FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1983113172> ?p ?o ?g. }

• W1983113172 endingPage "62" @default.
• W1983113172 startingPage "53" @default.
• W1983113172 abstract "NCB5OR is a novel flavoheme reductase with a cytochrome b5-like domain at the N-terminus and a cytochrome b5 reductase-like domain at the C terminus. Ncb5or knock-out mice develop insulin deficient diabetes and loss of white adipose tissue. Ncb5or−/− mice have impairment of Δ9 fatty acid desaturation with elevated ratios of palmitate to palmitoleate and stearate to oleate. In this study we assess the role of the endoplasmic reticulum (ER) stress response in mediating lipotoxicity in Ncb5or−/− mice. The ER stress response was assessed by induction of BiP, ATF3, ATF6, XBP-1, and C/EBP homologous protein (CHOP). Exposure to palmitate, but not oleate or mixtures of oleate and palmitate induced these markers of ER stress to a much greater extent in Ncb5or−/− hepatocytes than in wild-type cells. In contrast, Ncb5or−/− and Ncb5or+/+ hepatocytes were equally sensitive to ER stress imposed by increasing concentrations of tunicamycin. In order to assess the role of ER stress in vivo, we prepared mice that lack both NCB5OR and CHOP, a proapoptotic transcription factor important in the ER stress response. Onset of hyperglycemia in the Chop−/−;Ncb5or−/− mice was delayed two weeks beyond that observed in Chop+/+;Ncb5or−/− mice. Taken together these results suggest that ER stress plays a critical role in palmitate-induced lipotoxicity both in vitro and in vivo. NCB5OR is a novel flavoheme reductase with a cytochrome b5-like domain at the N-terminus and a cytochrome b5 reductase-like domain at the C terminus. Ncb5or knock-out mice develop insulin deficient diabetes and loss of white adipose tissue. Ncb5or−/− mice have impairment of Δ9 fatty acid desaturation with elevated ratios of palmitate to palmitoleate and stearate to oleate. In this study we assess the role of the endoplasmic reticulum (ER) stress response in mediating lipotoxicity in Ncb5or−/− mice. The ER stress response was assessed by induction of BiP, ATF3, ATF6, XBP-1, and C/EBP homologous protein (CHOP). Exposure to palmitate, but not oleate or mixtures of oleate and palmitate induced these markers of ER stress to a much greater extent in Ncb5or−/− hepatocytes than in wild-type cells. In contrast, Ncb5or−/− and Ncb5or+/+ hepatocytes were equally sensitive to ER stress imposed by increasing concentrations of tunicamycin. In order to assess the role of ER stress in vivo, we prepared mice that lack both NCB5OR and CHOP, a proapoptotic transcription factor important in the ER stress response. Onset of hyperglycemia in the Chop−/−;Ncb5or−/− mice was delayed two weeks beyond that observed in Chop+/+;Ncb5or−/− mice. Taken together these results suggest that ER stress plays a critical role in palmitate-induced lipotoxicity both in vitro and in vivo. Genome scans have uncovered multiple loci that are linked to both type 1 (1Bartsocas C.S. Gerasimidi-Vazeou A. Genetics of type 1 diabetes mellitus.Pediatr. Endocrinol. Rev. 2006; 3: 508-513PubMed Google Scholar, 2Alizadeh B.Z. Koeleman B.P. Genetic polymorphisms in susceptibility to Type 1 diabetes.Clin. Chim. Acta. 2008; 387: 9-17Crossref PubMed Scopus (23) Google Scholar) and type 2 (3Doria A. Patti M.E. Kahn C.R. The emerging genetic architecture of type 2 diabetes.Cell Metab. 2008; 8: 186-200Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar) diabetes. In addition a number of other gene products play essential roles in β-cell function and viability. NCB5OR, also known as b5+b5R and Cyb5r4, is a novel, highly conserved NAD(P)H flavoheme reductase that contains a cytochrome b5-like domain at the N-terminus and a cytochrome b5 reductase-like domain at the C terminus (4Zhu H. Qiu H. Yoon H.W. Huang S. Bunn H.F. Identification of a cytochrome b-type NAD(P)H oxidoreductase ubiquitously expressed in human cells.Proc. Natl. Acad. Sci. USA. 1999; 96: 14742-14747Crossref PubMed Scopus (89) Google Scholar, 5Zhu H. Larade K. Jackson T.A. Xie J. Ladoux A. Acker H. Berchner-Pfannschmidt U. Fandrey J. Cross A.R. Lukat-Rodgers G.S. et al.NCB5OR is a novel soluble NAD(P)H reductase localized in the endoplasmic reticulum.J. Biol. Chem. 2004; 279: 30316-30325Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). It is