Opposite cross-talk by oleate and palmitate on insulin signaling in hepatocytes through macrophage activation

Chronic low grade inflammation in adipose tissue during obesity is associated with an impairment of the insulin signaling cascade. In this study, we have evaluated the impact of palmitate or oleate overload of macrophage/Kupffer cells in triggering stress-mediated signaling pathways, in lipoapoptosis, and in the cross-talk with insulin signaling in hepatocytes. RAW 264.7 macrophages or Kupffer cells were stimulated with oleate or palmitate, and levels of M1/M2 polarization markers and the lipidomic profile of eicosanoids were analyzed. Whereas proinflammatory cytokines and total eicosanoids were elevated in macrophages/Kupffer cells stimulated with palmitate, enhanced arginase 1 and lower leukotriene B4 (LTB4) levels were detected in macrophages stimulated with oleate. When hepatocytes were pretreated with conditioned medium (CM) from RAW 264.7 or Kupffer cells loaded with palmitate (CM-P), phosphorylation of stress kinases and endoplasmic reticulum stress signaling was increased, insulin signaling was impaired, and lipoapoptosis was detected. Conversely, enhanced insulin receptor-mediated signaling and reduced levels of the phosphatases protein tyrosine phosphatase 1B (PTP1B) and phosphatase and tensin homolog (PTEN) were found in hepatocytes treated with CM from macrophages stimulated with oleate (CM-O). Supplementation of CM-O with LTB4 suppressed insulin sensitization and increased PTP1B and PTEN. Furthermore, LTB4 decreased insulin receptor tyrosine phosphorylation in hepatocytes, activated the NFκB pathway, and up-regulated PTP1B and PTEN, these effects being mediated by LTB4 receptor BTL1. In conclusion, oleate and palmitate elicit an opposite cross-talk between macrophages/Kupffer cells and hepatocytes. Whereas CM-P interferes at the early steps of insulin signaling, CM-O increases insulin sensitization, possibly by reducing LTB4.This work was supported by Ministerio de Economía y Competitividad, Spain, Grants SAF2012-33283 and SAF2013-48201-R; Comunidad de Madrid Grant S2010/BMD-2423 (Spain); an European Foundation for the Studies of Diabetes and Amylin Paul Langerhans grant; and the Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM, Instituto de Salud Carlos III, Spain).Peer reviewe

Opposite cross-talk by oleate and palmitate on insulin signalling in hepatocytes by direct effects or through macrophage activation

Resumen del póster presentado al XII International Symposium on Insulin Receptors and Insulin Action: "New Opportunities for the Prevention and Treatment of Diabetes in the XXI Century", celebrado en Barcelona (España) del 7 al 9 de noviembre de 2013.[Background and Objectives]: Obesity is associated with a low-grade inflammation in adipose tissue that contributes to defects in the critical nodes of insulin signaling in peripheral tissues. We investigated the impact of saturated (palmitate, P) and unsaturated (oleate, O) fatty acid (FA) overload in stress signaling and insulin signaling in hepatocytes focusing on the effects elicited by direct FA stimulation or through macrophage/kupffer activation. In addition, the effect of several lipid species derived from FA on hepatic insulin signaling was evaluated. [Methods]: Mouse hepatocytes were treated with O, P or the combination of both. Raw 264.7 rnacrophages or kupffer cells were stimulated with O or P and after 24 h mRNA levels of pro-inflammatory cytokines were analyzed and the condition medium (CM) was added to hepatocytes. Activation of STAT3, stress kinases, ER stress markers and insulin signaling was assessed. Lipidomic analysis was performed in CM from O or P-loaded Raw 264.7 cells. Lipid species were used individually to treat macrophages or hepatocytes and evaluate insulin signaling. [Results and Conclusions]: P, but not 0, activated stress kinases in both hepatocytes and macrophages. Whereas insulin signaling was reduced in hepatocytes directly treated with P or CM of P-stimulated macrophages/kupffer cells, insulin hypersensitivity was observed in hepatocytes treated with O or CM of O-stimulated macrophages/kupffer cells. Moreover, O prevented the direct negative effects of P on insulin signaling in hepatocytes. Interestingly, CM of O-treated rnacrophages showed reduced LTB4 and 14,15 DHET levels compared to the CM of P treatment. These lipid species reverted the effects of O on hepatic insulin signaling. In conclusion, O and P elicit opposite cross-talks on hepatic insulin signaling either directly or through macrophage activation. Whereas P interferes at early steps (IFUIRSl) through the activation of stress kinases, O elicits insulin sensitizing effects and prevents the deleterious effects of P possibly by the reduction in LTB4 and 14,15 DHET lipid mediators. We are evaluating the rnechanims responsible of the insulin sensitizing effects of O in hepatocytes.Peer Reviewe

