology)-domain-containing proteins. One key enzyme in their synthesis, PPIP5K (diphosphoinositol pentakisphosphate kinase) 1/2, can phosphorylate InsP6 and 5-InsP7 to 1-InsP7 and InsP8 respectively. Stephen Shears's laboratory reported in this issue of the Biochemical Journal that PPIP5K1's unexpectedly high affinity for PtdIns(3,4,5)P3, which is synthesized at the plasma membrane, provides a recruitment mechanism for this enzyme in response to growth factor receptor activation. In competition experiments, they observed that PtdIns(3,4,5)P3 binding to PPIP5K1 could be displaced by inositol pyrophosphates and that PPIP5K1 substrates were more potent inhibitors than PPIP5K1 products. Those findings reveal a mechanism for localized depletion of InsP6 and 5-InsP7 at the plasma membrane and further translocation of PtdIns(3,4,5)P3-binding PH-domain-containing proteins.

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SWAP-70 Identifies a Transitional Subset of Actin Filaments in Motile Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e03-01-0043 ◽

2003 ◽

Vol 14 (8) ◽

pp. 3242-3253 ◽

Cited By ~ 34

Author(s):

Pirta Hilpelä ◽

Pia Oberbanscheidt ◽

Penelope Hahne ◽

Martin Hund ◽

Georg Kalhammer ◽

...

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Pleckstrin Homology Domain ◽

Cellular Organization ◽

Ph Domain ◽

Subcellular Targeting ◽

Actin Organization ◽

Cell Protrusion ◽

Motile Cells ◽

Phosphoinositide 3 Kinase

Functionally different subsets of actin filament arrays contribute to cellular organization and motility. We report the identification of a novel subset of loose actin filament arrays through regulated association with the widely expressed protein SWAP-70. These loose actin filament arrays were commonly located behind protruding lamellipodia and membrane ruffles. Visualization of these loose actin filament arrays was dependent on lamellipodial protrusion and the binding of the SWAP-70 PH-domain to a 3′-phosphoinositide. SWAP-70 with a functional pleckstrin homology-domain lacking the C-terminal 60 residues was targeted to the area of the loose actin filament arrays, but it did not associate with actin filaments. The C-terminal 60 residues were sufficient for actin filament association, but they provided no specificity for the subset of loose actin filament arrays. These results identify SWAP-70 as a phosphoinositide 3-kinase signaling-dependent marker for a distinct, hitherto unrecognized, array of actin filaments. Overexpression of SWAP-70 altered the actin organization and lamellipodial morphology. These alterations were dependent on a proper subcellular targeting of SWAP-70. We propose that SWAP-70 regulates the actincytoskeletonasaneffectororadaptorproteininresponsetoagoniststimulatedphosphatidylinositol (3,4)-bisphosphate production and cell protrusion.

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Ceramide dissociates 3′-phosphoinositide production from pleckstrin homology domain translocation

Biochemical Journal ◽

10.1042/bj3540359 ◽

2001 ◽

Vol 354 (2) ◽

pp. 359-368 ◽

Cited By ~ 69

Author(s):

Suzanne STRATFORD ◽

Daryll B. DEWALD ◽

Scott A. SUMMERS

Keyword(s):

Growth Factor ◽

Binding Proteins ◽

Cellular Proliferation ◽

Pleckstrin Homology Domain ◽

Ph Domain ◽

Pleckstrin Homology ◽

Lipid Kinase ◽

Ph Domains ◽

Phosphoinositide 3 Kinase ◽

Synthase Inhibitor

Numerous hormones, cytokines and transforming oncogenes activate phosphoinositide 3-kinase (PI-3K), a lipid kinase that initiates signal transduction cascades regulating cellular proliferation, survival, protein synthesis and glucose metabolism. PI-3K catalyses the production of the 3′-phosphoinositides PtdIns(3,4)P2 and PtdIns(3,4,5)P3, which recruit downstream effector enzymes to the membrane via their pleckstrin homology (PH) domains. Recent studies have indicated that another signalling lipid, the sphingolipid ceramide, inhibits several PI-3K-dependent events, including insulin-stimulated glucose uptake and growth-factor-stimulated cell survival. Here we show that ceramide analogues specifically prevent the recruitment of the PtdIns(3,4,5)P3-binding proteins Akt/protein kinase B (PKB) or the general receptor for phosphoinositides-1 (GRP1). Specifically, the short-chain ceramide derivative C2-ceramide inhibited the platelet-derived growth factor (PDGF)-stimulated translocation of full-length Akt/PKB, as well as truncated proteins encoding only the PH domains of Akt/PKB or GRP1. C2-ceramide did not alter the membrane localization of the PH domain for phospholipase Cδ, which preferentially binds PtdIns(4,5)P2, nor did it affect the PDGF-stimulated production of PtdIns(3,4)P2 or PtdIns(3,4,5)P3. Interestingly, a glucosylceramide synthase inhibitor, 1-phenyl-2-decanoylamino-3-morpholinopropan-1-ol (PDMP), shown previously to increase intracellular ceramide concentrations without affecting PI-3K [Rani, Abe, Chang, Rosenzweig, Saltiel, Radin and Shayman (1995) J. Biol. Chem. 270, 2859–2867], recapitulated the inhibitory effects of C2-ceramide on PDGF-stimulated Akt/PKB phosphorylation. These studies indicate that ceramide prevents the translocation of certain PtdIns(3,4,5)P3-binding proteins, despite the presence of a full complement of PtdIns(3,4)P2 or PtdIns(3,4,5)P3. Furthermore, these findings suggest a mechanism by which stimuli that induce ceramide synthesis could negate the fundamental signalling pathways initiated by PI-3K.

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