localized in the endoplasmic reticulum (ER) and is ubiquitously expressed, with relatively high mRNA levels in the pancreas, heart, and kidney. Ncb5or−/− mice are glucose intolerant at ∼4 weeks of age and at ∼6 weeks of age develop frank diabetes (6Xie J. Zhu H. Larade K. Ladoux A. Seguritan A. Chu M. Ito S. Bronson R.T. Leiter E.H. Zhang C.Y. et al.Absence of a reductase, NCB5OR, causes insulin-deficient diabetes.Proc. Natl. Acad. Sci. USA. 2004; 101: 10750-10755Crossref PubMed Scopus (42) Google Scholar). Serum insulin levels decrease with age, mirroring the reduced insulin content and progressive loss of β-cells in pancreatic islets. In addition, following birth, Ncb5or−/− mice develop lipoatrophy with progressive loss of white adipose tissue. This metabolic defect is observed even when diabetes has been prevented by early insulin treatment and pancreatic islet transplantation (7Larade K. Jiang Z. Zhang Y. Wang W. Bonner-Weir S. Zhu H. Bunn H.F. Loss of NCB5OR results in impaired fatty acid desaturation, lipoatrophy and diabetes.J. Biol. Chem. 2008; 283: 29285-29291Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). In keeping with the above-mentioned homology with classical cytochrome b5 and cytochrome b5 reductase, we investigated whether NCB5OR might provide an alternative source of electrons for fatty acid desaturation and recently reported that, indeed, Ncb5or−/− mice have a defect in the Δ-9 pathway (7Larade K. Jiang Z. Zhang Y. Wang W. Bonner-Weir S. Zhu H. Bunn H.F. Loss of NCB5OR results in impaired fatty acid desaturation, lipoatrophy and diabetes.J. Biol. Chem. 2008; 283: 29285-29291Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). We found that the triglycerides, diacylglycerides, free fatty acids, and cholesterol esters in the liver of knock-out mice have 50–80% reduction in the ratios of palmitoleate to palmitate and oleate to stearate. This finding led to the hypothesis that diabetes in Ncb5or−/− mice is due at least in part to toxic effects of saturated fatty acids. Although it was not technically feasible to carry out in vitro experiments on isolated pancreatic β-cells, we did show that the viability of isolated Ncb5or−/− hepatocytes was markedly impaired and apoptosis enhanced by incubation with palmitate but not with oleate or a mixture of palmitate and oleate (7Larade K. Jiang Z. Zhang Y. Wang W. Bonner-Weir S. Zhu H. Bunn H.F. Loss of NCB5OR results in impaired fatty acid desaturation, lipoatrophy and diabetes.J. Biol. Chem. 2008; 283: 29285-29291Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). There is growing evidence that in a number of cell types (8Borradaile N.M. Buhman K.K. Listenberger L.L. Magee C.J. Morimoto E.T. Ory D.S. Schaffer J.E. A critical role for eukaryotic elongation factor 1A–1 in lipotoxic cell death.Mol. Biol. Cell. 2006; 17: 770-778Crossref PubMed Scopus (120) Google Scholar, 9Borradaile N.M. Han X. Harp J.D. Gale S.E. Ory D.S. Schaffer J.E. Disruption of endoplasmic reticulum structure and integrity in lipotoxic cell death.J. Lipid Res. 2006; 47: 2726-2737Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar), including pancreatic β-cells (10Diakogiannaki E. Welters H.J. Morgan N.G. Differential regulation of the endoplasmic reticulum stress response in pancreatic beta-cells exposed to long-chain saturated and monounsaturated fatty acids.J. Endocrinol. 2008; 197: 553-563Crossref PubMed Scopus (102) Google Scholar), the ER is a target of lipotoxicity induced by palmitate. Saturated fatty acids have been shown to induce stress in the ER, triggering a set of intracellular events initially designated the unfolded protein response (11Kaufman R.J. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls.Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1944) Google Scholar, 12Mori K. Tripartite management of unfolded proteins in the endoplasmic reticulum.Cell. 2000; 101: 451-454Abstract Full Text Full Text PDF PubMed Scopus (790) Google Scholar, 13Zhang K. Kaufman R.J. Signaling the unfolded protein response from the endoplasmic reticulum.J. Biol. Chem. 2004; 279: 25935-25938Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar). Pancreatic β-cells are particularly prone to ER stress (14Harding H.P. Ron D. Endoplasmic reticulum stress and the development of diabetes: a review.Diabetes. 