Role of hepatocyte S6K1 in palmitic acid-induced endoplasmic reticulum stress, lipotoxicity, insulin resistance and in oleic acid-induced protection

The excess of saturated free fatty acids, such as palmitic acid, that induces lipotoxicity in hepatocytes, has been implicated in the development of non-alcoholic fatty liver disease also associated with insulin resistance. By contrast, oleic acid, a monounsaturated fatty acid, attenuates the effects of palmitic acid. We evaluated whether palmitic acid is directly associated with both insulin resistance and lipoapoptosis in mouse and human hepatocytes and the impact of oleic acid in the molecular mechanisms that mediate both processes. In human and mouse hepatocytes palmitic acid at a lipotoxic concentration triggered early activation of endoplasmic reticulum (ER) stress-related kinases, induced the apoptotic transcription factor CHOP, activated caspase 3 and increased the percentage of apoptotic cells. These effects concurred with decreased IR/IRS1/Akt insulin pathway. Oleic acid suppressed the toxic effects of palmitic acid on ER stress activation, lipoapoptosis and insulin resistance. Besides, oleic acid suppressed palmitic acid-induced activation of S6K1. This protection was mimicked by pharmacological or genetic inhibition of S6K1 in hepatocytes. In conclusion, this is the first study highlighting the activation of S6K1 by palmitic acid as a common and novel mechanism by which its inhibition by oleic acid prevents ER stress, lipoapoptosis and insulin resistance in hepatocytes.This work was supported by grants SAF2012-33283 (Spanish Ministry of Economy and Competitiveness), Comunidad de Madrid S2010/BMD-2423 (Spain), EFSD and Amylin Paul Langerhans Grant, Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM, Instituto de Salud Carlos III, Spain) to A.M.V. A. G-R receives support from CIBERDEM. S.C.K. is supported by grants II12/00002 (Instituto de Salud Carlos III, ISCIII) and BFU2012-38867 (Spanish Ministry of Economy and Competitiveness). J.M. is supported by grant P1/0021 (Instituto de Salud Carlos III, ISCIII).Peer Reviewe

Studies of naturally occurring friedelane triterpenoids as insulin sensitizers in the treatment type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) is a rapidly expanding public epidemic and frequently results in severe vascular complications. In an attempt to find anti-diabetic agents, we report herein on the isolation, structural elucidation and bioactivity of nine friedelane-type triterpenes (1-9) and twenty two known ones (10-31) from the root barks of Celastrus vulcanicola and Maytenus jelskii. Their structures were elucidated on the basis of spectroscopic analysis, including 1D and 2D NMR techniques. Two compounds from this series (1 and 3) exhibited increased insulin-mediated signalling, which suggests these friedelane triterpenes have potential therapeutic use in insulin resistant states. © 2012 Elsevier Ltd. All rights reserved.This work was supported by the Agencia Canaria de Investigación, Innovación y Sociedad de la Información (C200801000049), and PCI-Iberoamérica (A/023081/09, AECID) projects, and by grants SAF2009-08114 (AMV) MICINN (Spain) and CIBERDEM (ISCIII, Spain).Peer Reviewe

Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD

This work is licensed under a Creative Commons Attribution-NonCommercialNoDerivs 3.0 Unported License.-- et al.The pathogenic mechanisms underlying the progression of non-alcoholic fatty liver disease (NAFLD) are not fully understood. In this study, we aimed to assess the relationship between endoplasmic reticulum (ER) stress and autophagy in human and mouse hepatocytes during NAFLD. ER stress and autophagy markers were analyzed in livers from patients with biopsy-proven non-alcoholic steatosis (NAS) or non-alcoholic steatohepatitis (NASH) compared with livers from subjects with histologically normal liver, in livers from mice fed with chow diet (CHD) compared with mice fed with high fat diet (HFD) or methionine-choline-deficient (MCD) diet and in primary and Huh7 human hepatocytes loaded with palmitic acid (PA). In NASH patients, significant increases in hepatic messenger RNA levels of markers of ER stress (activating transcription factor 4 (ATF4), glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP)) and autophagy (BCN1) were found compared with NAS patients. Likewise, protein levels of GRP78, CHOP and p62/SQSTM1 (p62) autophagic substrate were significantly elevated in NASH compared with NAS patients. In livers from mice fed with HFD or MCD, ER stress-mediated signaling was parallel to the blockade of the autophagic flux assessed by increases in p62, microtubule-associated protein 2 light chain 3 (LC3-II)/LC3-I ratio and accumulation of autophagosomes compared with CHD fed mice. In Huh7 hepatic cells, treatment with PA for 8 h triggered activation of both unfolding protein response and the autophagic flux. Conversely, prolonged treatment with PA (24 h) induced ER stress and cell death together with a blockade of the autophagic flux. Under these conditions, cotreatment with rapamycin or CHOP silencing ameliorated these effects and decreased apoptosis. Our results demonstrated that the autophagic flux is impaired in the liver from both NAFLD patients and murine models of NAFLD, as well as in lipid-overloaded human hepatocytes, and it could be due to elevated ER stress leading to apoptosis. Consequently, therapies aimed to restore the autophagic flux might attenuate or prevent the progression of NAFLD.We acknowledge the following grant support: SAF2012-33283 (MINECO, Spain), Comunidad de Madrid S2010/BMD-2423, EFSD and Amylin Paul Langerhans Grant and Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM, ISCIII, Barcelona, Spain) to AMV.; SAF2010-16037, SAF2013-43713-R (MINECO) and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD, ISCIII) to PMS. RD12/0042/0019 (ISCIII) and S2010/BMD-2478 (Comunidad de Madrid) to LB, PI 13/01299 and Fundación Mutua Madrileña 2012 to C G-M and AIRC IG-2012 to GMF.Peer Reviewe

Studies of naturally occurring friedelane triterpenoids as insulin sensitizers in the treatment type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) is a rapidly expanding public epidemic and frequently results in severe vascular complications. In an attempt to find anti-diabetic agents, we report herein on the isolation, structural elucidation and bioactivity of nine friedelane-type triterpenes (1-9) and twenty two known ones (10-31) from the root barks of Celastrus vulcanicola and Maytenus jelskii. Their structures were elucidated on the basis of spectroscopic analysis, including 1D and 2D NMR techniques. Two compounds from this series (1 and 3) exhibited increased insulin-mediated signalling, which suggests these friedelane triterpenes have potential therapeutic use in insulin resistant states. © 2012 Elsevier Ltd. All rights reserved.This work was supported by the Agencia Canaria de Investigación, Innovación y Sociedad de la Información (C200801000049), and PCI-Iberoamérica (A/023081/09, AECID) projects, and by grants SAF2009-08114 (AMV) MICINN (Spain) and CIBERDEM (ISCIII, Spain).Peer Reviewe

Heme-regulated eIF2α kinase modulates hepatic FGF21 and is activated by PPARβ/δ deficiency