2002; 51: S455-S461Crossref PubMed Google Scholar, 15Araki E. Oyadomari S. Mori M. Endoplasmic reticulum stress and diabetes mellitus.Intern. Med. 2003; 42: 7-14Crossref PubMed Scopus (137) Google Scholar, 16Laybutt D.R. Preston A.M. Akerfeldt M.C. Kench J.G. Busch A.K. Biankin A.V. Biden T.J. Endoplasmic reticulum stress contributes to beta cell apoptosis in type 2 diabetes.Diabetologia. 2007; 50: 752-763Crossref PubMed Scopus (660) Google Scholar, 17Huang C.J. Lin C.Y. Haataja L. Gurlo T. Butler A.E. Rizza R.A. Butler P.C. High expression rates of human islet amyloid polypeptide induce endoplasmic reticulum stress mediated beta-cell apoptosis, a characteristic of humans with type 2 but not type 1 diabetes.Diabetes. 2007; 56: 2016-2027Crossref PubMed Scopus (341) Google Scholar). An ER chaperone protein, BiP (also known as GRP78) serves as sensor and master regulator of the ER stress response. In the unstressed cell, BiP binds to three effector transmembrane ER proteins, the kinase and endoRNase IRE-1, the basic leucine-zipper activated transcription factor 6 (ATF-6), and the double stranded RNA-activated protein kinase-like ER kinase (PERK). When unfolded protein accumulates in the ER, it binds competitively to BiP, thereby freeing up IRE-1, PERK, and ATF-6 so that they homodimerize and are activated by transphosphorylation. Activated IRE-1 has RNase activity that cleaves an mRNA precursor of X-box binding protein (XBP)-1, enabling a frameshift that encodes mature XBP-1. This transcription factor regulates a subset of ER-resident chaperone genes that are essential for protein folding, maturation, and degradation in the ER, including CHOP (C/EBP homologous protein) (18Wang X.Z. Lawson B. Brewer J.W. Zinszner H. Sanjay A. Mi L.J. Boorstein R. Kreibich G. Hendershot L.M. Ron D. Signals from the stressed endoplasmic reticulum induce C/EBP-homologous protein (CHOP/GADD153).Mol. Cell. Biol. 1996; 16: 4273-4280Crossref PubMed Scopus (610) Google Scholar). ATF-6 regulates a group of genes encoding ER-resident molecular chaperones and genes encoding folding enzymes. In this paper, we have investigated the contribution of the ER stress response to enhanced lipotoxicity in Ncb5or−/− hepatocytes by assaying expression of BiP, mature XBP-1, CHOP, ATF-6, and a related transcription factor ATF-3 (19Pan Y. Chen H. Siu F. Kilberg M.S. Amino acid deprivation and endoplasmic reticulum stress induce expression of multiple activating transcription factor-3 mRNA species that, when overexpressed in HepG2 cells, modulate transcription by the human asparagine synthetase promoter.J. Biol. Chem. 2003; 278: 38402-38412Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), all of which are upregulated during ER stress. In addition, we have assessed the in vivo contribution of CHOP expression to the development of diabetes in Ncb5or−/− mice. Anti-GRP78/BiP (GL-19) antibody was obtained from Sigma (St Louis, MO); anti-SCD1 (S-15) and anti-cytochrome b5 (H-114), anti-XBP-1 (M-186), anti-CHOP (GADD153 B-3), and anti-ATF3 (H-90) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-β-actin antibody from Ambion, Inc. (Austin, TX). Palmitic acid, oleic acid, fatty acid free BSA and tunicamycin were purchased from Sigma. FFA stock solutions were prepared in 5% FFA-free BSA as previously described (7Larade K. Jiang Z. Zhang Y. Wang W. Bonner-Weir S. Zhu H. Bunn H.F. Loss of NCB5OR results in impaired fatty acid desaturation, lipoatrophy and diabetes.J. Biol. Chem. 2008; 283: 29285-29291Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) and stored in liquid nitrogen to prevent oxidation. The final concentration of fatty acid was measured using a NEFA-HR [2] kit (Wako Chemicals, Richmond, VA), and adjusted to 5 mM. Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. In each reaction, 1μg total mRNA was used as template to perform RT-PCR using the Access RT-PCR system (Promega, Madison, WI). Primers used for RT-PCR include: XBP-1 forward: 5′-TTA CGG GAG AAA ACT CAC GGC-3′, XBP-1 reverse: 5′-GGG TCC AAC TTG TCC AGA ATG C-3′(20Lin J.H. Li H. Yasumura D. Cohen H.R. Zhang C. Panning B. Shokat K.M. Lavail M.M. Walter P. IRE1 signaling affects cell fate during the unfolded protein response.Science. 2007; 318: 944-949Crossref PubMed Scopus (1074) Google Scholar), Chop forward: 5′- GAA AGC AGA ACC TGG TCC ACG T-3′, Chop reverse: 5′-ATG TGC GTG TGA CCT CTG TTG- 3′ (21Shirakawa K. Maeda S. Gotoh T. Hayashi M. Shinomiya K. Ehata S. Nishimura R. Mori M. Onozaki K. Hayashi H. et al.CCAAT/enhancer-binding protein homologous protein (CHOP) regulates osteoblast differentiation.Mol. Cell. Biol. 2006; 26: 6105-6116Crossref PubMed Scopus (72) Google Scholar). For analysis of Xbp-1 splicing, a 289 bp amplicon was generated from unspliced Xbp-1; a 263 bp amplicon was generated from spliced Xbp-1. PCR products were separated on a 2.5% agarose gel and stained with ethidium bromide. For quality control, the RNA samples were analyzed by 1.5% agarose electrophoresis. The cDNA was synthesized using a High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA). Each PCR reaction (1μg total RNA) was performed using optical 96-well reaction plates (Applied Biosystems) and the ABI Prism 7300 sequence detection machine employing SYBR Green PCR Master Mix (Applied Biosystems). The primer sequences used were as follows: GAPDH forward: 5′-TGT GTC CGT CGT GGA TCT GA-3′; GAPDH reverse: 5′-CCT GCT TCA CCA CCT TCT TGA-3′; GADD153(Chop) forward: 5′-ATG AAG GAG AAG GAG CAG GAG AA-3′; GADD153(Chop) reverse: 5′-CTT GGT GCA GGC TGA CCA TG-3′; ATF3 forward: 5′-CGC CAT CCA GAA TAA ACA CC-3′; ATF3 reverse: 5′-GCAGGCACTCTGTCTTCTCC-3′; ATF6 forward: 5′-GGA TTT GAT GCC TTG GGA GTC AGA C-3′; ATF6 reverse: 5′-ATT TTT TTC TTT GGA GTC AGT CCA T-3′. All measurements were carried out in 20 μl volumes in triplicate, using mouse GAPDH mRNA levels for normalization. Average Ct values were recorded in triplicate and mRNA-level differences were calculated assuming that every cycle doubled the fluorescent intensity. To verify specificity, the products were analyzed by their melting curves and gel electrophoresis. Cells were collected at specified time points during the incubations. After washing with PBS, cells were lysed in RIPA lysis buffer (Santa Cruz Biotechnology). Lysates were cleared by centrifugation and after determining protein concentration of the supernatants, 30 μg samples were prepared and separated on SDS-PAGE, transferred to polyvinylidene fluoride membrane (Millipore), and immunoblotted with the indicated primary antibody. β-actin protein expression was used as an internal loading standard. Signals were detected by ECL Plus Western Blot Detection System (Piscataway, NJ) and exposed on Fuji Medical X-ray film (Fujifilm Corporation, Tokyo, Japan). Mouse hepatocytes were prepared from 3- to 4-week-old wild-type and Ncb5or−/− male and female animals by the two-step perfusion procedure using Liver Perfusion Medium (Invitrogen) and Liver Digestion Medium (Invitrogen) separately as described previously (7Larade K. Jiang Z. Zhang Y. Wang W. Bonner-Weir S. Zhu H. Bunn H.F. Loss of NCB5OR results in impaired fatty acid desaturation, lipoatrophy and diabetes.J. Biol. Chem. 2008; 283: 29285-29291Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). After isolation, hepatocytes were cultured in Williams’ medium E (Invitrogen) containing penicillin/streptomycin and glutamine for 12 h before any treatment. The method we are currently using for hepatocyte isolation is considerably improved over that which we had previously employed and reported (7Larade K. Jiang Z. Zhang Y. Wang W. Bonner-Weir S. Zhu H. Bunn H.F. Loss of NCB5OR results in impaired fatty acid desaturation, lipoatrophy and diabetes.J. Biol. Chem. 2008; 283: 29285-29291Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), both in yield and cell viability. We are now able to culture wild-type hepatocytes over a 24 h incubation period with <20% cell loss. After preincubation, cells were treated in Williams’ medium E containing: fatty acid free BSA only (control) or with different concentrations of palmitate or oleate as indicated. Adequate fatty acid free BSA was supplemented into the medium and adjusted to a final concentration of 0.5%. Wild-type cells treated for 12 h with tunicamycin (1μg/ml) were used as a positive control. In the tunicamycin sensitivity experiment, after preincubation, isolated cells were treated for 12 h with vehicle (DMSO) control or different concentrations of tunicamycin (0.2, 1, and 5 μg/ml). Isolated hepatocytes were analyzed following incubation with fatty acid to detect apoptosis by using PE-Annexin V staining (ApoAlert Annexin V Apoptosis Kit, Clontech, Palo Alto, CA), and an in situ cell