Fibroblast growth factor 21 (FGF21), a peptide hormone with pleiotropic effects on carbohydrate and lipid metabolism, is considered a target for the treatment of diabetes. We investigated the role of peroxisome proliferator-activated receptor (PPAR) b/d deficiency in hepatic FGF21 regulation. Increased Fgf21 expression was observed in the livers of PPARb/d-null mice and in mouse primary hepatocytes when this receptor was knocked down by small interfering RNA (siRNA). Increased Fgf21 was associated with enhanced protein levels in the heme-regulated eukaryotic translation initiation factor 2a (eIF2a) kinase (HRI). This increase caused enhanced levels of phosphorylated eIF2a and activating transcription factor (ATF) 4, which is essential for Fgf21-induced expression. siRNA analysis demonstrated that HRI regulates Fgf21 expression in primary hepatocytes. Enhanced Fgf21 expression attenuated tunicamycin-induced endoplasmic reticulum stress, as demonstrated by using a neutralizing antibody against FGF21. Of note, increased Fgf21 expression in mice fed a high-fat diet or hepatocytes exposed to palmitate was accompanied by reduced PPARb/d and activation of the HRI-eIF2α-ATF4 pathway. Moreover, pharmacological activation of HRI increased Fgf21 expression and reduced lipid-induced hepatic steatosis and glucose intolerance, but these effects were not observed in Fgf21-null mice. Overall, these findings suggest that HRI is a potential target for regulating hepatic FGF21 levels.This study was partly supported by funds from the Spanish Ministry of the Economy and Competitiveness (SAF2015-65267-R to Á.M.V., SAF2014-55725 to F.V., and SAF2012-30708 and SAF2015-64146-R to M.V.-C.) and the European Union European Regional Development Fund. CIBERDEM and CIBEROBN are Instituto de Salud Carlos III Health Institute projects. T.Q.-L. is supported by a CONACyT (National Council for Science and Technology in Mexico) PhD scholarship. W.W. is supported by start-up grants from the Lee Kong Chian School of Medicine, Nanyang Technological University, and by the Région Midi-Pyrénées, France.Peer Reviewe

Pivotal role of protein tyrosine phosphatase 1B (PTP1B) in the macrophage response to pro-inflammatory and anti-inflammatory challenge

Inhibition of protein tyrosine phosphatase 1B (PTP1B) has been suggested as an attractive target to improve insulin sensitivity in different cell types. In the present work, we have investigated the effect of PTP1B deficiency on the response of human and murine macrophages. Using in vitro and in vivo approaches in mice and silencing PTP1B in human macrophages with specific siRNAs, we have demonstrated that PTP1B deficiency increases the effects of pro-inflammatory stimuli in both human and rodent macrophages at the time that decreases the response to alternative stimulation. Moreover, the absence of PTP1B induces a loss of viability in resting macrophages and mainly after activation through the classic pathway. Analysis of early gene expression in macrophages treated with pro-inflammatory stimuli confirmed this exacerbated inflammatory response in PTP1Bdeficient macrophages. Microarray analysis in samples from wild-type and PTP1B-deficient macrophages obtained after 24 h of pro-inflammatory stimulation showed an activation of the p53 pathway, including the excision base repair pathway and the insulin signaling pathway in the absence of PTP1B. In animal models of lipopolysaccharide (LPS) and D-galactosamine challenge as a way to reveal in vivo inflammatory responses, animals lacking PTP1B exhibited a higher rate of death. Moreover, these animals showed an enhanced response to irradiation, in agreement with the data obtained in the microarray analysis. In summary, these results indicate that, although inhibition of PTP1B has potential benefits for the treatment of diabetes, it accentuates pro-inflammatory responses compromising at least macrophage viability.This work was supported by grants BFU2011-24760 and SAF2012-33283 from MINECO, S2010/BMD-2378 and S2010/BMD-2423 from Comunidad de Madrid and FIS-RIC RD06/0014/0025 and EFSD and Amylin Paul Langerhans Grant. Ciberdem and Ciberehd are funded by the Instituto de Salud Carlos III.Peer Reviewe

SIRTI is not required for the beneficial effects of resveratrol treatment on insulin sensitivity in diabetic mice