death detection kit (Fluorescein, Roche Applied Science). Cells were washed with cold PBS and incubated with PE-Annexin V. PE-Annexin V stained positive cells were determined by fluorescence microscopy (Nikon Eclipse TE300 microscope) using a rhodamine filter. TUNEL positive cells were identified and counted as described previously (7Larade K. Jiang Z. Zhang Y. Wang W. Bonner-Weir S. Zhu H. Bunn H.F. Loss of NCB5OR results in impaired fatty acid desaturation, lipoatrophy and diabetes.J. Biol. Chem. 2008; 283: 29285-29291Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The isolated hepatocytes were cultured in 6-well culture dishes and treated accordingly. At appropriate times, the liver cells that were attached to the bottom of the wells were fixed with 2.5% glutaraldehyde, 2% paraformaldehyde, and 0.02% picric acid in 0.1 M cacodylate buffer. While still in the wells, the cells were post fixed with 1% osmium tetroxide and 1.5% potassium ferrocyanide for one h. After washing in water and 0.5% maleate buffer (pH 5.2), the cells were treated with 1% uranyl acetate in maleate buffer and washed with water. The cells in the wells were dehydrated with graded cold ethanol, warmed to room temperature, and while in the last absolute ethanol dehydration step, each well was scored with a fine dissecting needle into tiny squares of 1 to 2 mm. In the next step, when propylene oxide was added, the solution was repeatedly flushed with the pipette until the cells were detached from the culture dish and transferred to a centrifuge tube and centrifuged into a pellet. The pellet was then treated like a piece of tissue and embedded in Epon Araldite. Thin sections were placed on bare grids and stained with saturated uranyl acetate in equal parts of acetone. Following lead citrate and staining, sections were examined and images were digitally recorded with a JEOL 1200 EX electron microscope. All procedures involving animals were approved by the Animal Care and Use Committee of Brigham and Women’s Hospital and Children’s Hospital in Boston. Mice were housed in a pathogen-free barrier facility on a 12 h light/12 h dark cycle on a standard rodent chow (Prolab Isopro RMH 3000 5P76). Ncb5or−/− mice, in which exon 4 was replaced with a Pgk-Hyg expression cassette, have been described previously (6Xie J. Zhu H. Larade K. Ladoux A. Seguritan A. Chu M. Ito S. Bronson R.T. Leiter E.H. Zhang C.Y. et al.Absence of a reductase, NCB5OR, causes insulin-deficient diabetes.Proc. Natl. Acad. Sci. USA. 2004; 101: 10750-10755Crossref PubMed Scopus (42) Google Scholar). Ncb5or-deficient mice used in the present study were established on a virtually pure C57BL/6 (B6) background (>12 generations). Chop+/− mice on a B6 background (22Zinszner H. Kuroda M. Wang X. Batchvarova N. Lightfoot R.T. Remotti H. Stevens J.L. Ron D. CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum.Genes Dev. 1998; 12: 982-995Crossref PubMed Scopus (1690) Google Scholar) were obtained from the Jackson Laboratory (Bar Harbor, ME). Ncb5or+/− heterozygotes bred into a pure B6 background were crossed with homozygous Chop KO mice (Chop−/−), generating Chop+/−;Ncb5or+/− and Chop+/−;Ncb5or+/+ mice. Chop+/−;Ncb5or+/− mice were crossed with Chop+/−;Ncb5or+/− or Chop−/−;Ncb5or+/− mice, producing each of the genotypes in this study, including Chop+/+;Ncb5or+/+, Chop+/+;Ncb5or−/−, Chop−/−;Ncb5or+/+, and Chop−/−;Ncb5or−/−. Mice were genotyped by PCR using the following primers: i) Ncb5or: 5′-GTG AAC ACT AAT GCA TAC TCC CAG TCT GTG ATG C-3′; 5′-TGG AAC AGC AGG CTT CAC AGC- 3′; and 5′-GCG CCT ACC GGT GGA TGT GGA A-3′; ii) Chop forward: 5′- GCA GCC ATG GCA GCT GAG TCC CTG CCT TCC-3′; Chop reverse: 5′- CAG ACT CGA GGT GAT GCC CAC TGT TCA TGC– 3′. The thermal cycle reaction was performed as follows: 94°C for 2 min, followed by 35 cycles at 94°C for 15 s, 55°C for 30 s, 68°C for 2 min, and an additional step of 72°C for 7 min at the end. Blood glucose concentration was measured using the One Touch blood glucose monitoring system (Lifescan). Intraperitoneal glucose tolerance tests (GTT) were performed after a 10 to 12 h fast (2 mg dextrose/g body weight). Blood samples for GTT were obtained from tail veins at 0, 15, 30, 60, and 120 min after injection. Tissue for analysis was fixed in either buffered 4% paraformaldehyde solution or Bouin’s solution, embedded in