Resumen del póster presentado a la Conferencia del Centro Nacional de Investigaciones Cardiovasculares (CNIC) titulada: "Energy homeostasis and metabolic disease", celebrada en Madrid del 7 al 8 de noviembre de 2014.[Objectives]: Resveratrol, a plant-derived polyphenolic compound, is a potent activator of the histone deacetylase Sirtl. In fact, some studies have demonstrated that both resveratrol treatment and moderate increase of Sirtl levels improved insulin sensitivity. Mice with complete deletion of insulin receptor substrate (IRS) 2 develop hyperglycaemia, impaired hepatic insulin signalling and elevated gluconeogenesis. The elevated expression and activity of PTPIB in their liver block insulin receptor (IR)/IRSI -mediated insulin signalling. Accordingly, PTPI B inhibition by resveratrol treatment improves peripheral insulin sensitivity of these diabetic mice. As the mechanisms underlying the beneficial effects of resveratrol are not totally elucidated, we have investigated if resveratrol action in controlling insulin resistance is mediated by Sirt l activation. [Methods]: In the present study we will attempt to amilorate the peripheral insulin resistance of two different diabetic models (IRS2-deficient mice and streptozotocin (STZ)-induced diabetic mice) using resveratrol treatment or over-expressing Sirt I. [Results]: Resveratrol treatment improved systemic insulin sensitivity in hyperglycemic IRS2-deficient mice but did not change glucose tolerance due to the inability to revert beta cell failure. Conversely, moderate over-expression of Sirtl in IRS2-deficient mice did recover neither peripheral insulin resistance nor glucose intolerance. In both liver and muscle of IRS2-deficient mice levels of PTPIB were increased. Resveratrol treatment of IRS2-deficient mice significantly decreased PTPIB mRNA and inhibited its enzymatic activity in both tissues, thereby restoring IRSI -mediated insulin signalling. Conversely, moderate over-expression of Sirtl did not normalize PTPIB levels and, consequently, insulin signalling remained impaired. These results were confirmed in STZ-diabetic mice since resveratrol treatment improved peripheral insulin sensitivity and restored hepatic insulin signalling, but these effecrs were not recovered by the moderate over-expression of Sirt l. [Conclusions]: Our results suggest that the effects of resveratrol on insulin action in diabetic mice are not mediated by Sirt l activation.Peer Reviewe

Loss of protein tyrosine phosphatase 1B increases IGF-I receptor tyrosine phosphorylation but does not rescue retinal defects in IRS2-deficient mice

et al.[Purpose]: Mice with deletion of insulin receptor substrate (IRS) 2 develop type 2 diabetes and photoreceptor degeneration. Loss of protein tyrosine phosphatase 1B (PTP1B) in diabetic IRS2-/- mice restores insulin sensitivity and normalizes glucose homeostasis. Since insulinlike growth factor (IGF)-IR promotes survival of photoreceptors and is a substrate of PTP1B, we investigated IGF-IR-mediated survival signaling and visual function in PTP1B-/- and double mutant IRS2-/-/PTP1B-/- mice. [Methods]: IGF-IR-mediated Akt signaling was evaluated in IGF-I-stimulated retinal explants. Histologic and electroretinogram analysis was performed in wild-type (WT), IRS2-/-, PTP1B-/-, and the double mutant IRS2-/-/PTP1B/ mice. [Results]: IGF-I stimulated the tyrosine phosphorylation of its receptor and Akt activation in retinal explants of WT mice. In PTP1B-/- retinal explants, these responses were enhanced. Conversely, in retinas from IRS2-/- mice, expression of PTP1B was increased, coincident with decreased IGF-I-mediated Akt serine 473 phosphorylation. PTP1B deletion in IRS2 -/-mice also enhanced IGF-IR tyrosine phosphorylation but, unexpectedly, did not rescue Akt activation in response to IGF-I. One potential explanation is that PTEN was increased in retinas of IRS2-/- and IRS2-/-/PTP1B-/- mice. Histologic evaluation revealed alterations in various structures of the retina in IRS2-/- and IRS2-/-/PTP1B-/- mice, specifically in the outer nuclear layer (ONL) and retinal outer segments (ROS). Electroretinogram (ERG) analysis confirmed that PTP1B deficiency did not restore visual function in IRS2-/- mice. [Conclusions]: Although loss of PTP1B enhances tyrosine phosphorylation of the IGF-IR in retinal explants of IRS2-/- mice, Akt activation remains defective owing to elevated PTEN levels and, thus, structural and functional visual defects persist in this model. © 2013 The Association for Research in Vision and Ophthalmology, Inc.Supported by Grants SAF2012-33283, SAF2010-21879-C02-01 (MINECO, Spain), NEURORET-DIAB from CIBERDEM (Centro de Investigacion Biomedica en Red de Diabetes y Enfermedades Metabolicas Asociadas, Instituto Salud Carlos III, Spain), Comunidad de Madrid S2010/BMD-2423, and The European Consortium for the Early Treatment of Diabetic Retinopathy (278040 PCOLSMALL), a project funded by the FP7 Program of the European Community. AIA, JR-C, and AG-R hold postdoctoral contracts from CIBERDEM. ThPeer Reviewe