paraffin, and sectioned for analysis. Slides were stained with hematoxylin and eosin (H-E) for islet identification and analysis. Insulin was detected by guinea pig anti-human insulin (Linco) diluted 1:100, followed by incubation with peroxidase-conjugated AffiniPure goat anti-rabbit Ig (H+L) (Jackson ImmunoResearch) diluted 1:1000. A 3,3-diaminobenzidine tetrahydrochloride (DAB) staining kit (Vector) was used as the substrate for peroxidase. Sections were counterstained by hematoxylin. All of the error bars shown in the figures are mean ± standard deviation. Statistical significance was calculated by the Student’s t-test. We previously reported that primary hepatocytes from Ncb5or−/− (KO) mice had impaired viability and enhanced apoptosis, analyzed by TUNEL, following 1 h and 4 h exposure to 1.0 mM palmitate but not to 1.0 mM oleate or an equimolar mixture of the two fatty acids (7Larade K. Jiang Z. Zhang Y. Wang W. Bonner-Weir S. Zhu H. Bunn H.F. Loss of NCB5OR results in impaired fatty acid desaturation, lipoatrophy and diabetes.J. Biol. Chem. 2008; 283: 29285-29291Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). With a greatly improved method for cell isolation, we are now able to obtain cells at much higher yields and improved viability, allowing longer incubation times. Supplementary Fig. I shows responses to lower, more physiologic levels of palmitate: 0.1, 0.25, and 0.5 mM, following a 12 h incubation. Increased cell death, as assessed by trypan blue exclusion, and enhanced apoptosis, as determined by Annexin V staining and TUNEL, were induced in KO but not in wild-type (WT) cells by a 12 h exposure to as little as 0.1 mM palmitate. Primary hepatocytes are inherently heterogeneous. Even with an improved isolation method, 25% of WT and KO cells were nonviable in the absence of exposure to palmitate and following incubation with 0.5 mM palmitate, 65% of KO cells were nonviable. The experiments reported below pertain to cells that remained viable after 12 h and 24 h incubations. The ER stress response in cells generally includes induction of a number of transcripts including spliced XBP-1 and the CHOP transcription factor. Tunicamycin, an agent that causes a marked impairment in N-linked glycosylation of proteins, is commonly used as a positive control to trigger the ER stress response. As shown in Fig. 1A, tunicamycin induced both the spliced XBP-1 transcript and Chop mRNA in WT hepatocytes. Treatment with 0.5 mM palmitate induced both the spliced XBP-1 (Fig. 1A) and Chop mRNA (Fig. 1A and B) in KO cells but not in WT cells. In contrast, treatment with 0.5 mM oleate failed to induce either the spliced XBP-1 or Chop transcripts, a result consistent with the impairment of cell viability and enhancement of apoptosis imposed by saturated palmitate [supplementary Fig. I and Larade et al. (7Larade K. Jiang Z. Zhang Y. Wang W. Bonner-Weir S. Zhu H. Bunn H.F. Loss of NCB5OR results in impaired fatty acid desaturation, lipoatrophy and diabetes.J. Biol. Chem. 2008; 283: 29285-29291Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar)] but not by monounsaturated oleate (7Larade K. Jiang Z. Zhang Y. Wang W. Bonner-Weir S. Zhu H. Bunn H.F. Loss of NCB5OR results in impaired fatty acid desaturation, lipoatrophy and diabetes.J. Biol. Chem. 2008; 283: 29285-29291Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). In like manner, as shown in Figs. 1C and D, palmitate induced mRNA expression of two other markers of ER stress, ATF3 and ATF6, in KO cells but not in WT cells. In contrast, no significant induction was noted in either WT type or KO cells following exposure to oleate. We also examined markers of ER stress at the protein level. As shown in Fig. 2A, following incubation with 0.5 mM palmitate, BiP, the master regulator of the ER stress response (see above), was induced in KO cells but not in WT cells. As expected, treatment of wild-type hepatocytes with tunicamycin (1 μg/ml for 12 h) also induced BiP along with the other markers of ER stress shown in this figure. The level of BiP protein was further enhanced in KO cells by a longer (24 h) incubation (not shown). Palmitate incubation also induced expression of the 50 kDa N-terminal cleavage product of ATF6 (23Chen X. Shen J. Prywes R. The luminal domain of ATF6 senses endoplasmic reticulum (ER) stress and causes translocation of ATF6 from the ER to the Golgi.J. Biol. Chem. 2002; 277: 13045-13052Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar), as well as CHOP and ATF3. In like manner, as shown in Fig. 2B, palmitate induced XBP-1 protein in KO but not in WT hepatocytes. Stearoyl CoA desaturase-1 (SCD-1) and microsomal cytochrome b5 (Cyt b5) protein expression were induced somewhat by palmitate in both KO and WT cells whereas expression in WT cells was not affected by tunicamycin. KO and WT hepatocytes were incubated for 12 h with 0, 0.1, 0.25, and 0.5 mM palmitate. No significant induction of the spliced XBP-1 mRNA, nor of Chop, ATF3, or ATF6 mRNAs were noted with 0.1 mM palmitate whereas exposure to 0.25 and 0.5 mM palmitate resulted in robust induction of all four markers of ER stress (Fig. 3). This dose response closely mimics the effect of increasing levels of palmitate on cell viability and apoptosis shown in Supplementary Fig. I. We examined ultrastructure of hepatocytes from three WT and three KO mice. Cells were incubated for 4, 12, and 24 h with 0.5 mM palmitate in 0.5% albumin versus the same concentration of lipid-depleted albumin. The integrity of the majority of the cells remained fully intact in all samples. Not surprisingly, WT and KO cells incubated in palmitate contained increased numbers of lipid droplets, many of which appeared crystalline. In all samples mitochondrial morphology and number were normal. No abnormalities were seen in cells incubated for 4 h and 12 h. Following 24-h incubation in palmitate, a substantial fraction of KO cells had only small amounts of rough ER with distension of the cisternae and decreased numbers of polyribosomes (Fig. 4B). In these cells, there were increased amounts of agranular or smooth surfaced ER, which were tubular in form. These changes were considerably less prominent in both KO cells treated with 0.5% albumin (Fig. 4A) and in WT cells treated with palmitate (Fig. 4C). A 12 h exposure of KO hepatocytes to 0.5 mM palmitate induced Chop mRNA as shown in Figs. 1A, 1B, and 5A, lane 2. However, addition of increasing concentrations of oleate to the palmitate solution resulted in a reduction in Chop mRNA expression (Fig. 5A). At equimolar concentrations of palmitate and oleate (0.5 mM each), the expression of Chop mRNA was indistinguishable from that seen in cells not exposed to added fatty acids. In like manner, as shown in Fig. 5B, coincubation with increasing amounts of oleate gradually lowered the induction of CHOP and XBP-1 protein. ER stress imposed by palmitate can be reversed by subsequent exposure to oleate. KO hepatocytes were first incubated for 24 h in 0.5 mM palmitate and then incubated for an additional 12 h in either 0, 0.1, 0.25, or 0.5 mM oleate. As shown in Fig. 5C, the induction of ATF6 mRNA was gradually abolished by the addition of increasing concentrations of oleate. As mentioned above, inhibition of N-linked glycosylation by the agent tunicamycin impairs proper folding and trafficking of glycoproteins and is frequently used to assess cytologic and biochemical markers of the ER stress response. We exposed isolated hepatocytes to 12 h incubations in increasing concentrations of tunicamycin (0, 0.2, 1.0, and 5.0 μg/ml) and, as shown in Fig. 6, observed dose-dependent increases in expression of Chop, ATF3 and ATF6 mRNAs and no difference between KO and WT cells in their sensitivity to tunicamycin. As shown in Supplementary Fig. IIA, following 12-h incubation of WT hepatocytes with increasing concentrations of palmitate (0.1, 0.25 and 0.5 mM), Ncb5or mRNA expression was enhance" @default.
• W1983113172 created "2016-06-24" @default.
• W1983113172 creator A5006526982 @default.
• W1983113172 creator A5033134478 @default.
• W1983113172 creator A5048068295 @default.
• W1983113172 creator A5052103601 @default.
• W1983113172 creator A5058999827 @default.
• W1983113172 creator A5061636400 @default.
• W1983113172 creator A5079586983 @default.
• W1983113172 date "2010-01-01" @default.
• W1983113172 modified "2023-10-17" @default.
• W1983113172 title "The flavoheme reductase Ncb5or protects cells against endoplasmic reticulum stress-induced lipotoxicity" @default.
• W1983113172 cites W1570888466 @default.
• W1983113172 cites W1594016328 @default.
• W1983113172 cites W1970121621 @default.
• W1983113172 cites W1976829005 @default.
• W1983113172 cites W1982871405 @default.
• W1983113172 cites W1986836799 @default.
• W1983113172 cites W1997413214 @default.
• W1983113172 cites W2000601548 @default.
• W1983113172 cites W2024475665 @default.
• W1983113172 cites W2029642537 @default.
• W1983113172 cites W2042186381 @default.
• W1983113172 cites W2056689556 @default.
• W1983113172 cites W2062574758 @default.
• W1983113172 cites W2063363508 @default.
• W1983113172 cites W2069924169 @default.
• W1983113172 cites W2076597761 @default.
• W1983113172 cites W2089894176 @default.
• W1983113172 cites W2099125910 @default.
• W1983113172 cites W2100236190 @default.
• W1983113172 cites W2100347985 @default.
• W1983113172 cites W2107601804 @default.
• W1983113172 cites W2108554272 @default.
• W1983113172 cites W2110098472 @default.
• W1983113172 cites W2116287353 @default.
• W1983113172 cites W2116380331 @default.
• W1983113172 cites W2117881262 @default.
• W1983113172 cites W2122647692 @default.
• W1983113172 cites W2129630264 @default.
• W1983113172 cites W2133122560 @default.
• W1983113172 cites W2133771702 @default.
• W1983113172 cites W2153965694 @default.
• W1983113172 cites W2159466552 @default.
• W1983113172 cites W2163725144 @default.
• W1983113172 doi "https://doi.org/10.1194/jlr.m900146-jlr200" @default.
• W1983113172 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2789786" @default.
• W1983113172 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19609006" @default.
• W1983113172 hasPublicationYear "2010" @default.
• W1983113172 type Work @default.
• W1983113172 sameAs 1983113172 @default.
• W1983113172 citedByCount "23" @default.
• W1983113172 countsByYear W19831131722012 @default.
• W1983113172 countsByYear W19831131722013 @default.
• W1983113172 countsByYear W19831131722014 @default.
• W1983113172 countsByYear W19831131722015 @default.
• W1983113172 countsByYear W19831131722016 @default.
• W1983113172 countsByYear W19831131722017 @default.
• W1983113172 countsByYear W19831131722018 @default.
• W1983113172 countsByYear W19831131722019 @default.
• W1983113172 countsByYear W19831131722021 @default.
• W1983113172 crossrefType "journal-article" @default.
• W1983113172 hasAuthorship W1983113172A5006526982 @default.
• W1983113172 hasAuthorship W1983113172A5033134478 @default.
• W1983113172 hasAuthorship W1983113172A5048068295 @default.
• W1983113172 hasAuthorship W1983113172A5052103601 @default.
• W1983113172 hasAuthorship W1983113172A5058999827 @default.
• W1983113172 hasAuthorship W1983113172A5061636400 @default.
• W1983113172 hasAuthorship W1983113172A5079586983 @default.
• W1983113172 hasBestOaLocation W19831131721 @default.
• W1983113172 hasConcept C134018914 @default.
• W1983113172 hasConcept C134651460 @default.
• W1983113172 hasConcept C139447449 @default.
• W1983113172 hasConcept C158617107 @default.
• W1983113172 hasConcept C181199279 @default.
• W1983113172 hasConcept C185592680 @default.
• W1983113172 hasConcept C2777391703 @default.
• W1983113172 hasConcept C44353895 @default.
• W1983113172 hasConcept C511355011 @default.
• W1983113172 hasConcept C55493867 @default.
• W1983113172 hasConcept C86803240 @default.
• W1983113172 hasConcept C95444343 @default.
• W1983113172 hasConceptScore W1983113172C134018914 @default.
• W1983113172 hasConceptScore W1983113172C134651460 @default.
• W1983113172 hasConceptScore W1983113172C139447449 @default.
• W1983113172 hasConceptScore W1983113172C158617107 @default.
• W1983113172 hasConceptScore W1983113172C181199279 @default.
• W1983113172 hasConceptScore W1983113172C185592680 @default.
• W1983113172 hasConceptScore W1983113172C2777391703 @default.
• W1983113172 hasConceptScore W1983113172C44353895 @default.
• W1983113172 hasConceptScore W1983113172C511355011 @default.
• W1983113172 hasConceptScore W1983113172C55493867 @default.
• W1983113172 hasConceptScore W1983113172C86803240 @default.
• W1983113172 hasConceptScore W1983113172C95444343 @default.
• W1983113172 hasIssue "1" @default.
• W1983113172 hasLocation W19831131721 @default.
• W1983113172 hasLocation W19831131722 @default.
• W1983113172 hasLocation W19831131723 @default.

• next